ATPases - Springer Link

2 downloads 0 Views 1018KB Size Report
Pitfalls of ATPase studies. 3. AT1 ~ hydrolysis and synthesis. 4. Structure-function relationships in transport ATPases. 5. Type and specificity of interaction with ...

REVIEW Folia Mierobiol. 27, 195--210 (1982)

ATPases: Common and Unique Features within a Group of Enzymes K. Smv.~.R Department of Cell Physiology. Institute of Microbioloyy, Czechoslovak Academy of Sciences, 142 20 Prague 4 Received July 2, 1981

ARSTRACIF. A n a t t e m p t is m a d e to s u r v e y A T P a s e s w i t h respect to features c o m m o n to all or some of t h e m a n d features peculiar to each individual e n z y m e of the group. Clues are presented for a t e n t a t i v e classification of A T P a s e s a n d a simple s y s t e m is suggested for t h e designation of interaction of A T P a s e s w i t h ions which is often used as tim m a i n feature for identification of individual ATPases.

Contents 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.

Introduction Pitfalls of A T P a s e studies AT1~ hydrolysis a n d s y n t h e s i s Structure-function relationships in t r a n s p o r t A T P a s e s T y p e a n d specificity of interaction with ions Designation of A T P a s e s S u b s t r a t e specificity a n d o p t i m u m p H F o r m a t i o n of a stable p h o s p h o e n z y m e intermediate T y p e a n d sequence of individual A T P a s e reacti~a steps Molar mass, structure, s u b u i n t s Localization with regard to m e m b r a n e s t r u c t u r e s Sensitivity to inhibiters, activators a n d other substances l:r in the cell or organelle Chins for possible A T P a s e classification

1. Introduction

ATPases* (adenosinetriphosphatases, A T P phosphohydrolases, EC are enzymes with a central position in the bioenergetics of organelles, ceils, tissues and whole organisms. T h e y serve as primary agents in energy transductions and interconversions and catalyze the utilization of chemical energy for osmotic and electrical work necessary to transport substances vectoriMly across membranes and build their concentration and electrochemical gradients, for mechanical work done b y various types of contractile elements and devices, for chemical reactions and mechano* The t e r m A T P a s e is n o t r e c o m m e n d e d b y Enzyme Nomenclature 1978 (International U n i o n of Biochemistry; Academic Press, l~TewY o r k - - S a n F r a n c i s c o - - L o n d o n 1979), a l t h o u g h , e.g., dGTPase, EC, is r e c o m m e n d e d in the same book, b u t it is in general use a n d will t h u s be used t h r o u g h o u t this review.


Vol. 27


T.~BLE I. Examples of multiplicity of ATPase activities in biological sources


ATPa.qe type


~euroblastome cells in culture

(Mg, Ca; Ca; Ca) (Mg; K ; - - )

transport of Ca ~ (?) contr~tile properties (?)


(Mg; Ca; H ) (Mg, M n ; -- ; Mg, M n )

energy transduction transport of Mg 2 ~ a n d M n 2§


(Ca; -- ; Ca) (Mg; K ; 0) (Mg; Na-~ K ; N a + K )

transport of Ca 'z~ contractile properties transport of Na* and K +

(Mg; (Mg; (Mg; (Mg;

energy transduction transport of Ca ~ transport of HCO3-



Ituman erythrocytes

Diatom ~.'itzschia alba

Ca; H ) Ca; Ca) H C O a ; -- ) -- ; - - )

(Mg; Ca; Ca) (Ca; K ; 0) (Mg; 0; 0) (Mg, Ca; Na§ (Mg; (Mg; (Mg; (~'lg; (Ca; (Mg;

0; - - ) N a ; -- ) Na, K; --) I-[CO3 : -- ) -- ; - - ) K ; --)



transport of Ca 2+ shape maintenance (?) shape maintenance (?) transport of ~N~a4 and K + --------

Localization ---

thylakoid membrane outer envelope ---

inner membrane inner membrane outer membrane matrix spcctrin (?) s p e c t r i n (?)

membrane membrane plasma membrane plasma membrane plasma membrane plasma membrane mitochondria Golgi apparatu~

chemical processes inherent in D N A replication, etc. On the other hand, they are instrumental in conserving various types of energy (chemical, electrical, osmotic, light) in a universally utilizable chemical form by being capable of synthesizing A T P as the chief high-energy compound of the cell. Among the best-known representatives of ATPases are the F1F0 complex (see p. 197) of mitochondria, bacteria, and chloroplasts, the Na~-K-ATPase of animal cells, the Ca-ATPase of the muscle sarc~plasmic reticulum and erythrocytes, the actomyosin ATPase of muscle, and the ATPase of gastric mucosa. The common knowledge of ATPases does not extend much beyond these well-known enzymes; yet, in reality, ATPases are both much more numerous and variegated. An apparent lack of unifying features may be responsible for the fact that the systematic classification and designation of these enzymes is, after several decades of research, still in a rudimentary state. Rather than to survey the factual knowledge of individual ATPases, the purpose of this review is to point out the features in which individual ATPases differ and those they have in common. 2. Pitfalls of A TPase studies The detailed study of the ATPase equipment of even a simple biological object such as erythrocyte, mitochondrion or chloroplast is stymied by several factors. One of them is the multiplicity of ATPase activities in these objects (eft Table I). Thus human erythrocytes seem to contain at least three ATPase activities activated by magnesium and one or two activated by calcium; the functions of these enzymes include, according to various authors (Avissar et al. 1975; Schatzmann 1975; MirSevovs et al. 1977; MirSevovs 1979), active transport of Ca 2+, Na + and K +, control of






HO--P--O r 0

]nucleotidyltransferoses (EC 2.7.7) / \ \ATP diphosphotose (EC

HO--P"LO ~ A T P phosphoribosyltronsferose (EC o p~4~-~. ,pho.photr.naf.r..., (EC 2.?.xcl. 2.7.7) HO i



F{o. 1. Sites of a t t a c k of v a r i o u s e n z y m e s on A T P molecule.

passive fluxes of Na T and K + across the membrane, maintenance of the biconcave cell shape, etc. Mitoehondria and chloroplasts contain, apart from the well-known FiF0-ATPases, also other ATPases with different functions. Thus the mitoehondrial matrix seems to contain an ATPase different from the MFIF0 complex residing in the inner mitochondrial membrane (Le Deaut et al. 1972) whereas the outer mitochondrial membrane contains a HCO3-ATPase functioning presumably in the transport of hydrogen carbonate (Grisolia and Mendelson 1974). The outer chloroplast membrane or envelope contains a (Mg, Mn)-activated ATPase different from the CFIF0 complex (Joyard a n d Deuce 1975). As seen from Table I, neuroblastoma cells in culture contain two ATPase activities (Stefanovic et al. 1974) whereas the cells of the diatom Nitzschia alba contain five or six different ATPase activities (Okita et al. 1976). Table I does not include more complex systems such as muscle, nerve tissue, animal and plant secretory tissues or organs, etc. The ATPases in Table [ are designated in the way described in detail below. Another complicating factor in ATPase studies is the difficult identification of separate ATPases. In an organellc or cell exhibiting several ATPase activities, it is rarely exactly known if these activities belong to different enzymes or if t h e y are an expression of an altered activity of one and the same enzyme under changed assay conditions. I n addition, even when individual ATPases have been exactly distinguished a n d defined, it is not an easy task to attribute to t h e m defined functions in the cell. This is w h y the results from different laboratories concerning the same ATPase are often controversial. Despite all these drawbacks it is still possible, on the basis of the extensive experimental material available, to form certain conclusions on features common to all ATPases and on features unique to the individual enzymes of the ATPase family. These features are listed in Table I I and each of t h e m will be briefly discussed in turn. 3. A T P hydrolysis and synthesis

The major feature various ATPases have in common is their ability to catalyze AT1) hydrolysis (and A T P synthesis). ATPases belong to a large family of enzymes catalyzing the cleavage of one of the P - 0 bonds in ATP. Fig. 1 shows the sites of


Vol. 27


TA~L~ II. Eaqential features of ATPases Features Common to all or some ATPases

ATP hydrolysis (and synthesis) structure.function relationships (in transport ATPases)

Peculiar to individual ATPases

type and specificity of interaction with ions substrate specificity and p H optimum formation of a stable phosphoenzyme intermediate type and sequence of reaction steps molar mass, structure localization with regard to membrane structures sensitivity to inhibiters, activators and other substances role in cell or organelle

attack on ATP by various ATP-splitting enzymes (Mildvan 1979). Together with phosphotransferases, ATPases split the ~,-phosphate off the molecule of ATP. The ATPase reaction may be written in short as ATPase

ATP ~- H20 :-------- ADP + phosphate The reaction may occur spontaneously (AGo' = 29 kJ/mol) but is greatly enhanced in the presence of an ATPase. Several mechanisms have been devised for the reaction, including (a) nucleophilic substitution bimolecular (SN2) in which, during ATP synthesis, water formation and phosphorylation take place in one step (Mitchell 1974); (5) nucleophilic substitution monomolecular (SN1) with an oxygen anion (O u-) translocation (Mitchell 1977); (c) mechanism including changes in the eonformational state of the enzyme (Boyer et al. 1977). The actual details of the reaction may differ between individual ATPases. The coupling of the ATPase reaction with chemical, mechanical or osmotic work is very tight, reversible, and has a high efficiency (50--70 %). F~F0-ATPases, which perform the conversion of energy of transmembrane H + gradients into ATP synthesis, contain special devices, so-called inhibitory protein subunits, IF1, which prevent immediate splitting of the produced ATP and an active transport of H + against the prevailing gradient. 4. Structure-function relationships in transport A TPases In the case of transport ATPases, the requirement for two basic functions, i.e. ATP hydrolysis and transport of ions across a membrane, give rise to certain unifying principles. All major transport ATPases (F1F0-ATPase, Na -~ K-ATPase, Ca-ATPase) consist of an integral membrane protein which is rather hydrophobic, and a peripheral component which is often a glycoprotein (Kyte 1972; Andreu et al. 1978) containing glucose, ribose, mannose, fueose, rhamnose, glucosamine, galactosamine, and sometimes sialic acid. The enzymes have the following common structural-functional features: a) Active site for the bindir~g and hydrolysis of the substrate nucleotide, located mostly on the peripheral subunit; b) One or more binding sites for the ions which determine the selectivity of ion translocation; c) A pore spanning the membrane, or an ionophoretic unit in the integral part of the enzyme complex, which is the site of the actual translocation of the ion(s) across the membrane:




d) Gate controlled presumably by conformational changes in the molecule of the enzyme; e) Different effective affinities for the transported ion(s) at the two membrane faces. 5. Type and specificity of interaction with ions Like all ATP-attacking enzymes of Fig. 1, ATPases require for ATP hydrolysis the presence of Mg 2+, Ca 2+, Mn 2+ or some other ion. The role of these activating ions, as determined by NMI~ and by studies of stable metal-ATP complexes (Mildvan 1979), is in the formation of metal-enzyme-nucleotide complexes facilitating the ATPase reaction. The types of complexes coming into consideration for ATPase reaction are : /M (a) linear metal bridge ( E - - M - - S ) ; (b) cyclic metal bridge (E ! ); (c)inner-sphare \S ligand bridge ( E - - M ( L ) - - S ) ; (d) double metal bridge (E--M(L)S--M) (E enzyme, M metal, S substrate, L ligand). The activating ions are thus bound to the enzyme at the nucleotide-binding sites and participate directly in the nucleotide hydrolysis. Apart from this complexing and coordinating function, Mg 2+, but sometimes also Ca 2+, Mn 2+, Zn 2+ and other ions, are necessary for particular steps in the ATPase reaction sequence such as the dephosphorylation of Ca-ATPase phosphoenzyme intermediate, etc. I n contrast, stimulating ions (often identical with the translocated ones) are bound to the enzymes at distinct binding sites different from the nucleotide binding site(s). Yet their binding affects the ATPase reaction, probably via conformational changes in the enzyme. The third category of interaction is translocation of ions by ATPases; the most common activating, stimulating and translocated ions are listed in Table III. Individual categories of interaction of ATPases with ions can be defined as follows :

a) An ATPase is activated by an ion if it is inactive in the absence of the ion. Since it is not always simple to ensure completely ion-free conditions and since some ATPases exhibit partial reactions even in the absence (apparent) of ions, the sufficient working definition is t h a t an activating ion causes an increase in basal ATPase activity even if present alone, i.e. in the absence of other ions. b) A stimulating ion causes an additional rise in ATPase activity in the presence of an activating ion but has no stimulating or activating effect if present alone. This is the ease of, e.g., Na + and K + ions in the Na-{-K-ATPase, K + in the Ca-ATPase of cardiac membrane vesicles (Jones et al. 1977) or HCOa- ion in the HCOa-ATPase (Iritani and Wells 1976) and others. TABLE I H . The most common ATPase-activating, ATPa~e-stimulating and ATPase-translocated ions a Ions Activating

Mg ~+,

Ca 2+,

Mn 2+,

Zn 2e,

Co ~+,

Ni 2§


Ca ~+,

Na +,

K +,

Mg ~ ,

Mn ~+, HCO3-, CI-,


H +,

Ca 2+,

Na +,

K +,

Mg 2~,

Mn 2+,

tiCOaSO4 2-, blOa-,


HCOa-, C1-

a The sequences reflect roughly the frequency with which the ion is found in individual types of interaction in various ATPases.

[email protected]

K. S I G L E R

Vol. 27

c) The definition of a translocated ion is self-evident. It should be noted t h a t usually, b u t not always, the translocated ion is identical with the stimulating one (cf. Table I).

6. Designation of A T P a s e s A convenient w a y of designation reflecting the above facts is, e.g., (A; S; T ) - A T P a s e , where A stands for activating ion(s), S for stimulating ion(s) and T for translocated one(s). The valency signs with individual ions are optional; in most cases t h e y m a y be (and actually are) omitted without any loss of informative value. The designation reflects various situations in the following way: 1. I f the ionic interactions are unknown, then the appropriate space contains a dash : (-- ; -- ; Ca)-ATPase or (HCO3; -- ; --)-ATPase; 2. In the (proven) absence of ionic interaction of given type, the appropriate space contains zero sign: (Mg; Ca; 0)-ATPase -- nontransport ATPase (0; 0; --)-ATPase -- ion-independent ATPase (cf. Table I); 3. I f the stimulation b y several ions is independent, t h e y are written in space S with commas : (Mg; HCOs, S04; H)-ATPase, whereas if the stimulation is synergistic, the designation if (Mg; N a - ~ K ; N a - ~ K ) - A T P a s e ; 4. In the case of the most common transport ATPases the designation can be further shortened to, e.g., N a ~ - K - A T P a s e , Ca-ATPase, Mn-ATPase, CI-ATPase, etc., the indicated ion being the translocated one, not the activating or stimulating one. Thus, Mg-ATPase denotes an enzyme translocating Mg 2+, not activated b y it. To denote newly discovered ATPases as Mg 2+ (activated) is meaningless since most ATPases are activated b y Mg 2+. The designation should not include the action of nonionic agents, except where already currently used as in, e.g., EDTA-ATPase. Thus the preferred notation would be, e.g., N-acetyl-L-glutamate-dependent (HCO3; - - ; - - ) A T P a s e (Grisolia and Mendelson 1974) or DNA-stimulated (Mg,-Mn; --; 0)-ATPase (Assairi and Johnston 1979). I f the incomplete or trivial names such as Mg-ATPase are used in the hitherto accepted way, or if the author does not wish to elaborate further on the ionic relations of the ATPase, the term "Mg-ATPase activity" should be used. The system does not include effects such as inhibition b y ions. Table IV lists some ATPases from different sources and compares their original designation with that deduced from relevant data on the basis of our system. The t y p e of interaction of ATPase with ions is sometimes hard to assess. This is especially true for plant ATPases which were often found to be stimulated b y KC1, KBr, K N 0 3 , KeSO4, potassium acetate, NaC1, NaBr, l~bCl, LiC1, NH4C1, CsC1, Tris. .HC1, choline chloride, t e t r a m e t h y l a m m o n i u m chloride, succinate, malate, etc. (Kylin and K~hr 1973; Dodds and Ellis 1966; Fischer and Hodges 1969). In F1F0-ATPases the translocated ion, H +, does not stimulate the enzyme. However, these ATPases are stimulated b y a number of other ions including Na +, K +,




TABLE IV. Conventional rs. unified ATPase n o t a t i o n l~ame used b y author

(A; S; T)-ATPasea



Adenosine triphosphatase Coupling factor

(Mg; 0; I-I) (Mg, Ca; 0; H)

r a t liver mit~chondria Chromatium strain D

Membrane ATPase Low-molar mass ATPase Membrane ATPase

(Mg, Mn; 0; H) (Mg; --; H) (Ca, Zn; 0; H)

wheat mitochondria

Mg~+-ATPase K+-stimulated ATPase H+/K+-ATPase

(Mg; -- ; H) (Ca; K; I-t} (Mg; Ca; K, H)

synaptic vesicles gastric mueosa hog gastric microsomes

(Ca 2+ ~- Mg 2+) -stimulated ATPase (Ca~++Mg~+)-ATPase

( - - ; Ca, Mg; Ca) (Mg, Ca; Ca; Ca)

h u m a n erythroeytes h u m a n erythroeytes

(Mg, Ca; Ca; Ca) (Ca; K; Ca)

h u m a n erythrocytes cardiac m e m b r a n e

( -- ; Ca; Ca) (Ca, Mg; - - ; Ca) (Mg; Ca; Ca)

smooth muscle brain

Thorens (1979) D o u g h t y (1978) Robinson (1978)

(Mg; N a - b K ; N a + K )

ox b r a i n

Skou (1974)

(Mg; N a + K ; Na-t-K)

r a t brain microsomes

Harris and Stahl (1976)

(Mg; N a - ~ K ; Na~-K) (Mg; Na-~ K; Na~-K)

ox b r a i n dog renal medulla

Klodos a n d Skou (1976) Inagaki et al. (1974)

(Mg; HCO3; --)

r a t uterus

I r i t a n i a n d Wells (1975)

Ca~+-ATPase Ca2+-ATPase Ca2+-ATPase w i t h o u t requirement for Mg 2+ Ciliary Ca-ATPase (Mg-bCa)-ATPase (Na + ~- K+).dependent ATPase (Na + + K+)-stimutated ATPase (Na +, K+)-activated ATPase

VeiUonella alcalascens Bacillus subtilis


T r a n s p o r t ATPase Bicarbonate -stimulated ATPase Bicarbonate-stimulated ATPase CI=]HCOa--ATPase

(Mg; HCOa; HCOa, H) r a b b i t renal tubule (HCOa; C1; H) goldfish gills

Mg 2+-ATPase

(Mg; -- ; -- )

h u m a n granulocytes

Metal ion-independent ATPase

(0; 0; --)

dog renal cortex

Tyler and Webb (1979) Gepshtein a n d Carmeli (1977) Yoshimura (1978) T u p p y a n d Sperk (1976) Serrahima-Zieger a n d Monteil (1978) Toll a n d Howard (1978) R a y (1978) Proverbio and Michelangeli (1978) S c h a t z m a n n (1977) Farrance a n d Vincenzi (1977) Scharff and Foder (1977) Jones et al. (1977)

Liang a n d Sacktor (1976) DeRenzis a n d Bornanoni (1977) Smolen a n d ~reissmann (1978) Suzuki (1974)

a A activation ion(s), S stimulating ion(s), T transloeated ion(s).

HCO3-, SO4 ~-, SCN-, NOa- and other anions (Recktenwald an4 Hess 1977; Selwyn 1968). The major representative of anion-stimulated ATPases is the HCOa-ATPase found in many secretory tissues (gastric mucosa, uterus, brain cortex, pancreas, liver lysoseines, fish gills, salt glands of some birds). However, recent studies vy Van Amelsvoort et al. (1977a, b, 1978) showed that the apparent presence of HCOa--stimulated ATPases in some preparations was actually due to mitochondrial contamination. 7. Substrate specificity and optimum p H The separation and identification of separate ATPases in multi-ATPase systems is often based on their pH optima and substrate specificities. The specificity towards the hydrolyzed nucleotide varies widely: some ATPases, such as the H-ATPase of the yeast plasma membrane (Serrano 1978; Ahlers et al. 1978; Willsky 1979), the K, H-ATPase of the gastric mucosa (Sachs et al. 1976) or the DNA-dependent ATPase


K. S I G L E R

Vol. 27

Mg, AT P





Mg, K

FIo. 2. Simplified reaction sequence of Ca-ATPase ( Y a m a d a et al. 1970); indexes i and e denote intra- a n d extracellular ions; E -- enzyme.

ATP ~ /K





K~--/ EB-P





E~p7.1E~p_ADP ADP FIo. 3. Simplified reaction sequence of N a + K - A T P a s e (Karlish et al. 1978); indexes i and e denote intraa n d extracellular ions; Ea, Eb -- Na+-selective a n d K+-selective forms of ATPase.







K i ,Mg,He,P




FIG. 4. Simplified r e a c t i o n s e q u e n c e of K , H - A T P a s e (Sachs 1977); i n d e x e s i a n d e d e n o t e i n t r a - a n d e x t r a cellular ions; E l , E., -- s u b u n i t s 1 a n d 2 of A T P a s e ; curling arrow d e n o t e s s h i f t of s u b u n i t s in t h e m e m b r a n e .

of calf t h y m u s (Assair and Johnston 1979) strongly prefer ATP over any other nucleotide. Others, such as the Ca-ATPase of sarcoplasmic reticulum, prefer purine nucleotides (ATP, GTP, ITP) over pyrimidine ones (UTP, CTP). in still others, A T P may be readily replaced with any other nucleotide (the F1F0-ATPase of mitochondria and chloroplasts) or even other phosphates such as acetylphosphate, carbamylphosphate or 4-nitrophenylphosphate, with an almost complete preservation of the ion-transporting ability. The practically equal affinity for all nucleotides irrespective of the type of their bases, found in some ATPases (the ATPase of the avian myeloblastosis virus; Schneider and Beaudreau 1979) resembles closely the properties of NTPases (nucleoside triphosphatases, EC such as that found, e.g., in the vesicular stomatitis virus (Roy and Bishop 1971). The pH optimum of various ATPases is usually in the range of 5.0--9.0. The experimentally assessed p H o p t i m u m depends, among other things, on the conditions of ATPase preparation, purification and assay. F1Fo-ATPases have in most cases a pH o p t i m u m of 8.0--8.5 while plasma membrane A~Pases have a lower p i t optimum (5.5--7.0). This difference is often used to detect and eliminate mitochondrial contamination of membrane ATPase preparations. The pH o p t i m u m may be fairly sharp, as in the Ca-ATPase of the alveolar macrophage plasma membrane (Gennaro et al. 1979), or it may be less distinct, covering 1--2 p H units (as in the Ca-ATPase of erythrocytes; Schatzmann 1975). Some ATPases may have more than one pH o p t i m u m (e.g. dynein ATPase; Nakamura and Masuyama 1977). 8. Formation of a stable phosphoenzyme intermediate The ATP-binding and catalytic site of Ca-ATPase and N a + K - A T P a s e are known t o have a similar structure formed by t h e tripeptide (Ser,Thr)-Asp-Lys and also by cysteine, 1--2 histidine (Ca-ATPase) and arginine (Na+K-ATPase) residues (De


K. S I G L E R

Vol. 27

]?ont et al. 1977; Jorgensen 1975). The y-phosphate of A T P is bound to the enzyme as an acylphosphate b y a covalent bond to the ~-carboxyl group od the aspartyl residue. These two ATPases, as well as some others (the y e a s t plasma membrane ATPase -- Willsky 1979) form a relatively stable (at least at acid pH) high-energy phosphorylated intermediate; it is still not quite certain whether one or two phosphoenzyme intermediates arc formed (Shikegawa and Akowitz 1979). In contrast, F1F0-AT]?ases are much less defined as to the structure of the catalytic site. I t is assumed to involve arginine, tyrosine and lysine residues localized on subunit ~, histidine residues from subunit ~ and NI-I2-froups from subunit 7 (Godinot et al. 1979). The kind of AT]? bond to the site is still unknown. Moreover, the binding and splitting (or synthesis) of A T P is known to include several sites capable of so-called tight binding of AT]? and ADP, which change their nucleotide-exchange ability on energization of the enzyme. No stable phosphoenzyme akin to t h a t of Na ~ K - A T P a s e or Ca-ATPase has been found to be formed b y these enzymes. 9. Type and sequence of individual A T P a s e reaction steps The t y p e and sequence of separate steps in the AT]?ase reaction, as well as the t y p e and number of reaction intermediates involved, are currently a matter of dispute (for actomyosin ATPase, see, e.g., the review b y Taylor 1979). This uncertainty is reflected in the dissimilarity of proposed reaction schemes for different ATPases. Figs. 2 - - 4 show simplified reaction sequences for three of the best-known ATPases, the Ca-AT]?ase of sarcoplasmic reticulum ( Y a m a d a et al. 1970), the N a ~ - K - A T P a s e (Karlish et al. 1978) and the K,H-AT]?ase of the gastric mucosa (Sachs 1977). The schemes differ in the sequence of individual steps (ATP binding, ion binding, ion transfer, liberation of ADP, liberation of phosphate, release of ions), whether due to actual differences in the reaction sequences, or merely due to the still incomplete knowledge of the actual reaction events. Moreover, some of the ATPase are oligomeric enzymes composed of two or mose identical monomers (cf. below). Different reaction events are then thought to take place on different monomers, contributing to additional complexity of the reaction schemes. 10. Molar mass, structure, subunits Many AT]?ases are composed of several identical monomers; each of these monomeric units m a y in turn be composed of several subunits. Thus Na~-K-AT]?ase is considered to be a dimeric e n z y m e , each of the monomers consisting of an ~ and a subunit (e.g., l~epke 1977). Ca-ATPase from the protozoan Tetrahymena pyriformis is also a dimer (Doughty 1978) while the soluble AT]?ase from the cytoplasm of this organism is a trimer composed of identical monomeric units of M ~ 26 kg/mol (Chua et al. 1976). The humber of subunits in each monomer m a y vary; the Fl-factor of mitochondrial ATPase is composed of five t y p e s of subunits, some of them also being oligomeric. In addition, the ATPase complex, m a y also contain a n u m b e r of auxiliary components, chiefly with regulatory functions, which are more or less loosely connected with the AT]?ase proper. In some ATPasc these components m a y b y quite numerous; thus the actomyocin A T P a s e complex, besides the main ATPase components myosin and actin, involves tropomyosin, troponin, I-protein, and a-, ~- and 7-actinins. As shown below, m a n y AT]?ases also require for their activity the presence of activators of cofactors t h a t do not form an integral part of the enzyme itself. Table V compares several AT]?ases from this standpoint. Individual ATPases have also different affinities for other membrane components, particularly lipids. Thus


A T P A S E S (I~]~V]EW)


TABLE V. Molar m a s s a n d s u b s t r u c t u r e o f s o m e A T P a s e s Enzyme


Myosin ATPase D y n e i n ATPa~e (14S d y n e m ) Virus ATPase Na, K-ATPase H - A T P a s e (F1F0)


AT1)-dependent "DNA-gyrase" DNA-dependent ATPase Ca-ATPase Ion-independent ATPase Ribosomal ATPase

Escherichia coli, Micrococcus luteus

Molar m a s s kg/mol

Presumed number of s u b u n i t s

600-- 800


600 650 500a 450 450 450 190

-2X2 10-- 12 10-- 12 10-- 12 2

115 102 55 32

2 2-- 3 ---

protozoan axonemes avian myeloblastosis virus muscle, n e r v e mitochondria chloroplasts thermophilic bacteria

calf t h y m u s sarcoplasmic reticulum dog renal cortex

Bacillus stearothermophilus

a I n c l u d i n g lipids.

N a + K - A T P a s e is known to require phospholipids for its full activity and the removal of 75 ~/o of phospholipids bound to the ATPase protein leads to inactivation. The best reactivation of delipidated enzyme is achieved with phosphatidylserine, phosphatidylglycerol and phosphatidylinositol. N a + K - A T P a s e from human erythrocytes is also sensitive to membrane cholesterol (Seiler and Fiehn 1976), the enzyme from plant cells requires choline phospholipids and sulpholipids (Kylin et al. 1972) whereas the enzyme from the avian salt gland requires sulphatides (Karlsson et al. 1974). The minimum number of bound lipids is 20--30 molecules per ATPase molecule. Ca-ATPases have similar requirements for lipids while FiF0-ATPases have very slight requirement for lipids, even though some activation with phosphatidylethanolamine, phosphatidylglycerol and cardio]ipin has also been observed. 11. Localization with regard to membrane structures

Even though some ATPases are soluble, such as the cytoplasmic ATPase from Tetrahymena pyriformis (Chua et al. 1976) or the mitochondrial matrix enzyme (Le Deaut et al. 1972), most ATPases are associated with membrane or other (dynein, actomyosin) structures. Most membrane ATPases are situated with the catalytic site inside the cell or organelle; however, some ATPases such as the enzyme from Ehrlich ascites tumor cells (tr and Agren 1975), glial cells (De Pierre and Karnovsky 1974), polymorphonuclear leukocytes (Agren et al. 1971; Harlan et al. 1977) avian myeloblastosis virus (Schneider and Beaudreau 1979), tumour rat liver cells in culture (Karasaki and Okigahi 1976) or human granulocytes (Smolen and Weissmann 1978) are oriented in the opposite direction and are thus capable of hydrolyzing extracellular ATP. 12. Sensitivity to inhibitors, activators and other substances

One of the most widely used tools for distinguishing between separate ATPases is their sensitivity to inhibitors. Table VI summarizes the most frequently used inhibitors and indicates the sensitivity of the best-known ATPases to them. I t should be borne in mind t h a t t h e sensitivity of ATPases to individual agents is not constant but that it depends on many factors such as experimental conditions, state of the enzyme, and even the metabolic state of the cell or tissue under study. Thus the

Vol. 27


TA]aT.E V I . A c t i o n

of some


on ATPases



H-ATPase mitoehondria chloroplasts bacteria Na + K -ATPase Ca-ATPase sarcoplasmic reticulum protozoa erythrocytes "DNA-gyrase" DNA-dependent ATPase a +


+ + + +


+ + + 0 0

-- no effect, 0 not

+ +

+ + + 0



+ + + .

---0 0

+ + -0 0

---0 0

+ 0 0 0 0

0 . 0 0 0

~-+ .

. 0


. 0 0 0

+ -+ .

+ + +



+ + + 0



0 . 0 0 0


+ + -0 0

+ + + 0 0

+ + -0 0

---+ --

---+ --

0 0 0


sensitivity of brain N a + K - A T P a s e towards vanadate seems to depend on the electrical stimulation of the nerve tissue, the sensitivity of MFjFo-ATPase towards oligomycin depends on the intact bond of fac.tor F1 to the membrane component F0 and also on catabolite repression (Lloyd and Edwards 1976), etc. According to their sensitivity to inhibitors, ATPases may be divided into several groups. F1F0 ATPases including MF1F0 of mitochondria, BFIF0 of bacteria, CF1F 2 of chloroplasts, TF1F0 of thermophilic bacteria like PS3, AF1F0 (algal coupling factors found in thermophilic blue-green algae or cyanobacteria such as Mastigocladus laminosus; Binder and Bachofen 1979), and, possibly, GFIF0 found in the plasma membrane of chromaffin granules or catecholamine storage vesicles (Apps and Schatz 1979), are sensitive to dicyclohexylcarbodiimide, oligomycin (except for CF1Fo), and a number of other agents, such as uncouplers, the antibiotic Die-9, efrapeptin, chlorohexidine, aurovertin, venturicidin, quercetin, citreoviridin, spegazzinine, clofibrate, phlorizin, phenylglyoxal, and bathophenanthroline. However, they differ in their requirement for activation by heat or trypsin, in their requirements for Ca 2+ (AF1Fo), etc. Likewise, Ca-ATPases from different sources (erythrocytes, endoplasmic reticulum, sarcoplasmic reticulum, protozoan plasma membrane, mitotic spindle vesicles, zymogen granules or parotid glands) are usually inhibited by La 3+ ions and by ruthenium red (with the exception of the enzyme from endoplasmic reticulum). Sensitivity to ouabain and vanadate is the main feature of N a + K - A T P ases from different sources and the sensitivity of other ATPases, e.g. the yeast plasma membrane enzyme (Willsky 1979), to these agents points to their similarity with the N a + K - A T P a s e of animal cells. Other inhibitors of N a + K - A T P a s e include N-ethylmaleimide, azide, lectins, quercetin, polyamines (spermine, spermidine), chelating agents, and other substances. ATPases participating in DNA replication ("DNA-gyrase", "DNA-topoisomerase", ribosomal ATPase, "primases", "unwinding enzymes", etc.) are usually sensitive to fusidie acid, novobiocin, nalidixic acid and oxolinic acid, even though not necessarily to all these agents (Mizuuchi et al. 1978). One should not confuse the ATPases functional in DNA and RNA replication with enzymes such as DNA polymerase




(EC and RNA polymerase (EC which, though requiring also ATP, are in fact nueleotidyltransferases, and with other ATP-requiring non-ATPase enzymes. At the other end of the scale of agents affecting ATPases are activators and cofactors which are necessary for full function of these enzymes. Most of these modifiers have a regulatory function, adjusting probably the level of ATPase activity to the momentary demands of the cell and, in the case of highly specialized ATPases active only in selected events (production of secretagogues, cell division, muscle contraction), promoting the switch-on and switch-off of the ATPase action. Thus, according to the source, HCOs--stimulated ATPases are stimulated by 17-~-estradiol or by carbonate dehydratase (EC (Iritani and Wells 1973, 1976), whereas Na~-KATPases of adipocytes, salivary glands, pancreatic aeinar cells, etc., are sensitive to adrenalin, oxytocin, insulin, triiodothyronine, ~-adrenergic agents, etc. (Koketsu and Ohta 1976; Grinstein and Erlij 1974; Lo et al. 1976; Rogus et al. 1977). The ATPase from rat brain microtubules is stimulated by 6S rubulin (Ihara et al. 1979), actomyosin ATPase requires actin, actin ATPase requires protamine (Ferri et al. 1979). Similarly "DNA-gyrase" requires the presence of double-strand DNA for A T P hydrolysis (Mizuuchi et al. 1978), whereas DNA-dependent ATPase of calf t h y m u s requires the presence of single-strand DNA (Assairi and Johnston 1979). Many CaATPases are stimulated by soluble cytoplasmic protein activators like calmodulin, the protein activator of erythrocytes, parvalbumin, troponin C, and others. 13. Role in the cell or organelle

Activation by various co-factors and regulatory agents is closely connected with the role of individual ATPases in the given organism. Despite the diversity of their tasks, ATPases may be basically divided into several groups. Transport ATPases are primarily ion-translocation devices and may also assist in secretion of hormones, nenrotransmitters, cell division, etc. The second group, mechanochemical ATPases, are primarily connected with contractile elements and their function (actin, myosin, tubulin, dynein, and others). The third major group of ATPases are closely related with DNA replication and include "primases" (Kolodner and Richardson 1978), "mobile promoters" of single-strand DNA transformation into the replicative form (Ueda et al. 1978), stimulators of DNA polymerase activity (Barry and Alberts 1972), ATP-dependent unwinding enzymes and DNA "helicases" (Scott and Kornberg 1978, Abdel-Monem et al. 1977), deoxyribonucleases (Wilcox and Smith 1976; Bs et al. 1979), "DNA-gyrases" (Mizuuchi et al. 1978), "DNA-topoisomerases" (Liu et al. 1979), and others. The fourth group of ATPases includes enzymes that have still other functions (luminescence, invasion of host cells by viruses, etc.). 14. Clues for possible A T P a s e classification

However, diverse and inconstant, the above features of ATPases may be used for classifying the enzymes of this group. For instance, the following set of characteristics may be used: A. Function 1. Transport 2. Mechanochemical 3. Replication 4. Other B. Formation of a stable phosphoenzyme



Vol. 27

C. Stimulation by translocated ion(s) D. Stimulation by nonionic co-factors E. Sensitivity to inhibitors 1. Oligomycin and/or dicyelohexylcarbodiimide 2. Ouabain and/or vanadate 3. Ruthenium red and/or La a+ ions 4. Novobiocin, nalidixie and/or oxolinic acid. This set of features may be supplemented by others, e.g. by characteristics concerning molar mass, subunit arrangement, etc. It may also be considerably extended by including more (and more specific) inhibitors. A combination of these features would then be sufficient to characterize practically any ATPase. Tentatively groups of classes of ATPases may be characterized. )'or instance~ enzymes of group I would be characterized by the F~F0 arrangement and by characteristics A1 and E l . ATPases of group II might include enzymes with features A1, B, C, (D), E2 and E3, and would be represented by N a + K - A T P a s e , Ca-ATPase and other transport ATPases. Group III might include ATPases with features A2, D, among them aetomyosin and dynein ATPases. Enzymes of group IV might exhibit characteristics A3, D, E4 and would include enzymes functional in DNA replication. Using this or another similar system, the designation and nomenclature of ATPases could be improved to provide more information about the individual enzymes under study. REFERENCES 2~LBDEL-MONEI~ :~[., DURWALD I~., H O F F M A N - B E R L I N G I-I. : Eur.J.Biochem. 79, 39 (1977). AGP.EN G . K . , POETEN J . , :RoNQUIST G., WESTERMARK B . : J.Cell Physiol. 78, 171 (1971). AtKLERS J . , AH~ E . , SEYFA~TH A. : Mol.Cell.Biochem. 22, 39 (1978). AND~EU J . M . , ~ V ~ T H R . , MU~OZ E. : F E B S Letters 86, 1 (1978). A1-Ps D . K . , SCHATZ G. : Eur.J.Bioehem. 1{}9, 411 (1979). ASSIA~I L . M . , JOHNSTON I . R . : Eur.J.Biochem. 99, 71 (1979). AVISSAR ~N., DE u162 A., BEN-SHATJL Y . , COHEN I. : Bioehim.Biophys.Acta 375, 35 (1975). B~NFALVI G., CSVZI S., OHLBAUI~ A., ANTONI F. : Acta Biochim.Biophys.Acad.Sci.Hung. 14, 53 (1979). BARRY J . , ALBE~TS B. : Proc.Nat.Acad.Sei. USA 69, 2717 (1972). B:~UE~LEIN E. : Energy Conservation in Biological Membranes (G. Schiller, M. K l i n g e n b e r g , E d s ) , p. 237. Springer Verlag, Berlin 1978. BINDER A., BAeHOFEE R . : F E B S Letters 104, 66 (1979). BoYEI~ P . D . , GI~ESSER M., VIN](LER C., :HACKNEY D., CHOATE G. : Structure and Function of Energy-Transducing M e m b r a n e s (K. v a n D a m , B . F . v a n Gokler, E d s ) , p, 261. Elsevier, A m s t e r d a m 1977. C~UA B., ELSON C., Sm~AGo E. : Biochem.Biophys.Res.Commun. 69, 641 (1976). DEI~ERRE J . , KAENOVSKu M . L . : J.Biol.Chem. 249, 7121 (1974). D E P O N T J . J . H . : H . M . , SCHOOT B.M., V A N IOBOOYEN-VAN E E D E N A., BONTING S.L.: Biochim.Biophys.Acta 482, 213 (1977). DERENZIS G., BO~NANCIN M. : Biochim.Biophys. Acta 467, 192 (1977). DODDS J . J . A . , ELLXS R . J . : Biochem.J. 161, 31P (1966). DOUGHTY M.J. : Compar.Biochem.Physiol. 66B, 339 (1978). FA~RANCE M.L., VINCENZI F . L . : Biochim.Biophys.Acta 471, 59 (1977). FE~I~I A., MAGRI E . , GRAZI E. : Biochem.J. 183, 475 (1979). I~SCHER J . D . , HODGES T . K . : Plant Physiol. 44, 385 (1969). G E N N ~ O R . , MOTTOLA C., SCHNEIDER C., RO~EO D. : Biochim.Bjophys. Acta 567, 238 (1979). GEPSHTEIN A., CA-~ELI C. : Eur.J.Bioehem. 74, 463 (1977). GODINOT C., PENIN F . , GAUTHE~ON D.C. : Arch.BiocMm.Biophys. 192, 225 (1979). GI~INSTEIN S., ERJ~IJ D. : Nature 2~l, 57 (1974). G~SOL~A S., MENDELSON J. : Bioehem.Biophys.Res.Commun. ~8, 968 (1974). H ~ J ~ A N J . , DECHATELETT L . R . , IVERSON D . B . , McCALL C.E. : Infect.Immun. 1~, 436 (1977). H~s B L E . , STAHL W . L . : Biochim.Biophys.Acta 426. 325 {1976). ImAF~ Y . , FUJH T., ARAI T., T ) ~ A K A R . , K A Z ~ O Y . : J.Biochem. 86, 587 (1979). INAGAKI C., LINDENI~AYEI~ G . E . , SCHWARTZ A . : J.Biol.Chem. 249, 5135 (1974).



[email protected]

]~ITANI N . , WELLS W . W . : Arch.Bioehem.Biophys. 164, 347 (1973). IRITANI N . , WELLS W . W . : Biochim.Biophys.Acta 436, 863 {1976). J o N E s L . R . , B E s c ~ t t . R . , WATANABE A.M. : J.Biol.Chem. 252, 3315 (1977). JO~GENSEN P . L . : Quart.Rev.Biophys. 7, 327 (1975). JOYA~D J . , DOVCE R . : F E B S Letters 5 1 , 3 3 5 (1975). ~SAKI S., O~:IGA~ T . : Cancer Res. 36, 4491 (1976). KAPJ~ISH S . J . D . , YATES D . W . , GLyl~N I.l~. : Bioehim.Biophys,Acta 525, 252 (1978). K A ~ S S O N K . A . , SA~UELSSO~ B . E . , STEEN G.O. : Eur.J.Biochem. 46, 243 (1974). KLODOS ] . , SKOU J.C. : Bioehim.Biophys.Acta 3 9 1 , 4 7 4 (1975). KOKETSV K . , ORTA Y. : Life Sci. 19, 1009 (1976). KOLOD~:~ R . , RICHAaDSOlV C.C. : J.Biol.Chem. 253, 574 (1978). KYLIN A., KUYPER P . J . C . , HAIVSSOlV G. : Physiol.Plant. 26, 271 (1972). KY'L1N A., K h ~ M. : PhysioI.Plant. 28, 452 (1973). KY~E J.: J.Biol.Chem. ,947, 7642 (1972). LE BEAUT J . Y . , LEDI~ M., MANnEL P., MARFING M. : F E B S Letters 20, 153 (1972). L ~ w G C.T., SACXTO~ B. : Areh.Bjochem.Bgophys. 176, 285 (1976). L ~ L . F . , L I e C~.CH., A ~ E ~ T S B.M. : Nature ~)Bl, 456 (1979). LLOYD D . , EDWAm)S S . W . : Biochem.J. 160, 335 (1976). L o C.S., AUGUST T . R . , LI~E~MA-N U . A . , E])EL~AN I.S. : J.Biol.Chem. 251, 7826 (1976). MILI)VAN A . S . : Adv.Enzymol. 49, 103 (1979). i~IRSEVOV-~ L., VIKTORA L., ~I~ON0Vl A.- Blur 34, 133 (1977). M~(~EVOV-~ L. : D S c T h e s i s . I n s t . H e m a t o l . B l o o d T r a n s f u s i o n , P r a g u e 1979. M~TCHELL P . : F E B S Letters 43, 189 (1974). : M I s c a L L P. : F E B S Letters 8~, 9 (1977). M~Z~VCH~ K . , O'DEA M.I~I., GELLE~T M. : Proc.Nat.Aead.Sci.USA 7~, 5960 (1978). NAXA~V~A K . I . , MASUYAMA E. ! Bioehim.Biophys.Acla 4 6 1 , 6 6 0 (1977). O ~ T A T., SV~L~VA~ C.W., V O L C ~ 1 B . E . : Plant Sci. Letters 6, 129 (1976). ~I~OVE~B~O F., M~C~LANGEL~ F . : J.Membr.BioL 42, 301 (1978). RAY T . K . : ~ E B S Letters 92, 49 (1978). RECKTE~VCALD D., HESS B. : F E B S Letters 76, 25 (1977). REe~:E K . H . R . : Biochemistry of Membrane Transport (G. S e m e n z a , E. Carafoli, Eds),) F E B S S y m p o s i a :No. 42, p. 363. S p r i n g e r V e r l a g , B e r l i n 1977. ROBINSON J . D . : F E B S Letters 87, 261 (1978). R o G u s E . M . , CHENG L.C., Z~E~LE~ K . : Biochim.Biophys.Acta 464, 347 (1977). RONQUIST G., ~C-RE~ G . K . : Cancer Re8. 3~, fi402 (1975). R o Y P., B~sHoP D . L . H . : Biochim. Biophys.Acta 235, 191 (1971). SACHS G., CHANG H . H . , RAEON E . , S C H A C ~ A ~ R . , LE~U~ M., SACCOMAN~ G.: J.Biol.Chem. 251, 7690 (1976). SACRS G. : Rcv.Physiol.Biochem.Pharmacol. 79, 133 (1977). SC~A~F~ O., FODE~ B. : Biochim.Biophys.Acta 483, 416 (1977). SCHATZ~A~N H . J . : Current Topics in Membranes and Transport (F. B r o n n e r , A. Kleinzeller, E d s ) , Vol. 6, p. 126. A c a d e m i c Press, :New Y o r k 1975. SCHATZMANN H . J . : J.Membr.Biol. 3~, 149 (1977). SCtII~'E~I)EI~ R . P . , BEAUDaEAU G.S.: Arch.Biochem.Biophys. 196~ 475 (1979). SCOTT J . F . , [email protected] A . : J.Biol.Chem. 2~3, 3292 (1978). SE~LER D . , FIEH~- W . : Experimentia 32, 849 (1976). SELWYN M . J . : :Nature 219, 490 (1968). S~.~H~aA-Z~EGE]r M., MON~'E~L t-I. : Biochim.Biophys.Acta ~02, 445 (1978). S E ~ A N O R. : Mol.Cell.Biochem. 22, 51 (1978). S~XEGAWA M., AXOVC~TZA.A.- J.Biol.Chem. 2~4, 4726 (1979). S K o v J.C. : Biochim.Biophys. Acts 339, 234 (1974). S~OLEN J . E . , WE~SS~ANN G. : Bioehim.Biophys.Acta ~12, 525 (1978). STE~A-NOV~C V., C~ESn~LS~(~-T~EsKA J . , EEEL A., M A ~ ) E L P. : F E B S Letters 49, 43 (1974). S u z v ~ J. : J.Biochem. 76, 1327 (1974). TAYLO~ E . W . : CRC Critical Reviews in Biochem., April, 103 (1979). THO~ENS S. : F E B S Letters 98, 177 (1979). TOLL L., HOWARD B . D . : Biochemistry 17, 2517 (1978). T ~ e P Y H . , SPEaX G.: Eur.J.Biochem. 68, 13 (1976). TV~E~ D . D . , WEBB P . R . : Bioehem. J. 178, 289 (1979). UEDA K . , MoMAcKEN R., KO~,'~aERG A.sJ.Biol.Chem. 2~3, 261 (1978). VAN AME~SVOO~T J . M . M . , DE P o ~ J . J . H . H . M . , B O ~ T ~ G S.L. : Bioehim.Biophys.Acta 466, 283 (1977a). VAN A~nSVOORT J . J . M . , DE PONT J . J . H . H . M . , STOLS A.L.I:L, BO~T~NG S.L. : Biochim.Biophys.Acta 471, 78 (1977b).


K. S I G L E R

Vol. 27

VJ~" A~mLSVOOBT J.M.M., J ' A ~ S ~ J.W.C.M., DE PONT J.J.H.H.M., B o ~ m - ~ S.L. : Bioch~m.Biophys.Acta ~12, 296 (1978). W~5oox K.W., Sm'rH H.O.: J.Biol.Chem. P.~I, 6122 (1976). W~J~sKY G.R. : J.Biol.Chem. 254, 33"26 (1979). Y . ~ D A S., YA~AM~OTOT., TOgOMURA Y. : J.Biochem. 67, 789 (1970). Y0SHI~uttA F. : J.Biochem. 83, 1231 (1978).

Suggest Documents