Purealin, a novel activator of skeletal muscle ... - Wiley Online Library

Eur. J. Biochem. 167, 1-6 (1987)

0 FEBS 1987

Purealin, a novel activator of skeletal muscle actomyosin ATPase and myosin EDTA-ATPase that enhanced the superprecipitation of actomyosin Yoichi NAKAMURA, Masaki KOBAYASHI, Hideshi NAKAMURA, Houming WU, Jun’ichi KOBAYASHI and Yasushi OHIZUMI

Mitsubishi-Kasei Institute of Life Sciences, Tokyo (Received January 26/March 10, 1987) - EJB 87 0086

1. Purealin, a novel bioactive principle of a sea sponge Psammaplysilla purea, activated the superprecipitation of myosin B (natural actomyosin) from rabbit skeletal muscle. The maximum change in the turbidity increased with increasing purealin concentrations and was three times the control value in the presence of 50 pM purealin. 2. The ATPase activity of myosin B was also elevated to 160% of the control value by 10 pM purealin. On the other hand, purealin inhibited the myosin ATPase in the presence of 10 mM CaClz and 0.5 M KCl (CaZtATPase), and the concentration for the half inhibition was 4 pM. 3. On the other hand, purealin activated the myosin ATPase in the presence of 5 mM EDTA and 0.5 M KCl (EDTA-ATPase). The maximum activation by 10 pM purealin was 160% of the control value. 4. Furthermore, similar results concerning the modification of ATPase activities by purealin were obtained in myosin subfragment-1 instead of myosin. 5. These results suggest that purealin activates the superprecipitation of myosin B by affecting the myosin heads directly. It is also an interesting observation that there is a correlation between the activities of the myosin EDTA-ATPase and actomyosin ATPase of myosin B. Numerous marine natural products, such as tetrodotoxin, saxitoxin, sea anemone toxins [l - 31 and maitotoxin [4- 61, have been useful as tools for physiological and biochemical studies because of their actions on specific sites of the cell membrane. In the course of our survey on bioactive substances from marine sources, much attention has been given to compounds affecting the contractile apparatus. Recently we have isolated purealin, a novel natural product from an Okinawan sea sponge, Psammaplysilla purea, that modifies the activity of ATPases [7]. Purealin has been demonstrated to modify the activity of skeletal [8] and smooth [9] muscle myosin ATPase. The superprecipitation of actomyosin is generally accepted to be basically the same phenomenon in vitro as a contraction in skeletal muscle cells [lo]. It is of interest whether the activation by purealin is due to the direct effect on the myosin molecules or to the modulatory effect on the interaction between actin and myosin. We measured the effect of purealin on the myosin ATPase activities under various conditions. It is well known that the activity of myosin ATPase shows complicated patterns of dependence on cation concentration [l 1 - 131. The activity in the presence of EDTA (absence of divalent cations) and high concentrations of KCl is very high, and is referred to as ‘EDTA-ATPase’. The activity decreases as the divalent cation is raised to about 0.1 mM, but increases with further increasing concentrations of the cation. The activCorrespondence to Y. Ohizumi, Mitsubishi-Kasei Institute of Life Sciences, 11 Minamiooya, Machida, Tokyo, Japan 194 Abbreviations. S1, subfragment-1 of myosin; ICs0,the concentration for 50% inhibition. Enzymes. ATPase (EC 3.6.1.3); creatine kinase (EC 2.7.3.2); lactate dehydrogenase (EC 1.I .1.27); alkaline phosphatase (EC 3.1.3.1).

Br&r

H

Fig. 1 . Chemical structure of purealin

ity enhanced by concentrations of Ca2 of several millimolar is referred to as ‘Ca’+-ATPase’. Although the activity is low in the presence of millimolar concentrations of Mg2+, this activity is important considering the physiological conditions in muscle cells. It is referred to as ‘Mg2+-ATPase’.However, the relationship is not established between these three types of myosin ATPase and the actin-activated myosin ATPase, which is crucial to the contractile mechanism. In this paper we report for the first time that the EDTAATPase of skeletal muscle myosin but not the Ca2+-ATPase or Mg2+-ATPaseis activated by purealin, an activator of the superprecipitation of myosin B. A preliminary account of some of these findings has been presented elsewhere [8]. +

MATERIALS AND METHODS Preparation of purealin

The sea sponge Psammaplysilla purea was extracted with methanol and the methanolic extract was fractionated by partition between ethyl acetate and water. The ethyl-acetatesoluble portion was chromatographed on silica gel, anionexchange and Sephadex columns to give purealin (Fig. 1).

2 The purification method and the chemical character of this compound have been reported in detail elsewhere [7]. Purealin was dissolved in ethanol and 1 volume of the ethanol solution was added to the enzyme solution 5 min before the start of the enzyme reaction by adding the substrate to determine the activity. The same volume of ethanol was added for the control experiments.

50

1.5 MM purealin

Preparation of enzymes Myosin [14], myosin subfragment-1 (Sl) [15], myosin B [lo], and sarcoplasmic reticulum [I61 were prepared from rabbit skeletal muscle. The Ca2+,Mg2+-ATPase of sarcoplasmic reticulum was purified by deoxycholic acid treatment of sarcoplasmic reticulum [17]. Alkaline phosphatase from calf intestine (grade 11), creatine kinase from rabbit muscle and lactate dehydrogenase from hog muscle were purchased from Boehringer-Mannheim (Indianapolis, IN). Na', K ATPase from canine kidney (grade IV) was purchased from Sigma Chemical Co. (St Louis, MO). +

-1ogCpureaiinl

Assay of superprecipitation The superprecipitation of myosin B was induced by adding 0.03 mM ATP in 0.3 mg/ml myosin B, 0.2 mM CaC12,0.5 mM MgC12, 0.2 M KC1 and 40 mM Tris/maleate at pH 7.0 and 2 7 T , and the change in the absorbance at 660nm was followed. Assay of ATPases The conditions used were: for myosin or S1 Ca2+-ATPase, 0.05 mg/ml myosin or S1, 1 mM ATP, 10 mM CaC12, 0.5 M KCl and 20 mM Mops at pH 7.0; for myosin EDTA-ATPase, the same conditions as above except for 5 mM EDTA instead of CaCI2; for myosin Mg2+-ATPase, 1 mg/ml myosin or S1, 1 mM ATP, 5 mM MgC12,0.5 M KCl and 50 mM Tris/HCl at pH 7.4; for actomyosin ATPase, 0.1 mg/ml myosin B, 0.1 mM ATP, 1 mM EGTA, 1 mM MgC12, 1.1 mM CaC12, 50 mM KCl and 20mM Tris/maleate at pH 7.0; for Ca2+, Mg2+-ATPase from sarcoplasmic reticulum, 0.05 mg/ml enzyme, 1 mM ATP, 0.49 mM CaCl,, 0.5 mM EGTA, 5 mM MgC12, 90mM KCl and 50mM Mops at pH7.0; for Na+,K+-ATPase,0.05 mg/ml enzyme, 3 mM ATP, 140 mM NaCl, 14mM KC1, 5 m M MgC12 and 50mM Mops at pH 7.0. The mixture was preincubated in the absence of purealin and ATP at 30 "C for 5 min, followed by the addition of purealin and further preincubation for 5 min. In the control experiment 5 pl ethanol was added instead of purealin. The reaction was started by the addition of ATP and stopped by adding an equal volume of cold 10% trichloroacetic acid. The amount of inorganic phosphate liberated during the 1 - 5-min incubation was determined by the method of Martin and Doty [181. Assay of other enzymes The activity of creatine kinase was determined by measuring the amounts of creatine produced by the method of Eggleton et al. [19] in the presence of 0.5 pg/ml enzyme, 5 mM MgC12, 90 mM KCl and 50 mM Mops at pH 7.0 and 30°C. The activity of lactate dehydrogenase was determined by monitoring the decrease in the absorbance at 340 nm of NADH' in the presence of 0.1 pg/ml enzyme, 0.1 mM NADH', 0.33 mM sodium pyruvate and 100 mM potassium

Fig. 2. Activation of superprecipitation of myosin B by purealin. The absorbance change at 660 nm, induced by the addition of 30 FM ATP to 0.3 mg/ml myosin B suspension, was monitored in the presence of various concentrations of purealin and 0.5 mM MgCI,, 0.2 mM CaCI,, 0.2 M KCI and 40 mM Tris/maleate at pH 7.0 and 27°C. The inset shows the concentration dependence of purealin on the maximum change in the absorbance determined as the difference between the levels at 1 min and at time zero, extrapolated by a linear part of the increase

phosphate at pH 7.5 and 27°C. The alkaline phosphatase and K+-phosphatase activities, catalyzed by the Na+,K+-ATPase, were determined by monitoring the increase in the absorbance at 420 nm of the p-nitrophenol produced [20]. The conditions used were: for alkaline phosphatase, 1 pg/ml enzyme, 1 mM p-nitrophenylphosphate, 2 mM MgC12 and 100 mM Tris/HCl at pH 8.0 and 27°C; for K+-phosphate, 0.3 mg/ml enzyme, 3 mM p-nitrophenylphosphate, 3 mM MgC12, 20 mM KCl and 50 mM Tris/HCl at pH 7.5 and 27°C. RESULTS Activation of superprecipitation and actomyosin ATPase of myosin B The superprecipitation of the myosin B suspension was followed by measuring the turbidity. Fig. 2 shows the effect of purealin on the superprecipitation in the presence of 30 pM ATP, 0.3 mg/ml myosin B, 0.2 mM CaC12, 0.5 mM MgC12 and 0.2 M KCl at pH 7.0. After the addition of ATP, clearing occurred and then the turbidity increased for 30 - 50 s. In the presence of purealin the clearing was not affected but the increase in turbidity was enhanced markedly up to three times that of the control at 50 pM purealin. The inset shows the concentration dependence of purealin on the maximum turbidity change determined as the difference between the absorbance at 1 min and the clearing level. The effect of purealin on the actomyosin ATPase activity of myosin B was examined. The activity increased as the concentration of purealin was raised from 0.66 FM to 22 pM, but decreased when the purealin concentration was increased further from

3 I

0.3

0 u)

m t-

a

Table 1. Reversible effect of purealin on myosin ATPase Myosin (1 mg/ml) was preincubated with 10 pM purealin in the presence of 0.5 M KC1 and 50 mM Mops at pH 7.0 and 30°C for 5 min, and then 0.05 vol. of the preincubated solution was added to a reaction medium with or without 10 pM purealin containing 1 mM ATP, 0.5 M KCI and 5 mM EDTA or 10 mM CaC12.After an appropriate reaction time (2-4 min for EDTA-ATPase and 4- 8 min for C a 2 + ATPase) the reactions were stopped and the amounts of inorganic phosphate liberated were measured. The activities are shown as means k SD,n = 3 o r 4

a

L.

0.lk

o

h

I

6

I

Treatment

Enzyme activity Ca2+-ATPase

EDTA-ATPase

I

5

nmol mg-' min-'

-logCpurealinl

Fig. 3 . Activation of actomyosin ATPase of myosin B by purealin. The activities in the presence of various concentrations of purealin were measured under the conditions of 0.1 mM ATP, 0.1 mg/ml myosin B, 1.I mM CaCI2, I mM EGTA, 50 mM KCI, 1 mM MgC12,and 20 mM Tris/maleate at pH 7.0 and 30°C

None Purealin (10 pM) Purealin (10 pM) and x 20 dilution

152 f 0 46 & 4

324 f 18 5085 1

142 k 4

323 f 12

to 160% of the control value on increasing the concentration up to 22 pM, and then decreased to 130% when the concentration was further raised to 66 pM. On the other hand, the Ca2'-ATPase activity decreased as the concentration of purealin increased. The concentration for 50% inhibition was 3.7 pM. The value was independent of concentrations of myosin between 0.05 mg/ml and 0.5 mg/ml. The slope of the Hill plot (not shown) was 0.88. The Mg2+-ATPase activity was also inhibited by purealin, though the effect was small, i.e. about 70% of the activity still remained at 66 pM purealin. We could observe the effects of purealin on the myosin ATPase even in the presence of 1 mM ATP and 5 mM or 10 mM divalent cations or a chelating agent. Therefore, the modulations of the ATPase activities are due to the binding of purealin to myosin not to either divalent cations or ATP. The effect of a sulfhydryl-protecting reagent on the modulation by purealin was examined in order to exclude the possibility that purealin acted as a reductant on the cysteine residues of myosin, which might be oxidized during storage (see Discussion). In the presence of 1 mM dithiothreitol the -logCpurealinl similar activation of EDTA-ATPase and inhibition of Ca2+Fig. 4. Activation of EDTA-ATPase and inhibition of Ca2+-ATPase ATPase were observed (Fig. 4). and Mg2+-ATPaseof myosin by purealin. The EDTA-ATPase and CaThe effect of dilution of purealin was examined. After a .ATPaseactivities in the presence of various concentrations of purealin 5-min preincubation of 1 mg/ml myosin with 10 pM purealin, were measured under the conditions of 1 mM ATP, 0.05 mg/ml 20 volumes of the reaction solution containing 1 mM ATP myosin, 5 mM EDTA, (0, 0 )or 10 mM CaC12 ( A , A), 0.5 M KCI were added to measure the activities of EDTA-ATPase and and 20 mM Mops at pH 7.0 and 30°C. The activities with ( 0 ,A) or A ) 1 mM dithiothreitol were plotted. The Mg2+-ATPase Ca2+-ATPase. When the concentration of purealin was without (0, activity in the presence of various concentrations of purealin (0)was diluted to 0.5 pM, both of the activities returned to the origimeasured under the conditions of 1 mM ATP, 1 mg/ml myosin, 5 mM nal levels (Table 1). The change in the preincubation time (0.5 - 5 min) of myosin with purealin prior to the ATP addiI41gCl2,0.5 M KCI, and 50 mM Tris/HCI at pH 7.5 and 30°C tion did not influence the modulation of ATPase activities (data not shown). Thus, the modulation of myosin ATPase 22 pM to 66 pM (Fig. 3). The extent of maximal activation by purealin was due to the reversible binding to myosin. As is well known, the pH-activity curve of myosin Ca2+was 160% of the control value. ATPase is complex showing an activity maximum at about pH 6.5 and minimum at 7.5 [ll].We examined the activation Activation of EDTA-ATPase and inhibition of EDTA-ATPase and the inhibition of Ca2+-ATPase by of Ca2+-ATPase of myosin purealin over a wide range of pH from 5.5 to 8.5 (Fig. 5). The The effects of purealin were examined on the various types activation of EDTA-ATPase by 22 pM purealin was observed of myosin ATPase activities, Ca2+-ATPase, EDTA-ATPase, over the range of pH used although the percentage of the and Mg2+-ATPase. Fig. 4 shows the concentration depen- activation was relatively high at the lower pH (