Photoaffinity Labeling of (Na+K+)-ATPase with - Semantic Scholar

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Aug 25, 2016 - Joseph M. LowndesSQ, Mabel Hokin-NeaversonSQ, and Arnold E. Ruoholl. From the Departments of $Physiological Chemistry, §Psychiatry, ...
Val. 259, No. 16,Issue of August 25, pp. 10533-10538,1384 Printed in U.S.A.

THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1984 by The American Society of Biological Chemists, Inc.

Photoaffinity Labelingof (Na+K+)-ATPase with [1251]Iodoazidocymarin* (Received for publication, March 2, 1984)

Joseph M. LowndesSQ, Mabel Hokin-NeaversonSQ,and Arnold E.Ruoholl From the Departments of $Physiological Chemistry, §Psychiatry, and TiPharmacology, and the Wisconsin Psychiatric Institute, University of Wisconsin, Madison, Wisconsin 53706

A radioiodinated, photoactive cardiac glycoside de- subunits, have been identified but the nature and functionof rivative, 4’-(3-iodo-4-azidobenzenesulfony1)cymarin these peptides is alsounknown. Tritium-labeled photoactive derivativesof cardiotonic ste(IAC) was synthesized and used to label (Na+K+)-ATPase in crude membrane fractions. In the dark, IAC roids have been usedto specifically label the ouabain binding inhibited the activityof (Na+K+)-ATPase in electroplax site on (Na+K+)-ATPase. Specific labeling of the a subunit microsomes from Electrophoruselectricus with the occurred when the photoactive moiety of the photoaffinity same Isoas cymarin. [‘2SI]IACbinding, in the presence label was on the C-3 of digitoxigenin (3), the c-17 of digitoxof Mg2+and Pi, was specific, of high affinity(KO= 0.4 igenin (4),the C-19 of strophanthidin ( 5 ) , the rhamnose of PM), and reversible( k 1 = 0.11 min”) at 30 “C. At0 “C, ouabain (5, 6), or on the digitoxose of digitoxin monodigitox3 h, thus permitting oside (7). These results are consistentwith the ouabain bindthe complex was stable for at least washing before photolysis. Analysis of [‘251]IAC pho- ing site beinglocated primarilyonthe a subunit of the tolabeled electroplax microsomes by sodium dodecyl enzyme. However, specific labeling of the /3 subunit aswell as sulfate-polyacrylamide gel electrophoresis (SDSPAGE) (7-14%)showed that most of the incorporated the CY subunit was observed when lubrol-purified electric eel radioactivity was associated with the a (Mr = 98,000) enzyme was labeled with a probe which had the photoactive and ,8 (Mr= 44,000)subunits of the (Na+K+)-ATPase moiety farther away from the A ring of the steroid (i.e. 4“(ratio of CY to 0 labeling = 2.5). A higher molecular diazomalonyl-bisdigitoxoside or 4”’-diazomalonyl-digitoxin) weight peptide (100,000), similar inmolecular weight (7). Thus, the /3 subunit may also contribute to the structure to the brain a(+) subunit, and two lower molecular of the binding site, perhapsin the “sugar specific” region (8). weight peptides (12,000-15,000), which may be pro- A better understanding of the structure of the binding site teolipid, were also labeled. Two-dimensional gel elec- and in particular the interface between the a and /3 subunits suggest trophoresis (isoelectric focusing then SDS-PAGE, in the sitemay explain how the subunits interact and 10%)resolved the P subunit into 12 labeled peptides possible functions for the/3 subunit. ranging in PI from 4.3 to 5.5. When (Na+K+)-ATPase Two majordifficulties which limit investigations of the in synaptosomes from monkey brain cortex was pho- native structureof the cardiotonic steroid binding siteare: (i) tolabeled and analyzed by SDS-PAGE (7-14%),spe- the cardiotonic steroid photoaffinity labels that are presently cific labeling of the a(+), a, and ,8 subunits could be availableall contain tritium in the aglycon portion of the detected (ratio of a(+) plus a to 0 labeling = 35). The molecule withthephotoactive group attachedseparately results show that [‘261]IAC is a sensitive probe of the through an ester or amide bond. Loss of the aglycon due to cardiac glycoside binding site of (Na+K+)-ATPaseand hydrolysis of the ester or amide bond will result in loss of the can be used to detect the presenceof the a(+) subunit radiolabel. (ii)Purified(Na+K+)-ATPase which has been in crude membrane fractions from various sources. treated with detergents may be structurally altered especially with regards to the interactions between a and /3 subunits. Ideally, the native structureof the enzyme should be investigated in situ without the potential complications resulting Sodium and potassium ion gradients are maintained across from theuse of detergents. the plasma membrane of most animal cells by the enzyme To address the questionof topographical arrangement of a (Na+K’)-ATPase. This enzyme maintains the gradients by and /3 subunits around the ouabain binding site, particularly an ATP-dependent process in which sodium is transported in preparations of native, membrane-bound(Na+K+)-ATout of the cell and potassium is transported into the cell. The Pase, new photoaffinity labels are needed. These new photoenzyme is specifically inhibited by cardiotonic steroids such labels must have the radioactive and photoactive groups on asouabainandcymarin. (Na+K’)-ATPase,purifiedfrom the sameside of potentially hydrolyzable bonds so that radioseveral sources, consists of a t least two polypeptides: an CY activity is not lost from the protein during amino acid and subunit ( M , = 100,000) and a /3 carbohydrate-containing peptide analysis. They must also have sufficiently high spesubunit ( M , = 44,000)(1,2). The LY subunit contains the ATP cific radioactivity to detect thephotolabeled enzyme in crude phosphorylation site and the cardiac glycoside binding site. membrane preparations (i.e. in situ). The function of the /3 subunit is presently unknown. Small In this paperwe report the synthesis, characterization, and peptides ( M , = 10,000-12,000), referred to asproteolipid or y application of [’251]IAC’(Fig. 1) as a probe for the cardiac glycoside bindingsite of (Na+K+)-ATPasein microsomes

*This work was supported by National Institutes of Health Research Grant MH-26494. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

_ _ ~ The abbreviations used are: IAC, iodoazidocymarin; SDS-PAGE, sodium dodecyl sulfate-polyacrylamide gel electrophoresis; NP40, Nonidet P-40.

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_

~

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(lz5I/IAC Photolabeled (Na+K+)-ATPase

resolved by SDS-PAGE analysis of the photolyzed (Na+K+)ATPase. The catalytic a subunit (band b, Fig. 6A) and the glycoprotein p subunit (bandc, Fig. 6A), which were identified byCoomassie staining of the gel, were heavily labeled. In addition, a band of radioactivity (band a, Fig. 6A) was detected which had amolecular weight approximately 2000 greater than the (Y subunit. This polypeptide has a molecular weight which is similar to the a(+) subunit form of (Na+K+)-ATPase first observed in brine shrimp larvae (21) and in brain (18). A corresponding Coomassie stained band for the a(+) band IODOAZIDOCYMARIN was not visible, indicating that therewas little a(+) subunit FIG. 1. Structure of the photoaffinity label, iodoazidocyrelative to the a subunit of (Na+K+)-ATPase in eel electromarin. plax. The presenceof the a(+)subunit has not been previously from electric eel electroplax and in synaptosomes from mon- identified in the electric eel enzyme. Two specifically radiolabeled peptides of low molecular key brain cortex. Inelectroplax microsomes andinbrain weight were also identified (bands d and e, Fig. 6A). Based on of the synaptosomes, [ '''II]IAC was found to specifically label all the molecular weights, one or both of these peptides may be peptides ( a ( + ) ,CY, (3, and y subunits) which have previously been demonstrated by various methods to be associated with proteolipid components similar to those previously reported to be associated with (Na+K+)-ATPase(5, 6). The two pep(Na+K+)-ATPase. tides are referred to here as P-1 and P-2; they have, respecEXPERIMENTAL PROCEDURES~ tively, approximate molecular weights of 15,000 and 12,000. Eel electroplaxmicrosomes preincubatedwith ['251]IAC RESULTS AND DISCUSSION could be washed by dilution and sedimentation prior to phoThe data for the chemical characterization of IAC, the tolysis since thedissociation rate of the enzyme-IAC complex inhibition of (Na+K')-ATPase activity and binding of ["'I] A B IAC to the enzyme are presented in the Miniprint. Rationale for the Preparation and Use of IAC-The orthoiodoazidophenyl group used in this derivatization hasseveral advantages over photoactive groups used successfully in the past, i.e. the diazomalonyl group (3) and the meta-nitroazidophenyl group (5, 6). (i) The ortho-iodoazidophenyl group can be synthesized with "'1 a t a theoretical specific activity -aof 2200 Ci/mmol. (ii) After photolysis of the azide and sub- bsequent covalent insertionof the nitrene into the binding site, the radioactivity will remain with the peptide even if the sulfonyl ester bondwere to be hydrolyzed since theradioactive -Catom is part of the photoactive moiety rather than a part of the aglycon. (iii) The use of'1 also has the advantage that, since its emission energy is very high, direct and facile autoradiography of photolabeled material in electrophoretic gels can be performed. We used cymarin as the parent cardiotonic glycoside be'dcause it has a single hydroxyl group on its cymarose residue .e. which can be derivatized. Acetylation of cymarin at the 4' position of thesugar residue is known notto affect the dissociation rate constant of the glycoside (8) and derivati+ - + 4.7 24 zation at the same position with a diazomalonyl group has nM been shown not. to affect cymarin's ability to inhibit (Na+K')FIG. 6. ['2"I]IAC photoaffinity labeling of eel electroplax ATPase (20). The results presented in the Miniprint show microsomal membrane. General incubation and photolysis condithat modification of cymarin at the 4' position with 3-iodo- tions, and analysis by SDS-PAGE (7-14%) and autoradiography were 4-azidobenzene sulfonyl chloride did not interfere with either as described under "Experimental Procedures:" a, a(+) subunit; b, the inhibitory activityof cymarin or thehigh affinity binding subunit; c, 0 subunit; d, P-1; e, P-2. A , effect of ['251]IACconcentration on photolabeling.Microsomal membrane (0.29 mg of protein/sample) (Figs. 2 and 3). Photoaffinity Labeling of (Na+K+)-ATPase in Eel Electro- was incubated with ['251]IAC (specific activity = 7.7 Ci/mmol) at final of4.7,24, 65, and 250 nM in the presence (+) or plax Microsomal Membrane-The concentration dependence concentrations absence (-) of 0.1 mM ouabain. The samples were not washed before of [12sII]IACphotolabeling in the eel electroplax microsomal photolysis. The photolysistime was 6 s. Approximately 70pgof membrane is shown in Fig. 6A. Five radiolabeled bands were protein/sample were analyzed. B , photoaffinity labeling of samples (Y

Portions of this paper(including"Experimental Procedures," parts of "Results," Figs. 2-5, and Table I) are presented in miniprint at the end of this paper. Miniprint is easily read with the aid of a standard magnifying glass. Full size photocopies are available from the Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, MD 20814. Request Document No. 84M-0672, cite the authors, and include a check or money order for $4.80 per set of photocopies. Full size photocopies are also included in the microfilm edition of the Journal that is available from Waverly Press.

washed prior to photolysis. Microsomal membrane (0.38 mg of protein) was incubated with [Iz5I)IAC(specific activity = 6.0 Ci/mmol) at a final concentration of 0.15 p ~ in, the presence (+) or absence (-) of 0.1 mM ouabain. The microsomal membrane samples were diluted after incubation with ice-cold Type I1 medium and centrifuged in a Beckman Ti50 rotor a t 41,000 rpm for 60 min. The pellets were resuspended in 5 ml of ice-cold Type I1 medium and photolyzed for 6 s. Approximately 30pg of protein/sample were analyzed. Right, Coomassie protein stain of slab gel tracks. Left, autoradiogram of slab gel tracks.

['*'I]IAC Photolabeled (Na+K+)-ATPase a t 0 "C was very slow (Fig. 3). Fig. 6B shows that washed microsomes were essentially free of nonspecific radiolabeling although Coomassie staining of the gel shows that therewere a great number of peptides in these preparations (Fig. 6C). Washing did not affect thespecific radiolabeling of the a(+), a, 8, P-1, and P-2 bands indicating that these peptides are associated with the cardiotonic steroid binding site. The total photoincorporation of [12sII]IACa t a concentration of 0.16 nM into the five specifically labeled bands was calculated in a representative photolabeling experiment (gel not shown) to be 3.8 pmol of ["sII]IAC/mg of protein. Since bound and free ["'II]IAC were a t equilibrium at the time of photolysis, the numberof occupied sites was calculated based on the equilibrium binding presented in Fig. 3 and was 84.4 pmol/mg of protein. From these two values, the efficiency of specific photoincorporation was estimated as 4.5% for the photolysis conditions described in Fig. 6. Structure-activitystudies (8,22) ontheinhibition of (Na+K+)-ATPaseby cardiotonic steroids have suggested that there is a region in the binding siteof the enzyme which can interact specifically with the sugar moiety of a cardiotonic steroid. It is likely, then, the photolabels which have their photoactive group located on the sugarmoiety are covalently linked after photolysis to this sugar specific region of the binding site. In photolabeling experiments on eel electoplax (Na+K+)-ATPase withnitroazidophenyl-ouabain,Rogers and Lazdunski (5) showed specific radiolabeling of the a subunit of the enzyme and proteolipid ( M , = 12,000). Similar observations of specifically photolabeled proteolipid from pig and lamb kidney enzyme using nitroazidobenzoyl-ouabain were it was concluded reported by Forbush etal. (6). In these studies that there was a smallproteinassociatedwiththesugar specific binding site. On the other hand, Hall and Ruoho (19) showed ouabain protectable photolabeling of both the (Y and p subunits of the eel electroplax enzyme but no detectable labeling of the proteolipid with 4"'-diazomalonyl-digitoxin. They concluded that the two major subunits of the enzyme are in intimate contact in the region of the sugar specific binding site. The specific radiolabeling of a,8, and P-2, with [ 12s1] IAC is consistent with both of the conclusionsdescribed above for (Na+K+)-ATPase ineel electroplax microsomes. The differences observedbetween 4"'-diazomalonyl-digitoxin, nitroazidophenyl-ouabain,and IAC in the photolabeling of peptides from eel electroplax (Na+K+)-ATPasemay be related to the typeof photoreactive group used and the manner in which the group is attached to the cardiac glycoside. Specific['2sI]IAC incorporationintobothP-2andthe p subunit in addition to the (Y subunit may occur for a t least two reasons: (i) IAC possesses a hydrophobic photoreactive group which can partition into proteolipid and interact with P-2.Thephotoreactivegroups of IAC, nitroazidobenzoylnitroazidophenyl-ouabain are hydrophobic ouabain, and phenyl azides and may partition into proteolipid near the binding site. On the other hand, the photoreactive group of 4"'-diazomalonyl-digitoxin is less hydrophobic thanthe phenyl azide and may not partition as readily into hydrophobic regions. (ii)Thephotoreactive group of IAC maybe positioned favorably for reaction with the /3 subunit of the enzyme because the phenyl azide moiety in IAC is attached to cymarose which can hydrogen bond to the sugar specific region of the cardiac glycoside binding site of the enzyme. Studies on thedissociation rates of various cardiac monoglycosides from (Na+K+)-ATPase(8)have indicated that the3'a-hydroxyl or the 3'-a-methoxylgroups of the sugar moiety are important in stabilizing the ligand-enzyme complex, presumably through hydrogen bonding to the enzyme surface.

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The 3'-(~-hydroxylgroup of ouabain is not present in either nitroazidophenyl-ouabain or nitroazidobenzoyl-ouabainand the p subunit is not labeled with these compounds. On the other hand, the 3'-a-methoxylgroup of cymarin and the 3'a-hydroxyl group of digitoxin areintact in IAC and 4"'diazomalonyl-digitoxin, respectively, and they do photolabel the p subunit of (Na+K+)-ATPase. Two-dimensional Electrophoresis of Labeled Eel Electroplax-Eel electroplaxmicrosomal membrane was photolabeled with ['251]IACas described in Fig. 6B and thenanalyzed by two-dimensional gel electrophoresis with isoelectric focusing in the firstdimension. The results areshown in Fig. 7 and indicate that the radiolabeled band corresponding to the B subunit on SDS-PAGE gels was composed of approximately 12 individual peptides with PI values ranging from 4.3 to 5.5. There was nodetectablechange in the PI values of the peptides upon specific covalent modification with ['2sII]IAC since the radioactive spots (Fig. 7B) corresponded precisely with the Coomassie staining spots (Fig. 7A) in the molecular weight range of 44,000. This microheterogeneity of the p subunit agreeswith that reported by Marshall and Hokin (23);theyhad evidence that this heterogeneity is dueto variations in the sialic acid content of the subunit. Some of the protein from the electroplax membrane remained in the stackinggel of the firstdimension as indicated by the streak of Coomassie-stained protein on the right side of the SDS-polyacrylamide slab gel shown in Fig. 7A. This protein, which was specifically radiolabeled (Fig. 7B), probably contained the a subunit since only a small portion of the radiolabeled (Y subunit which was loaded onto the isoelectric focusing gel was resolved in both dimensions. Radiolabeled areas above the (Y subunit shown in Fig. 7B may consist of various combinations of a and p subunits which remained undissociated. IAC Photoaffinity Labeling of Brain (Na+K+)-ATPase-Iodinated photolabels are better suitedfor identifying low density binding sites in crude membrane preparations than are tritiated photolabels since '"I has high specific activity and emission energy (7). In order to test whether (Na+K+)-ATPase can be radiolabeled sufficiently for visualization in tissues where the amount of the enzyme relative to the total protein is lower than in eel electroplax, crude synaptosomes frommonkey brain were photolabeledwith ['2sI]IAC. The specific (Na+K+)-ATPase activity incrude monkey brain

*

60 43

27

t-

83

64

4 9

41

43

27

I 111

pH

60

d 84 39

*-

-

64

..

, '

14

4.1

FIG. 7. Two-dimensional electrophoresis of ['*'I]IAC phoA toaffinity labeled eel electroplax microsomal membrane. washed sample, prepared as described in Fig. 6B (no ouabain in preincubation), was analyzed by two-dimensional electrophoresis as described under "Experimental Procedures." A, Coomassie protein stain of the two-dimensional gel. The molecular weight standards X indicated are: myosin, 220; phosphorylase A, 94; catalase, 60; actin, 43; and lysozyme, 14 (Kendrick Laboratories, Madison, WI). The arrow indicates the isoelectric focusing standard vitamin Ddependent calcium binding protein (Mr = 27,000, apparent PI 4.2; Kendrick Laboratories). The pH gradient of the first dimension (isoelectric focusing) is indicated at the bottom of the autoradiogram. B, Two-month autoradiogram of the two-dimensional gel.

[1251]IAC

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Photolabeled

synaptosomes was less than 5% of that found in eel electroplax microsomes. Analysis of photolabeled brain synaptosomes by SDS-PAGE (Fig. 8, C-E) showed a radiolabeled peptide doublet (difference in M, = 2,000) in the molecular weight range of the (Y subunit of eel electroplax (Na+K’)-ATPase (Fig. 8, A-B). The labeling of both peptides was found to be very specific as indicated by ouabain protection and by the lack of label in other membrane proteins (compare the Coomassie profile in Fig. 8E with the photolabeling pattern in Fig. 80). Thus, [‘=I]IAC photolabeling of monkey brain cortex suggests that two forms of the catalytic subunit of (Na+K’)-ATPase, (u(+) and (Y, are present in primate brain. Sweadner (18) identified these two forms in brain from calf, dog, frog, mouse, and rat. She also found that rat brain axolemma contained the a(+) form and rat brain astrocytes contained the LYform. This suggests that the ratio of radiola-

beled a(+) to radiolabeled LYfor photolabeled brain (Na+K+)ATPase

shown

in Fig. 8D is probably

related

to the ratio

of

axolemma membrane to astrocyte membrane in the synaptosome preparation. A 3-week autoradiogram of the same gel used in Fig. 8D revealed specific labeling of a diffuse area (position c, Fig. 8C)

A

B

c

D

E

with

-b-

to be 35 to 1 for monkey

of eel

brain enzyme whereas the same ratio

was found to be 2.4 to 1 for eel electroplax enzyme. Thus, photoinsertion into the @ subunit of the enzyme appears to be a function

of both the type of cardiac

glycoside

photolabel

used and the type of (Na’K’)-ATPase labeled. In summary, a comparison of photolabeled crude membranes from eel electroplax and monkey brain indicates that the (Na+K+)-ATPase in these tissues appear to have the same binding-site specific peptides. A difference between the two sources of enzyme is in the efficiency of radiolabeling of the minor peptides (/9 subunit, P-l, P-2) relative to the cysubunit labeling. These results suggest that the topographical arrangement of the peptides in the native conformation of the enzyme

I. 2. 3.

/

6. -d-

7.

-e-

the tissue source. The detection

of an n(+)

REFERENCES Cantley, L. C. (1981) Curr. Top. Bioenerg. 11.201-237 Jergensen, P. L. (1982) Biochim. Biophys. Acta 694,27-68 Ruoho. A. E.. and Hall. C. C. (1980) Ann. N. Y. Acad. Sci. 242, QO- 103 Deffo, T., Fullerton, D. S., Kihara, M., McParland, R. H., Becker, R. R.. Simat, B. M., Fromm, A, H., Ahmed, K., and Schimerlik, M. I..( 1983) .Biochemistry 22.6303-6309 Rogers, T. B., and Lazdunski, M. (1979) Biochemistry 18, 135140 Forhush. B.. III, Kaplan, J. H., and Hoffman, J. F. (1978) Biochem&y’l7,3667-3676 Ruoho, A. E., Hall, C. C., and Rashidbaigi, A. (1983) Fed. Proc. 42,2837-2841

8.

9.

+

as the 6 subunit

microsomes as compared with monkey brain synaptosomes. The ratio of specific labeling of the catalytic subunits to the /3 subunit, determined from y-counting of gel slices, was found

5.

-

weight

peptides which were not ouabain protectable can be seen after this long exposure time. The efficiency of insertion into the glycoprotein subunit, relative to insertion into the catalytic (a(+) plus 0~) subunits, was not the same for eel electroplax

4.

+

molecular

form of the catalytic subunit in electroplax as well as in brain suggests that both forms may be present in many tissues. Further comparisons of native enzyme with respect to species and tissue type should be greatly facilitated through the use of [‘251]IAC and similar ortho-iodoazidophenyl cardiotonic steroid derivatives.

.-a-

-

the same

electroplax (Na+K+)-ATPase (Fig. 8, A-B). Some specific labeling of low molecular peptides, P-l and P-2, could be detected (positions d and e, Fig. 8C). Some other radiolabeled

may vary with

/L

-c-

(Na+K+)-ATPase

+

-

+

-

+

Rc. 8. [12”1]IAC

photoaffinity labeling of monkey cerebral cortex (Na+K’)-ATPase and comparison with eel electroplax (Na+K+)-ATPase labeling. General incubation and photolysis con-

ditions, and analysis hy SDS-PAGE (‘i-14%) and autoradiography were as described under “Experimental Procedures.” Monkey brain cortex synaptosomal membrane (0.27 mg of protein) or electroplax microsomal membrane (0.29 mg of protein) was incubated with [“51] IAC (specific activity = 5.5 Ci/mmol) at a final concentration of 0.15 PM in the presence (+) or absence (-) of 0.1 mM ouahain. The samnles were not washed prior to photolysis. Photolysis time was 10 s. Approximately 60 pg. of protein/sample were analyzed. The (Na’K+)-ATPase activity of the membranes was 0.2 pmol of Pi min-’ mg-’ of brain membrane protein and 4.4 qmol of Pi min-’ mg-’ of electroplax membrane proteins. u, a(+) subunit; 6, a subunit; c, @ subunit: d. P-l. e. P-2. A. Coomassie protein stain of the gel tracks of eel eIectroplax ‘microsomal membrane; B, 3-day autoradiogram of the eel microsome gel tracks; C, 2-week autoradiogram of the gel tracks of monkey cerebral cortex synaptosomal membrane; D, 3-day autoradiogram of the monkey synaptosome gel tracks; E, Coomassie protein stain of the monkey synaptosome gel tracks.

10. 11. 12. 13. 14. 15.

Yoda, A. (1974) Ann. N. Y. Acad. Sci. 242,598-616 Johnson, R. A., and Walseth, T. F. (1979) Adv. Cyclic Nuckotide Res. 10, 135-167 Rashidbaigi, A. (1982) Ph.D. dissertation, University of Wisconsin-Ma&son Yoda, A., and Yoda, S. (1981) Anal. Biochem. 110,82-88 Chee, P. Y., and Dahl, J. L, (1978) J. Neurochem. 30,1485-1493 Rosenthal, H. E. (1967) Anal. Biochem. 20, 525-532 Laemmli, U. K. (1970) Nature (Lond.) 227, 680-685 Lambin, P., Rochu, D., and Fine, J. M. (1976) Anal. Biochem. 74.567-575

O’Farrell, P. H. (1975) J. Biol. Chem. 250,4007-4021 17. Peterson. G. L. (1975) Anal. Biochem. 83.346-356 18. Sweadner, K. J. (1979) J. Biol. Chem. 254,6060-6067 19. Hall, C., and Ruoho, A. (1980) Proc. Nntl. Acad. Sci. U. S. A. 77, 16.

4529-4533 20.

Ruoho, A., and Kyte, J. (1974) Proc. Natl. Acud. Sci. U. S. A. 71, 2352-2356

21. Peterson, G. L., Ewing, R. D., Hootman, S. R., and Conte, F. P. (1978) J. Biol. Chem. 253,4762-4770 22. Wilson, W. E., Sivitz, W. I., and Hanna, L. T. (1970) Mol. Pharmacol. 6,449-459 23. Marshall, P. J., and Hokin, L, E. (1979) Biochem. Biophys. Res. Commun. 87,476-482

[1251]IACPhotolabeled (Na+K+)-ATPase

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Step 4. 4'-13-112511iodo-4-a.ldobenzene sulfonyl)-cymarln 111251iIACI. The Sulfonyichlorlde vas t o l u e n e e x t r a c t o f c r u3-11251110do-l-=rldobenzene de c o n c e n t r a t e d w l t h a g e n t l e ~ t r e a m o f N 2 t 0 5 0 0 #Y1a,9 tah ne n d transferred to a 6 x S O m m test tube and drled completely. Pyridine (freshly dlstllled and d r i e d over C ~ B I10 ~ J p l l w h l c h c o n t a l n e d 1 0 pIpole6 o f cymarin (dried for 24 hoUc6 under v a c u u m ln a drylng plstol. whlch was h e a t e d with refluxing ethanol) was added immediately. The Eeactlon tube was Stoppered, sealed Vlth parafilm, vottexed and left at room temperature for 24 hours. The product v a s p a r t l a l l y p u r i f i e d by T L C d e v e l o p e d r l t h b e n z e n e - a c e t o n i t r i l e 113:91. T h e r a d l a a c t i v e c o m p o u n d , ~ d e n t i f i e dby autoradlography, which chromatographed r l t h t h e same R f a s O Y t h e n t l C I A C was r e m o v e d f r o m t h e p l a t e a n d e x t r a c t e d with ethanol (total v o l u m e 6 m l l . The extract was redvced to a s m a l l volume wIth a gentle s t r e a m o f N 2 and further purified by TLC developed wlth Chlorof o r m - m e t h a n o l (97:3l. T h e i 1 2 5 1 1 1 A C b a n d was l d e n t l f l e d w ~ t hreference to authentic IAC and was extracted Ylth ethanol (total volume 6 m i l . The yield Of 112511 IAC from 3-11251110d~-4-aridobenrene Sulfonvl chloride v a s 58. In a d d l t i o n t o t h e t w o s o l v e n t s y s t e m s a l r e adescribed, dy chloroform-acetone 13:lI a n d t o l u e n e - m e t h a n o l (9:11 y e r e also used to test the purlty Of IL251l IAC by TLC. Thls compound was greater than 99% cadlopure in ail four solvent systems. Electroplax membranes. The electroplax membrane from Electrophorus electrlcus on photolabeling preparation used to study the effect Of llght exposure tlme was ci q l f t f r o m D C S . A t s u n o b u Y o d a a n d S h i z u k o Y o d a f r o m t h e D e p a r t m e n t ~n Pharmacology, U n l Y . Of Wisconsin, Madison. Eel electroplax membranes used other experiments were prepared a b PreYlouslY described (11).

.

of

.~

tubes. EleCtz&horuI electr;cus electroplax tissue which was Used to prepare electroplax microsomal membrane in our laboratory, was a generous g i f t f r o m D r . P r a n k L. S i e g e l , D e p a r t m e n t s O f P e d i a t r i c s a n d P h y a i o l o g i c a l C h e m i s t r y , Univ. O f W i s c o n s m , Madison. Monkey cerebral cortex was a generous gift from Dr. Bldeo Uno, Primate Research Center. Madison, W I .

Methods All synthetic steps Synthesla Of 4'-13-lodo-4-ar1dobenlene-eulfonylI-~ymar1n. w e r e c a r r i e d O u t under s u b d u e d l i g h t i n g c o n d i t i o n s . D r y c y m a r i n (100 mg, 0.182 mmolesl v a s d i s s o l v e d i n d r y p y r i d i n10.15 e ml, 1.58 m m o l e s l a n d a d d e d t o a s u s p e n s l o n o f 3 - i o d o - 4 - = r i d O b e n ~ e n e - . u l f a n y l c h l o r i d e (0.125 mg, 0.364 mmolesl I" 0.15 m 1 dcv Dvridine 1101. The c l e a r Yellowish-brown reaction was Pyridzne was allowed to proceed in-ti; dark at m o m temperatuFe for 2 bOUr8. removed with vacuum and the residue was dissolved In 1.0 m 1 Of aicetonitrlleT h l s s o l u t i o nwas a p p l i e d t oa l i l l c a g e l C o l u m n 126 x 1 . 5 c m ) benzene 1 l : l l . whlch had been pre-equxllbrated wlth acetonitrile-benzene (1:lI. The product w a s e l u t e d f r o m t h e C o l u m n w i t h 75 m l O f a c e t o n i t r i l e - b e n z e n e 1L:l). The solvent was removed under vacuum leaving an oil residue. Llght brown pcoduct 199.6 mg, 658 yield) was prccipltated from the oil wlth anhydrous ether. T h e ComPoYnd wda h o m m e n e o u s On ellica ael TLC develooed withacetonitrile-benzene ~~~

TABLE I PBYSICAL PROPERTIES OF IAC

Molecular weight: 855.7 g w1e-l Meltlng polnt: 148-1500 C Elemental analysis.: Calculated C 50.53 B 5.38 N 4.91

~

Blndlng to NaK-ATPase. Equlllbrlum bindlng Of IAC to NaK-ATPase I n micTosoma1 m e m b r a n e s was determined by rapld flltratlon using glass fiber fllters on a Mlllipore model 1225 aarnpllng manifold. The flnal assay volume o f 2 0 0 p i Contained T y p e I 1 m e d l u m (4 m M MgCi2. 3 n M T c l s - p h o s p h a t e . a n d 5 0m zole-BC1, PR 7.21, 9 5 pg membrane proteln. varying Concentriltvms of l s p e c i f i c a c t i v i t y = 0.29 C i / m n o l e l delivered I " d i m e t h y l f o r m a m i d e l f l n a l Concentration o f 18) and without and with 0.2 on ouabain. After lncubatlnq 6 0 minutes at 300 C. 5 ml of rce Cold Type I1 medlum was added to the assay tube a n d t h e c o n t e n t s were i m m e d l a t e l y d e c a n t e d O n t o a fllter. The fziter was w a s h e d w i t h 3 0 nl o f ~ c e c o l d T y p e 1 1 loedlum a n d l e f t on t h e vacuum f o r 5 minutea to dry. The amount of radloactlve iodine was d e t e r m z n e d w i t h a Packocd model A u t o Gamma 8OOC I-counter 170% countinq efficlencyl. Maxlmum Specific blndlng and the equlllhclum dissociation constant (Kg1 for IAC r e r e determined by Scatchard analysis 1131. T h e d l s s o c i a t i o n r a t e o f I A C f r o m e l e c t r omicrosomal plax membrane IPS a150 determined. Membrane (final proteln conc. 0.9 m g / m l l was pre' cuboted ln T y p e I 1 m e d l u m c o n t a i n i n g 1 % d i n e t h y l f o r m a m i d e a n d 0.55 pU lf'i5111Ac (specific activity 0.45 Ci/nrmolel. delivered in dirnethylformamlde. After 60 m i n u t e s a t 30° c , 5 0 u l a l i q u a r a were t a k e n a n d e a c h d i l u t e d I n t o 2 m l o f 0 . 1 m M Ouab.1" ~n T y p e I 1 m e d l u m . T h e d l l u t e d S a m p l e s w e r e ~ n c u b a t e d for v a r y l n g l e n g t h s o f t l mat e e l t h e r 3 0 0 Cor O o C. A t t h e e n d O f e a c h ~ n c u b a y e r e d e c a n t e d on t o a g l a s s flber filter on a t l O n t i n e . t h e d i l u t sarnplee ed M i l l l p O r e s a m p l i n g m a n i f o l d , W a s h e d 30 w I tmh1 O f i c e c o l d T yIp1e rnedlum and a n d d r i e d f o r 5 m i n u t e s on t h e vacuum. N o n s p e c l f 1 ~ b l n d x n g o f l a c t o t h e m e m b r a n e and to the filter was determined by the same protocol except that 0.1 nM Ouabain 1 6 6 present I n the preincubation. The amount of radioactive i o d ~ n e on the fllterr was determlned rlth a Packclrd model Auto Gamma 8OOc --counter.

$%:%

-

-

*Galbralth Laboratorlea, Knoxville. m.

Instruments, Inc., Galtheriburs. MDI, and we;. sent to Kendrlci Lab9 foc twodrmensional gel electrophoresis. Sample pellets for SDS-PAGE were dissolved in 80-100 p l o f a b u f f e r w h i c h c o n t a i n e d 75 m M T c i s - H C 1 I p R 6.8) 2.4% SDS, 10% g l y c e r ~ n a n d v h l c h w d b heated at 90° C for o n e minute.' I n some e x p e r i m e n t s , a n a l i q u o t v a s t a k e n f o r p r o t e i n determination 1171 p r ~ o r t o heatlng. T o e a c h s a m p l eY e r e a d d e d 5 p l O f 2 - m e r c a p t o e t h a n o l a n d 1 0 p l o f 0.05% bromophenol b l u e d y e m a r k e r . E l e c t r o p h o r e s l swas P e r f o r m e d O n a s i a b g e l w l t h a c r y l a n l d e c o n c e n t r a t i o n s O f3 % l n t h e s t a c k i n g g e l ( 2 x I4 1 0 . 1 5 cml and elther 9% or 7-11) I l l n e a r g r a d i e n t ) ~n t h e Separating g e l 117 x I 4 x 0.15 c ~ J . The Stacking gel contalned 0.08% N , N - m e t h y l e n e b l s a c r y l a n l d e . 0.1% N.N.N',N"terramethylethylenedlalnlne. 0.038 ammonium persulfate, 0.18 SDS, and 0.125 II T r l s - H C I IpR 6.81. T h e separating g e l c o n t a l n e d 0.24% Or 0.19-0.381 (linear gradient) N . N - r r e t h y l e n F b l S ~ c I y l a m l d e . 0.037% N.N,N',N'-tetranerhyle t h y l e n e d l a m l n e , 0 . 0 3 8 a r n n o n l ~ mp e r g u l f a t e . 0.1% SDS, 0.375 II T r l S - H C I IpH 8.81. T h e e l e c t r o d e b u f f e r c a n t c i l n e d 0.1% S D S , 0.1911 9lyclne. and 0.025 M a constant voltage of TClS-HCl (pH 8.21. S a m p l e s were stacked for 2 hour8 at 30 V o i r = a n d S e p a r a t e dfor approxilaately 2 days ata c o n s t a n t voltage o f 60 volts w ~ t ht h e P c o t e l n s moving 1" t h e d l r e c t l o n o f c a t h o d e t o a n o d e . T h e protein m o l e ~ u l a r ~ e l g h t s t a n d a r d sused were: bovlne serum albumin 166.2001, C a t a l a s e 157.500J. f u m a r a s e 148.5001, a n d a l d o l a s e 140,000). A f t e r e l e c t r o pharesis, the g e l 6 w e r e sralned for t w o hours rlth 0.1% ( r / v i Coomassle Blue-G ~n methanol-acetic a c l d - w a t e r (5:S:ll t h e n destained for 1.5 d a y s ~n a methanol-acetlc acld-later 17:3:301 SOlUtlDn COntalnlng 5% qlycerln. The gels wece drled under V ~ C Y Y O I vlth a Hoeffer slab gel drier and Sublected to autoradiography u51n9 Kodak X-OMAT film with a Uuponr Cronex intenslfylng s c r e e n a t - 8 O O C. I n some e x p e r l n e n t s t h e d r l e dgels, a f t e r a u t o r a d l o g r a p h y , w e r e silced lnto 2 mm pleces and the covalently incorporated radloactlve lodlne was determlned wlth a Packard model Auto Gamma 8OOc #-counter.

RKsuLrs

10538

I

["'IIIAC Photolabeled (Na+K+)-ATPase

T

I

'

I

03

0.1

A

0.8-

A

0P 0.6 -

.

0.4 0

3

0

0.3 0.6 0.9 IAC CONCENTRATION ( C Y )

1.2

1.5