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Effect of ion channel blockers on germination of Bacillus megaterium spores
FEMS Microbiology Letters 34 (1986) 211-214 Published by Elsevier
211
FEM 02397
Effect of ion channel blockers on germination of Bacillus megaterium spores (Sodium; potassium; calcium)
C. Mitchell, J.F. Skomurski and J.C. Vary Department of Biochemistry, University of Illinois at Chicago, Chicago, IL 60612, U.S.A.
1. SUMMARY We surveyed 23 drugs that can interact with membrane components, such as ion channels, for their effect on spore germination. The results showed that triggering of spore germination was inhibited by specific calcium (Ca2÷) potassium (K ÷) and sodium (Na ÷) channel blockers.
2. INTRODUCTION Although the mechanism for triggering bacterial spore germination is not known, one proposal suggests that membrane channels or pores may be involved [1]. Since it has been shown that K ÷, Na ÷ and Ca2+ are released early during germination [2], we have examined the effect of channel inhibitors on germination. This approach is not unusual either in the field of bacterial spores [3] or studies on calcium flux in eukaryotic cells [4].
3. MATERIALS AND METHODS
Bacillus megaterium QMB1551 was grown [5] and extracted with sodium dodecyl sulfate-dithiothreitol (SDS-DTT) as described [6]. Triggering of germination was as described [7,8] except that the concentrations of the trigger reagents were decreased to the minimum amounts for maximal
triggering of germination, determined from a dose-response curve for each reagent, as we have described for glucose [5]. The spore concentration used for absorbance loss was 200 #g/ml, whereas changes in conductivity and dipicolinic acid (DPA) release were at 5 mg/ml. For the latter two assays, the spores were removed by centrifugation at 5000 × g for 5 min and aliquots of the supernatant fractions were tested for conductivity (Radiometer conductivity meter type CDM-2) and DPA content [9]. Most chemicals were from Sigma Chemical Co. except nitrendipine, nifedipine and verapamil, which were gifts from H. Feinberg, amiloride from Y. Chan, and Y,4'-dichlorobenzamil from G. Kaczorowski. When ethanol or acetone was the solvent, the final concentration was < 1%, which had no effect on germination [10].
4. RESULTS AND DISCUSSION Verapamil, diltiazem and dihydropyridines such as nitrendipine and nifedipine are Ca2+-channel blockers in cardiac muscle, although the former two may act at different sites from the latter two [11,12] whereas yohimbine stimulates binding of dihydropyridine to Ca2+-channels [13]. Trifluoperazine inhibits Ca2+-channels in the sarcoplasmic reticulum [4]. In the above group (Table 1), only verapamil inhibited germination. As judged
0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies
212 b y a b s o r b a n c e , diltiazem, y o h i m b i n e a n d trifluop e r a z i n e i n h i b i t e d g e r m i n a t i o n , b u t b y conductivity a n d D P A , there was n o i n h i b i t i o n which was c o n f i r m e d m i c r o s c o p i c a l l y ; this d i s c r e p a n c y was a result of the different s p o r e concentrations. Of the next 5 c o m p o u n d s in T a b l e 1, Ca2+-channels are b l o c k e d b y tetracaine o r c h l o r p r o m a z i n e in the s a r c o p l a s m i c r e t i c u l u m [4,14], T M B - 8 in fibroblasts [15] a n d l a n t h a n u m or c a d m i u m in b r a i n a n d muscle [16]. A l l these c o m p o u n d s i n h i b i t e d triggering o f g e r m i n a t i o n , as j u d g e d b y three criteria ( T a b l e 1). I s o p r o t e r e n o l is a Ca2+-channel a c t i v a t o r ( a n d also stimulates p r o t e i n kinase C) while p r o p r a n o l o l antagonizes i s o p r o t e r e n o l activity [17], b u t neither c o m p l e t e l y i n h i b i t e d triggering of g e r m i n a t i o n . I n a d d i t i o n to the d a t a in T a b l e 1, which were o b t a i n e d with S D S - D T T e x t r a c t e d spores, the results were the s a m e with non-ext r a c t e d spores. Also, n o n e of the inhibitors were effective at c o n c e n t r a t i o n s b e l o w 0.1 m M . F r o m these results, one might speculate that spores d o n o t have d i h y d r o p y r i d i n e - s e n s i t i v e C a 2+ channels, c o m p a r a b l e to the L - t y p e channels o f c a r d i a c
muscle that are regulated b y c A M P - d e p e n d e n t p h o s p h o r y l a t i o n [18]. This w o u l d agree with the fact that spores d o n o t c o n t a i n c A M P [19]. H o w ever, other Ca2+-channel blockers d i d inhibit triggering of g e r m i n a t i o n which m a y i n d i c a t e the necessity for certain C a 2 + channels. U s i n g the s a m e a p p r o a c h as above, we tested o t h e r channel inhibitors. L i d o c a i n e a n d q u i n i d i n e are tertiary a m i n e s that inhibit N a + channels [20], while veratrine is an alkaloid mixture that can s t i m u l a t e N a + channels in certain cells [21]. F o r m o s t epithelial cells, a m i l o r i d e is the s t a n d a r d i n h i b i t o r o f the N a + / H + antiporter, as is quinid i n e in s o m e cases [22], a n d d i c h l o r o b e n z a m i l i n h i b i t s N a + / C a 2+ exchange [23]. Except for lidocaine, all of these c o m p o u n d s i n h i b i t e d triggering of g e r m i n a t i o n ( T a b l e 2) suggesting f u n c t i o n a l N a + channels. F o r K + channels, the 3 i n h i b i t o r s in T a b l e 2 inhibit v o l t a g e - d e p e n d e n t channels [24], b u t only quinine a n d t e t r a e t h y l a m m o n i u m ions i n h i b i t C a 2 + - i n d e p e n d e n t a n d / o r Ca2+-activated p o t a s s i u m channels [24,25]. Since quinine was the o n l y effective inhibitor, one might d e d u c e that a
Table 1 Effect of calcium channel inhibitors on germination Compound (mM) a
% loss in absorbance/30 min Glc Pro
Leu
Increase in conductivity b (btmho/mg)
Release of DPA b (txg/mg)
Control Verapamil (1.0) Diltiazem (1.0) Nitrendipine (1.0) Nifedipine (1.0) Yohimbine (1.0) Trifluoperazine (0.1) Tetracaine (1.0) TMB-8 (1.0) Chlorpromazine (0.1) Lanthanum (1.0) Cadmium (1.0) Isoproterenol (1.0) Propranolol (1.0)
59 2 6 + c + 3 11 4 1 9 7 14 31
67 4 15 + + 3 14 0 1 6 5 13 32
21 4 21 21 21 16 19 2 1 1 0 1 23 13
134 19 128 120 110 117 i 22 5 5 4 5 5 133 77
65 3 14 + + 2 13 2 2 7 4 45 31
a Heat-activated spores (200 pg/ml) were pre-incubated in 5 mM "Iris (pH 8) at 30°C with each compound at the indicated final concentrations and then germination was triggered at either 0.5 mM glucose (Glc), 10 mM proline (Pro) or 7 mM leucine (Leu). A 60 to 70% decrease in absorbance represented > 90% loss in heat resistance and DPA. b Germination was triggered by glucose as above (a) except the spores were at 5 mg/ml. The conductivity and DPA content were determined after 30 rain, as described in the text and expressed as p m h o or pg of DPA per mg of dry spores. c Some compounds interfered with absorbance measurements; + indicates > 80% and - indicates < 10%'phase dark spores after 30 rain as judged microscopically.
213
Ca2+-independent K + channel might be important in triggering germination. Finally, we investigated the effect of the chloride (CI-) channel inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) [26], quercetin, a protein kinase C inhibitor [27], and 2 membrane perturbants, indomethacin and n-decanol [28]. None of these compounds inhibited triggering of germination as judged by 3 criteria. While the above results seem intriguing, we would stress that the pharmacological activities of various chemicals on eukaryotic cells should be interpreted with caution with respect to their action on bacterial spores. For example, the concentrations required to inhibit spore germination were far above the pharmacologically active ranges for most of these drugs. While specific target sites for some of these chemicals have been well characterized, many others are known to have multiple effects. Trifluoperazine, verapamil and TMB-8 have been reported to inhibit ATPase activities in Mycobacterium phlei [29] and hog gastric membranes [30]. Also, TMB-8 may have no direct effect on Ca 2+ channels since it inhibits thrombo-
xane biosynthesis, causes membrane leakiness and protein kinase C activation (reviewed in [31]). Chlorpromazine and propranolol bind to phospholipids and inhibit phospholipase A and C in liver lysozomes [32]. Even the standard N a + / H + antiporter inhibitor, amiloride, has recently been shown to inhibit several types of protein kinases [33,34]. Finally, indomethacin, n-decanol, tetracaine and chlorpromazine have all been shown to have detergent-like properties on erythrocytes causing gross redistributions of membrane components [28]. In conclusion, we have observed some intriguing correlations which suggest that sodium, potassium and calcium ion channels may be necessary to trigger spore germination.
ACKNOWLEDGEMENTS This work was supported by Public Health Services grant AI 12678.
Table 2 Effect of sodium and potassium channel inhibitors on germination Compound (mM) a
Sodium inhibitors Control Lidocaine (0.5) Quinidine (0.5) Veratrine (0.025g) Amiloride (1.0) Dichlorobenzamil (1.0) Potassium inhibitors Quinine (0.5) Tetraethylammonium (1.0) 4-Aminopyridine (0.01) d Miscellaneous inhibitors DIDS (1.0) Quercetin (1.0) Indomethacin (110) n-Decanol (1.0) a b c d
~ loss in absorbance/30 min GIc
Pro
Leu
Increase in conductivity b (/tmho/mg)
Release of DPA b (/tg/mg)
59 62 1 17 4 _ c
65 61 3 11 0 _
67 46 3 1 3
21 14 1 1 1
134 106 4 3 4
-
1
3
1 59 66
3 66 62
1
1
3
68 71
20 23
126 120
68 + 60 47
69 + 60 46
71 + 43 44
23 20 20 26
144 134 126 133
Same as Table 1. Same as Table 1 Same as Table 1. At higher concentrations, 4-aminopyridine alone triggered germination.
214 REFERENCES [1] Vary, J.C. (1978) in Spores VII, (Chamblis, G. and Vary, J.C., Eds.), pp. 104-108. American Society for Microbiology, Washington, DC. [2] Swerdlow, B.M., Setlow, B. and Setlow, P. (1981) J. Bacteriol. 148, 20-29. [3] Lewis, J.C. and Jurd, L. (1972) in Spores V (Halvorson, H.O., Hanson, R.S. and Campbell, L.L., Eds.), pp. 384-389. American Society for Microbiology, Washington, DC. [4] Chamberlain, B.K., Volpe, P. and Fleischer, S. (1984) J. Biol. Chem. 259, 7547-7553. [5] Shay, L.K. and Vary, J.C. (1978) Biochim. Biophys. Acta. 538, 284-292. [6] Vary, J.C. (1973) J. Bacteriol. 116, 797-802. [7] Vary, J.C. (1975) J. Bacteriol. 121, 197-203. [8] Hsieh, L.K. and Vary, J.C. (1975) in Spores VI (Gerhardt, P., Castilow, R.N. and Sadoff, H.L., Eds.), pp. 465-471. American Society for Microbiology, Washington, DC. [9] Rotman, Y. and Fields, M.L. (1968) Anal. Biochem. 22, 168. [10] Racine, F.M., Dills, S.S. and Vary, J.C. (1979) J. Bacteriol. 138, 442-445. [11] Schramm, M., Thomas, G., Towart, R. and Franckowiak, G. (1983) Nature 303, 535-537. [12] Cantor, E.H., Kenessey, A., Semenuk, G. and Spector, S. (1984) Proc. Natl. Acad. Sci. USA 81, 1549-1552. [13] Miller, R.J. and Freedman, S.B. (1984) Life Sci. 34, 1205-1221. [14] Antoniu, B., Kim, D.H., Morii, M. and Ikemoto, N. (1985) Biochim. Biophys. Acta 816, 9-17. [15] Mix, L.L., Dinerstein, R.J. and Villereal, M.L. (1984) Biochim. Biophys. Acta 119, 69-75. [16] Nelson, M.T., French, R.J. and Krueger, B.K. (1984) Nature 308, 77-80.
[17] Horne, P., Triggle, D.J. and Venter, J.C. (1984) Biochem. Biophys. Res. Commun. 121, 890-898. [18] Nowycky, M.C., Fox, A.P. and Tsien, R.W. (1985) Nature 316, 440-443. [19] Setlow, P. (1973) Biochem. Biophys. Res. Commun. 52, 365-372. [20] Hondeghem, L.M. and Katzung, B.G. (1977) Biochim. Biophys. Acta 472, 373-398. [21] Catterall, W.A. (1980) Ann. Rev. Pharmacol. Toxicol. 20, 15-43. [22] Mahnensmith, R.L. and Aronson, P.S. (1985) J. Biol. Chem. 260, 12586-12592. [23] Kaczorowski, G.J., Barros, F., Dethmers, J.K. and Trumble, M.J. (1985) Biochem. 24, 1394-1403. [24] Decoursey, T.E., Chandy, K.G., Grupta, S. and Cahalan, M.D. (1984) Nature 307, 465-468. [25] Findlay, I., Dunne, M.J., Ullrich, S., Wollheim, C.B. and Peterson, O.H. (1985) FEBS Lett. 185, 4-8. [26] Smith, D.J., Bowman, B.J. and Iden, S.S. (1984) Biochem. Biophys. Res. Commun. 120, 964-972. [27] Friedman, Y., Poleck, T., Henricks, L. and Bruke, G. (1985) Biochem. Biophys. Res. Comm. 131, 971-980. [28] Maher, P. and Singer, S.J. (1984) Biochem. 23, 232-240. [29] Agarwal, N. and Kalra, V.K. (1984) Biochim. Biophys. Acta 764, 316-323. [30] Im, W.B., Blakeman, D.P., Mendlein, J. and Sachs, G. (1984) Biochim. Biophys. Acta 770, 65-72. [31] Simpson, A.W.M., Hallam, T.J. and Rink, T.J. (1984) FEBS Lett. 176, 139-143. [32] Hostetler, K.Y. (1984) Fed. Proc. 43, 2582-2585. [33] Besterman, J.M., May Jr., W.S., Levine, H., Crague Jr., E.J. and Cautrecasas, P. (1985) J. Biol. Chem. 260, 1155-1159. [34] Davis, R.J. and Czech, M.P. (1985) J. Biol. Chem. 260, 2543-2551.
211
FEM 02397
Effect of ion channel blockers on germination of Bacillus megaterium spores (Sodium; potassium; calcium)
C. Mitchell, J.F. Skomurski and J.C. Vary Department of Biochemistry, University of Illinois at Chicago, Chicago, IL 60612, U.S.A.
1. SUMMARY We surveyed 23 drugs that can interact with membrane components, such as ion channels, for their effect on spore germination. The results showed that triggering of spore germination was inhibited by specific calcium (Ca2÷) potassium (K ÷) and sodium (Na ÷) channel blockers.
2. INTRODUCTION Although the mechanism for triggering bacterial spore germination is not known, one proposal suggests that membrane channels or pores may be involved [1]. Since it has been shown that K ÷, Na ÷ and Ca2+ are released early during germination [2], we have examined the effect of channel inhibitors on germination. This approach is not unusual either in the field of bacterial spores [3] or studies on calcium flux in eukaryotic cells [4].
3. MATERIALS AND METHODS
Bacillus megaterium QMB1551 was grown [5] and extracted with sodium dodecyl sulfate-dithiothreitol (SDS-DTT) as described [6]. Triggering of germination was as described [7,8] except that the concentrations of the trigger reagents were decreased to the minimum amounts for maximal
triggering of germination, determined from a dose-response curve for each reagent, as we have described for glucose [5]. The spore concentration used for absorbance loss was 200 #g/ml, whereas changes in conductivity and dipicolinic acid (DPA) release were at 5 mg/ml. For the latter two assays, the spores were removed by centrifugation at 5000 × g for 5 min and aliquots of the supernatant fractions were tested for conductivity (Radiometer conductivity meter type CDM-2) and DPA content [9]. Most chemicals were from Sigma Chemical Co. except nitrendipine, nifedipine and verapamil, which were gifts from H. Feinberg, amiloride from Y. Chan, and Y,4'-dichlorobenzamil from G. Kaczorowski. When ethanol or acetone was the solvent, the final concentration was < 1%, which had no effect on germination [10].
4. RESULTS AND DISCUSSION Verapamil, diltiazem and dihydropyridines such as nitrendipine and nifedipine are Ca2+-channel blockers in cardiac muscle, although the former two may act at different sites from the latter two [11,12] whereas yohimbine stimulates binding of dihydropyridine to Ca2+-channels [13]. Trifluoperazine inhibits Ca2+-channels in the sarcoplasmic reticulum [4]. In the above group (Table 1), only verapamil inhibited germination. As judged
0378-1097/86/$03.50 © 1986 Federation of European Microbiological Societies
212 b y a b s o r b a n c e , diltiazem, y o h i m b i n e a n d trifluop e r a z i n e i n h i b i t e d g e r m i n a t i o n , b u t b y conductivity a n d D P A , there was n o i n h i b i t i o n which was c o n f i r m e d m i c r o s c o p i c a l l y ; this d i s c r e p a n c y was a result of the different s p o r e concentrations. Of the next 5 c o m p o u n d s in T a b l e 1, Ca2+-channels are b l o c k e d b y tetracaine o r c h l o r p r o m a z i n e in the s a r c o p l a s m i c r e t i c u l u m [4,14], T M B - 8 in fibroblasts [15] a n d l a n t h a n u m or c a d m i u m in b r a i n a n d muscle [16]. A l l these c o m p o u n d s i n h i b i t e d triggering o f g e r m i n a t i o n , as j u d g e d b y three criteria ( T a b l e 1). I s o p r o t e r e n o l is a Ca2+-channel a c t i v a t o r ( a n d also stimulates p r o t e i n kinase C) while p r o p r a n o l o l antagonizes i s o p r o t e r e n o l activity [17], b u t neither c o m p l e t e l y i n h i b i t e d triggering of g e r m i n a t i o n . I n a d d i t i o n to the d a t a in T a b l e 1, which were o b t a i n e d with S D S - D T T e x t r a c t e d spores, the results were the s a m e with non-ext r a c t e d spores. Also, n o n e of the inhibitors were effective at c o n c e n t r a t i o n s b e l o w 0.1 m M . F r o m these results, one might speculate that spores d o n o t have d i h y d r o p y r i d i n e - s e n s i t i v e C a 2+ channels, c o m p a r a b l e to the L - t y p e channels o f c a r d i a c
muscle that are regulated b y c A M P - d e p e n d e n t p h o s p h o r y l a t i o n [18]. This w o u l d agree with the fact that spores d o n o t c o n t a i n c A M P [19]. H o w ever, other Ca2+-channel blockers d i d inhibit triggering of g e r m i n a t i o n which m a y i n d i c a t e the necessity for certain C a 2 + channels. U s i n g the s a m e a p p r o a c h as above, we tested o t h e r channel inhibitors. L i d o c a i n e a n d q u i n i d i n e are tertiary a m i n e s that inhibit N a + channels [20], while veratrine is an alkaloid mixture that can s t i m u l a t e N a + channels in certain cells [21]. F o r m o s t epithelial cells, a m i l o r i d e is the s t a n d a r d i n h i b i t o r o f the N a + / H + antiporter, as is quinid i n e in s o m e cases [22], a n d d i c h l o r o b e n z a m i l i n h i b i t s N a + / C a 2+ exchange [23]. Except for lidocaine, all of these c o m p o u n d s i n h i b i t e d triggering of g e r m i n a t i o n ( T a b l e 2) suggesting f u n c t i o n a l N a + channels. F o r K + channels, the 3 i n h i b i t o r s in T a b l e 2 inhibit v o l t a g e - d e p e n d e n t channels [24], b u t only quinine a n d t e t r a e t h y l a m m o n i u m ions i n h i b i t C a 2 + - i n d e p e n d e n t a n d / o r Ca2+-activated p o t a s s i u m channels [24,25]. Since quinine was the o n l y effective inhibitor, one might d e d u c e that a
Table 1 Effect of calcium channel inhibitors on germination Compound (mM) a
% loss in absorbance/30 min Glc Pro
Leu
Increase in conductivity b (btmho/mg)
Release of DPA b (txg/mg)
Control Verapamil (1.0) Diltiazem (1.0) Nitrendipine (1.0) Nifedipine (1.0) Yohimbine (1.0) Trifluoperazine (0.1) Tetracaine (1.0) TMB-8 (1.0) Chlorpromazine (0.1) Lanthanum (1.0) Cadmium (1.0) Isoproterenol (1.0) Propranolol (1.0)
59 2 6 + c + 3 11 4 1 9 7 14 31
67 4 15 + + 3 14 0 1 6 5 13 32
21 4 21 21 21 16 19 2 1 1 0 1 23 13
134 19 128 120 110 117 i 22 5 5 4 5 5 133 77
65 3 14 + + 2 13 2 2 7 4 45 31
a Heat-activated spores (200 pg/ml) were pre-incubated in 5 mM "Iris (pH 8) at 30°C with each compound at the indicated final concentrations and then germination was triggered at either 0.5 mM glucose (Glc), 10 mM proline (Pro) or 7 mM leucine (Leu). A 60 to 70% decrease in absorbance represented > 90% loss in heat resistance and DPA. b Germination was triggered by glucose as above (a) except the spores were at 5 mg/ml. The conductivity and DPA content were determined after 30 rain, as described in the text and expressed as p m h o or pg of DPA per mg of dry spores. c Some compounds interfered with absorbance measurements; + indicates > 80% and - indicates < 10%'phase dark spores after 30 rain as judged microscopically.
213
Ca2+-independent K + channel might be important in triggering germination. Finally, we investigated the effect of the chloride (CI-) channel inhibitor, 4,4'-diisothiocyano-2,2'-disulfonic stilbene (DIDS) [26], quercetin, a protein kinase C inhibitor [27], and 2 membrane perturbants, indomethacin and n-decanol [28]. None of these compounds inhibited triggering of germination as judged by 3 criteria. While the above results seem intriguing, we would stress that the pharmacological activities of various chemicals on eukaryotic cells should be interpreted with caution with respect to their action on bacterial spores. For example, the concentrations required to inhibit spore germination were far above the pharmacologically active ranges for most of these drugs. While specific target sites for some of these chemicals have been well characterized, many others are known to have multiple effects. Trifluoperazine, verapamil and TMB-8 have been reported to inhibit ATPase activities in Mycobacterium phlei [29] and hog gastric membranes [30]. Also, TMB-8 may have no direct effect on Ca 2+ channels since it inhibits thrombo-
xane biosynthesis, causes membrane leakiness and protein kinase C activation (reviewed in [31]). Chlorpromazine and propranolol bind to phospholipids and inhibit phospholipase A and C in liver lysozomes [32]. Even the standard N a + / H + antiporter inhibitor, amiloride, has recently been shown to inhibit several types of protein kinases [33,34]. Finally, indomethacin, n-decanol, tetracaine and chlorpromazine have all been shown to have detergent-like properties on erythrocytes causing gross redistributions of membrane components [28]. In conclusion, we have observed some intriguing correlations which suggest that sodium, potassium and calcium ion channels may be necessary to trigger spore germination.
ACKNOWLEDGEMENTS This work was supported by Public Health Services grant AI 12678.
Table 2 Effect of sodium and potassium channel inhibitors on germination Compound (mM) a
Sodium inhibitors Control Lidocaine (0.5) Quinidine (0.5) Veratrine (0.025g) Amiloride (1.0) Dichlorobenzamil (1.0) Potassium inhibitors Quinine (0.5) Tetraethylammonium (1.0) 4-Aminopyridine (0.01) d Miscellaneous inhibitors DIDS (1.0) Quercetin (1.0) Indomethacin (110) n-Decanol (1.0) a b c d
~ loss in absorbance/30 min GIc
Pro
Leu
Increase in conductivity b (/tmho/mg)
Release of DPA b (/tg/mg)
59 62 1 17 4 _ c
65 61 3 11 0 _
67 46 3 1 3
21 14 1 1 1
134 106 4 3 4
-
1
3
1 59 66
3 66 62
1
1
3
68 71
20 23
126 120
68 + 60 47
69 + 60 46
71 + 43 44
23 20 20 26
144 134 126 133
Same as Table 1. Same as Table 1 Same as Table 1. At higher concentrations, 4-aminopyridine alone triggered germination.
214 REFERENCES [1] Vary, J.C. (1978) in Spores VII, (Chamblis, G. and Vary, J.C., Eds.), pp. 104-108. American Society for Microbiology, Washington, DC. [2] Swerdlow, B.M., Setlow, B. and Setlow, P. (1981) J. Bacteriol. 148, 20-29. [3] Lewis, J.C. and Jurd, L. (1972) in Spores V (Halvorson, H.O., Hanson, R.S. and Campbell, L.L., Eds.), pp. 384-389. American Society for Microbiology, Washington, DC. [4] Chamberlain, B.K., Volpe, P. and Fleischer, S. (1984) J. Biol. Chem. 259, 7547-7553. [5] Shay, L.K. and Vary, J.C. (1978) Biochim. Biophys. Acta. 538, 284-292. [6] Vary, J.C. (1973) J. Bacteriol. 116, 797-802. [7] Vary, J.C. (1975) J. Bacteriol. 121, 197-203. [8] Hsieh, L.K. and Vary, J.C. (1975) in Spores VI (Gerhardt, P., Castilow, R.N. and Sadoff, H.L., Eds.), pp. 465-471. American Society for Microbiology, Washington, DC. [9] Rotman, Y. and Fields, M.L. (1968) Anal. Biochem. 22, 168. [10] Racine, F.M., Dills, S.S. and Vary, J.C. (1979) J. Bacteriol. 138, 442-445. [11] Schramm, M., Thomas, G., Towart, R. and Franckowiak, G. (1983) Nature 303, 535-537. [12] Cantor, E.H., Kenessey, A., Semenuk, G. and Spector, S. (1984) Proc. Natl. Acad. Sci. USA 81, 1549-1552. [13] Miller, R.J. and Freedman, S.B. (1984) Life Sci. 34, 1205-1221. [14] Antoniu, B., Kim, D.H., Morii, M. and Ikemoto, N. (1985) Biochim. Biophys. Acta 816, 9-17. [15] Mix, L.L., Dinerstein, R.J. and Villereal, M.L. (1984) Biochim. Biophys. Acta 119, 69-75. [16] Nelson, M.T., French, R.J. and Krueger, B.K. (1984) Nature 308, 77-80.
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