Enzymic properties of a Ca2+-dependent protease in rat ventral prostate: differences in distribution between lobes of the prostatic complex

Soluble extracts of rat ventral prostate contain a calcium-dependent, neutral thiol protease which is separated from an endogenous inhibitor by DEAE-cellulose chromatography. The Ca2+-dependent protease had a high calcium requirement (half maximal activation at 0.19 mM CaCl2), a pH optimum in the neutral range (pH 7-8), and it was inhibited by increased ionic strength (30% inhibition at 0.2 M NaCl). Leupeptin and antipain were strong inhibitors of the enzyme. Ca2+-activated protease activities of the coagulating gland (anterior prostate) were about 40% of those of the ventral prostate and were not detectable in the dorsolateral prostatic lobe. There was no difference in specific activities of this enzyme in chromatographed extracts of prostatic lobes from young sexually mature adults and 12 month old retired breeders. In addition, Ca2+-dependent protease activity was not detectable in chromatograms of rat ventral prostate and coagulating gland secretions. Therefore, the Ca2+-activated protease does not appear to be a secretory protein and probably acts at some intracellular site(s).     

Phospholipid/Ca2+-dependent protein kinase activity in human parathyroid adenoma

We have examined the activities of phospholipid/Ca2+-dependent and cyclic AMP-dependent protein kinases of the parathyroid adenomas and the atrophic glands which were resected from three patients with primary hyperparathyroidism. Phospholipid/Ca2+-dependent protein kinase activity of atrophic parathyroid gland was exclusively present in cytosol fraction (90.7 +/- 12.3%). On the other hand, phospholipid/Ca2+-dependent protein kinase activity of parathyroid adenomas was 66.9 +/- 6.4% in cytosol and 33.1 +/- 6.4% in membrane fraction, suggesting a translocation of the enzyme from the cytosol to the membranes. Cyclic AMP-dependent protein kinase activity appeared to be higher in parathyroid adenoma than in atrophic parathyroid gland in both cytosol and membrane fractions.     

Activation of tyrosine hydroxylase by Ca2+-dependent neutral protease, calpain

Two molecular species of Ca2+-dependent neutral protease (calpains I and II) and its endogenous inhibitor (calpastatin) in cytosol fraction of bovine adrenal medulla were separated by hydrophobic interaction chromatography. Both calpains I and II, having low and high Ca2+ requirements for casein hydrolysis, respectively, were found to activate tyrosine hydroxylase(TH) that had been purified from cytosol fraction of bovine adrenal medulla. This activation of TH by calpain was inhibited by leupeptin and the endogenous inhibitor, calpastatin. The activated TH with calpain II, characterized by high-performance gel permeation chromatography, had a reduced Mr of 120,000 from the Mr of 230,000 of native enzyme.     

Activation of a calmodulin-dependent phosphatase by a Ca2+-dependent protease

A calmodulin-dependent protein phosphatase (calcineurin) was converted to an active, calmodulin-independent form by a Ca2+-dependent protease (calpain I). Proteolysis could be blocked by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, leupeptin, or N-ethylmaleimide, but other protease inhibitors such as phenylmethanesulfonyl fluoride, aprotinin, benzamidine, diisopropyl fluorophosphate, and trypsin inhibitor were ineffective. Phosphatase proteolyzed in the absence of calmodulin was insensitive to Ca2+ or Ca2+/calmodulin; the activity of the proteolyzed enzyme was greater than the Ca2+/calmodulin-stimulated activity of the unproteolyzed enzyme. Proteolysis of the phosphatase in the presence of calmodulin proceeded at a more rapid rate than in its absence, and the proteolyzed enzyme retained a small degree of sensitivity to Ca2+/calmodulin, being further stimulated some 15-20%. Proteolytic stimulation of phosphatase activity was accompanied by degradation of the 60-kilodalton (kDa) subunit; the 19-kDa subunit was not degraded. In the absence of calmodulin, the 60-kDa subunit was sequentially degraded to 58- and 45-kDa fragments; the 45-kDa fragment was incapable of binding 125I-calmodulin. In the presence of calmodulin, the 60-kDa subunit was proteolyzed to fragments of 58, 55 (2), and 48 kDa, all of which retained some ability to bind calmodulin. These data, coupled with our previous report that the human platelet calmodulin-binding proteins undergo Ca2+-dependent proteolysis upon platelet activation [Wallace, R. W., Tallant, E. A., & McManus, M. C. (1987) Biochemistry 26, 2766-2773], suggest that the Ca2+-dependent protease may have a role in the platelet as an irreversible activator of certain Ca2+/calmodulin-dependent reactions.     

Purification of the Ca2+-dependent proteinase inhibitor from bovine cardiac muscle and its interaction with the millimolar Ca2+-dependent proteinase

A protein inhibitor of the Ca2+-dependent proteinase has been purified from bovine cardiac muscle by using the following steps in succession: salting out 17,600 X gmax supernatants from muscle homogenates in 50 mM Tris acetate, pH 7.5, 4 mM EDTA between 25 and 65% ammonium sulfate saturation; eluting between 25 and 120 mM KCl from a DEAE-cellulose column at pH 7.5; salting out between 30 and 60% ammonium sulfate saturation; Ultrogel-22 gel permeation chromatography at pH 7.5; heating to 80 degrees C followed by immediate cooling to 0 degree C; 6% agarose gel permeation chromatography in 4 M urea, pH 7.5; and elution from a phenyl-Sepharose hydrophobic column between 0.7 and 0.5 M ammonium sulfate. Approximately 1.16-1.69 mg of purified Ca2+-dependent proteinase inhibitor are obtained from 1 kg of bovine cardiac muscle, fresh weight. Bovine cardiac Ca2+-dependent proteinase inhibitor has an Mr of 115,000 as measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a pI of 4.85-4.95, very little alpha-helical structure, a very low specific absorbance of 1.647 (A1% 280), and very low contents of histidine, tryptophan, phenylalanine, and tyrosine. Bovine cardiac Ca2+-dependent proteinase inhibitor probably contains a single polypeptide chain in nondenaturing solvents. One 115-kDa inhibitor polypeptide inactivates 10 110-kDa millimolar Ca2+-requiring proteinase (millimolar Ca2+-dependent proteinase) molecules in assays of purified proteins. Inhibition of millimolar proteinase by the proteinase inhibitor did not change in the pH range 6.2-8.6. The inhibitor requires Ca2+ to bind to millimolar Ca2+-dependent proteinase. The Ca2+ concentration required for one-half-maximum binding of millimolar Ca2+-dependent proteinase to the inhibitor was 0.53 mM, compared with a Ca2+ concentration of 0.92 mM required for one-half maximum activity of millimolar Ca2+-dependent proteinase in the absence of the proteinase inhibitor. Unless millimolar Ca2+-dependent proteinase is located subcellularly in a different place than the proteinase inhibitor or unless the proteinase/inhibitor interaction is regulated, millimolar proteinase could never be active in situ.     

The cytosol of human erythrocytes contains a highly Ca2+-sensitive thiol protease (calpain I) and its specific inhibitor protein (calpastatin)

The cytosol of human erythrocytes was found to contain a Ca2+-dependent thiol protease (calpain) and its specific inhibitor (calpastatin) by DEAE-cellulose chromatography at pH 8.0, although no proteolytic activity toward casein was detected in the unfractionated hemolysate. The protease required only 40 microM Ca2+ for 50% activation, indicating that it belongs to the highly Ca2+-sensitive type of calpain, namely, calpain I. It was not inactivated by heating at 58 degrees C for 10 min at pH 7.2, the optimal pH for its action on casein. The inhibitor comprised major and minor components, calpastatin H (Mr = 280,000) and caplastatin L (Mr = 48,000). Both were heat-stable proteins which were readily inactivated by tryptic digestion. The inhibition of erythrocyte calpain by erythrocyte calpastatin H or L was not due to sequestering of Ca2+ from the reaction medium by the inhibitor protein. The calpain preparation preferentially digests bands III and IVa of human erythrocyte membrane proteins, with little or no cleavage of the bands corresponding to spectrin.     

Effect of L-alpha-phosphatidylinositol on a vascular smooth muscle Ca2+-dependent protease. Reduction of the Ca2+ requirement for autolysis

Vascular smooth muscle contains large amounts of a Ca2+-dependent protease. Similar to a Ca2+-dependent protease previously purified from chicken gizzard smooth muscle (Hathaway, D. R., Werth, D. K., and Haeberle, J. R. (1982) J. Biol. Chem. 257, 9072-9077), the mammalian vascular muscle protease is a heterodimer consisting of 76,000- and 30,000-dalton subunits (IIa). The enzyme can undergo autolysis in the presence of Ca2+ to produce a smaller species consisting of 76,000- and 18,000-dalton subunits (IIb). Autolysis greatly reduces the Ca2+ dependence of catalytic activity. The autolytic species, IIb, was approximately 23-fold more sensitive to Ca2+ (K0.5 = 39 microM) than the native enzyme, IIa (K0.5 = 891 microM). In this communication, we report that phosphatidylinositol and to a lesser extent one metabolic derivative, dioleoylglycerol, stimulate autolysis of the vascular Ca2+-dependent protease by reducing the Ca2+ for autolysis from K0.5 = 680 microM in the absence of lipid to K0.5 = 87 microM in the presence of both phosphatidylinositol and dioleoylglycerol. Moreover, the reduction in the Ca2+ requirement for autolysis produced by the phosphatidylinositol was antagonized by the phospholipid-binding drug, trifluoperazine. In addition, the effect of phosphatidylinositol was specific for autolysis, and none of several phospholipids or derivatives tested altered the Ca2+ dependence or maximal rate for protein degradation of the autolytic product, IIb. Our results suggest that autolysis may be an important initial step in the activation of the Ca2+-dependent protease in vascular smooth muscle and that this step may be regulated by a combination of Ca2+ and phosphatidylinositol.     

Essential role of Ca2+ -dependent phospholipase A2 in estradiol-induced lysosome activation

The mechanism of lysosome activation by 17beta-estradiol has been studied in mussel blood cells. Cell treatment with estradiol induced a sustained increase of cytosolic free Ca2+ that was completely prevented by preincubating the cells with the Ca2+ chelator BAPTA-AM. Estradiol treatment was also followed by destabilization of the lysosomal membranes, as detected in terms of the lysosomes' increased permeability to neutral red. The effect of estradiol on lysosomes was almost completely prevented by preincubation with the inhibitor of cytosolic Ca2+ -dependent PLA2 (cPLA2), arachidonyl trifluoromethyl ketone (AACOCF3), and was significantly reduced by preincubation with BAPTA-AM. In contrast, it was virtually unaffected by preincubation with the inhibitor of Ca2+ -independent PLA2, (E)-6-(bromomethylene)tetrahydro-3-(1-naphtalenyl)-2H-pyran-2-one (BEL). The Ca2+ ionophore A-23187 yielded similar effects on [Ca2+](i) and lysosomes. Exposure to estradiol also resulted in cPLA2 translocation from cytosol to membranes, lysosome enlargement, and increased protein degradation. These results suggest that the destabilization of lysosomal membranes following cell exposure to estradiol occurs mainly through a Ca2+ -dependent mechanism involving activation of Ca2+ -dependent PLA2. This mechanism promotes lysosome fusion and catabolic activities and may mediate short-term estradiol effects.     

Ca2+-dependent proteolysis of the Ah receptor

We previously reported (J. Biol. Chem. (1986) 261, 6352-6465) that the photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, upon incubation with the liver cytosol fraction from C57BL/6 mice, labeled in a 1:1 ratio two peptides that had apparent molecular masses of 95 and 70 kDa and similar proteolytic fragmentation patterns. In the cytosolic fraction of Hepa 1 cells, a cloned murine hepatoma cell line, the product of photoaffinity labeling is almost exclusively a 95-kDa peptide which is rapidly hydrolyzed by a Ca2+-dependent proteinase to a 70-kDa peptide as well as other fragments. Thus, the ligand binding unit of the Ah receptor in C57BL/6 mouse liver and Hepa 1 cell is a 95-kDa peptide, and the 70-kDa fragment is a proteolytic artifact. The Ca2+-dependent proteinase which hydrolyzes the 95-kDa peptide has the properties of calpain II: (i) an absolute requirement for Ca2+, with maximal activity at 0.5 to 1.0 mM Ca2+; (ii) a pH optimum of 7.5 to 8.0; (iii) inhibition by EDTA, iodoacetamide, leupeptin and L-trans-epoxysuccinylleucylamido(4-guanidino)butane, but not by soybean trypsin inhibitor, aprotinin, or phenylmethanesufonyl fluoride. Upon chromatographic separation of the liver cytosol of C57BL/6 mice on DEAE-Sephacel, Ca2+-dependent proteinase activity (using casein or the labeled 95-kDa peptide as substrates) elutes with 0.25 M NaCl, and a specific proteinase inhibitor elutes with 0.15 M NaCl. Ca2+-dependent proteinase activity that hydrolyzes the 95-kDa peptide is found in the liver cytosols of several mammalian species.     

Identification of two proteins (actin-binding protein and P235) that are hydrolyzed by endogenous Ca2+-dependent protease during platelet aggregation

Our previous research has shown that the Ca2+-dependent protease within platelets is activated when platelets aggregate, resulting in the production of three polypeptides (Mr = 200,000, 100,000, and 91,000). We have now shown that these three polypeptides arise from the hydrolysis of actin-binding protein. An antibody against actin-binding protein raised in rabbits was shown to be specific for actin-binding protein on immunoblots of total platelet proteins. This antibody reacted with additional polypeptides of Mr = 200,000, 100,000, and 91,000 on immunoblots of the proteins of thrombin-activated platelets. Actin-binding protein was purified from fresh, human platelet concentrate and hydrolyzed with platelet-derived Ca2+-dependent protease; hydrolysis resulted in the appearance of three polypeptides with molecular weights and isoelectric points identical to those of the three polypeptides generated within intact, aggregating platelets. Production of these polypeptides was inhibited by leupeptin and by the chelation of Ca2+. Hydrolysis of actin-binding protein was observed at micromolar Ca2+ concentrations, demonstrating that the level of Ca2+ in aggregated platelets is sufficient to account for the hydrolysis of actin-binding protein by the Ca2+-dependent protease. P235 was also purified and tested for its susceptibility to the protease. It was hydrolyzed by the Ca2+-dependent protease, and two polypeptides (Mr = 200,000 and 46,000) were produced. Antibodies against P235 raised in rabbits reacted only with P235 on immunoblots of total platelet proteins. These antibodies also reacted with polypeptides of Mr = 200,000 and 46,000 on immunoblots of thrombin-activated platelets. These data show that both actin-binding protein and P235 are cleaved during thrombin-induced platelet aggregation and suggest that the activation of the Ca2+-dependent protease may permit reorganization of the platelet cytoskeleton in aggregating platelets.     

Regulation of the Ca2+-dependent neutral proteinases from rabbit liver by an endogenous inhibitor

An endogenous inhibitor of neutral Ca2+-dependent proteinases has been isolated from rabbit liver cytosol. The inhibitor is a heat-stable, 240-kDa, tetrameric protein. It is dissociated into its 60-kDa subunits by high concentrations of Ca2+ (0.1-1 mM), but not by lower concentrations in the physiological range. Inhibition of the 150-kDa proteinase of rabbit liver [Melloni, E., Pontremoli, S., Salamino, F., Sparatore, B., Michetti, M. and Horecker, B.L. (1984) Arch. Biochem. Biophys. 232, 505-512] requires the monomeric form of the inhibitor, and occurs only at the high concentrations of Ca2+ which also cause dissociation of the dimeric 150-kDa proteinase into its 80-kDa subunits. The molecular weight of the inactive proteinase-inhibitor complex was estimated by the equilibrium gel penetration method to be 140 kDa, suggesting that it contains one subunit of proteinase and one of inhibitor. The mechanism of interaction of the inhibitor with the 200-kDa proteinase at high concentrations of Ca2+ is identical to that observed for the 150-kDa proteinase, namely dissociation of both proteinase and inhibitor into subunits and formation of an inactive 160-kDa proteinase-inhibitor complex. However, unlike the 150-kDa proteinase, which does not interact with the inhibitor at low Ca2+ concentrations, the 200-kDa proteinase is also inhibited at low concentrations of Ca2+. Under these conditions, the high-molecular-weight complex (greater than 400 kDa) formed between the tetrameric inhibitor and the dimeric proteinase prevents conversion of the 200-kDa proenzyme to the active, low-Ca2+-requiring form.     

The appearance of a 34,000-dalton inhibitor of calpain (Ca2+-dependent cysteine proteinase) in rat liver after the administration of phenylhydrazine

A 34,000-dalton inhibitor of calpain (Ca2+-dependent cysteine proteinase) was found in the cytosol of anemic rat liver. When phenylhydrazine hydrochloride was continuously administered to rats, a 280,000-dalton calpain inhibitor that existed originally in the liver gradually disappeared within two weeks and, concomitantly, a 34,000-dalton inhibitor appeared. The purified 34,000-dalton inhibitor resembles 280,000-dalton inhibitor in that both are heat-stable proteins and do not inhibit papain and trypsin. Unlike the protomers of a 280,000-dalton inhibitor, 34,000-dalton inhibitor does not show any sign of self-association.     

Domain II of m-calpain is a Ca(2+)-dependent cysteine protease

Calpain, a Ca(2+)-dependent cytosolic cysteine protease, proteolytically modulates specific substrates involved in Ca(2+)-mediated intracellular events, such as signal transduction, cell cycle, differentiation, and apoptosis. The 3D structure of m-calpain, in the absence of Ca(2+), revealed that the two subdomains (domains IIa and IIb) of the protease domain (II) have an 'open' conformation, probably due to interactions with other domains. Although the presence of an EF-hand structure was once predicted in the protease domain, no explicit Ca(2+)-binding structure was identified in the 3D structure. Therefore, it is predicted that if the protease domain is excised from the calpain molecule, it will have a Ca(2+)-independent protease activity. In this study, we have characterized a truncated human m-calpain that consists of only the protease domain. Unexpectedly, the proteolytic activity was Ca(2+)-dependent, very weak, and not effectively inhibited by calpastatin, a calpain inhibitor. Ca(2+)-dependent modification of the protease domain by the cysteine protease inhibitor, E-64c, was clearly observed as a SDS-PAGE migration change, indicating that the conformational changes of this domain are a result of Ca(2+) binding. These results suggest that the Ca(2+) binding to domain II, as well as to domains III, IV, and VI, is critical in the process of complete activation of calpain.     

Activation of the Ca2+-ATPase of human erythrocyte membrane by an endogenous Ca2+-dependent neutral protease

Limited proteolysis of the plasma membrane calcium transport ATPase (Ca2+-ATPase) from human erythrocytes by trypsin produces a calmodulin-like activation of its ATP hydrolytic activity and abolishes its calmodulin sensitivity. We now demonstrate a similar kind of activation of the human erythrocyte membrane Ca2+-ATPase by calpain (calcium-dependent neutral protease) isolated from the human red cell cytosol. Upon incubation of red blood cell membranes with purified calpain in the presence of Ca2+ the membrane-bound Ca2+-ATPase activity was increased and its sensitivity to calmodulin was lost. In contrast to the action of other proteases tested, proteolysis by calpain favors activation over inactivation of the Ca2+-ATPase activity, except at calpain concentrations more than 2 orders of magnitude higher. Exogenous calmodulin protects the Ca2+-ATPase against calpain-mediated activation at concentrations which also activate the Ca2+-ATPase activity. Calcium-dependent proteolytic modification of the Ca2+-ATPase could provide a mechanism for the irreversible activation of the membrane-bound enzyme.     

Calcium-dependent and calcium-independent gelatinolytic proteinase activities of the rat ventral prostate and its secretion: characterization and effect of castration and testosterone treatment

Calcium-dependent and calcium-independent proteinase activities were detected in extracts of rat ventral prostate and its secretion by use of gelatin-containing SDS-PAGE zymography. Ca(2+)-independent proteinase activities of 22, 26, and 73-79 kDa and Ca(2+)-dependent activities of 58, 63, and 66 kDa were found in the adult gland. The 26- (most intense activity of gland) and 22-kDa activities were present in secretion and were not expressed in the undifferentiated gland of the 10-day-old animal. The Ca(2+)-dependent activities were also present in the secretion, where the 63-kDa form was more prominently expressed than the 58- and 66-kDa bands. The Ca(2+)-dependent and Ca(2+)-independent proteinase activities both responded to a broad range of pH values in the incubation media. The 73-79-kDa Ca(2+)-independent activities were sensitive to benzamidine and the Ca(2+)-dependent activities were inhibited by EDTA and EGTA. Both Ca(2+)-dependent and Ca(2+)-independent proteinase activities responded to androgenic manipulations. Castration was followed by the appearance of a 35-kDa Ca(2+)-independent proteinase (at 2 days) and a 43-kDa Ca(2+)-dependent proteinase (at 4 days). In the Ca(2+)-independent proteinase group, the 73-79-kDa activities were increased somewhat and the 22- and 26-kDa activities decreased after castration. The Ca(2+)-dependent proteinases of 58, 63, and 66 kDa increased in activity with castration, but activity of the 58-kDa form decreased again at 7 days after castration. Treatment of animals upon castration for 4 days with hydrocortisone prevented these changes in proteinase activities whereas treatment with actinomycin D or tranexamic acid did not. Testosterone propionate replacement therapy of rats castrated for 16 days stimulated the activities of the 22- and 26-kDa and 73-79-kDa Ca(2+)-independent and the 58- and 63-kDa Ca(2+)-dependent proteinases with 4 days of therapy. The activities of the 35-kDa Ca(2+)-independent and the 43-kDa Ca(2+)-dependent proteinases were repressed with 8 days of testosterone treatment. Thus, individual proteinases show differential changes in activity during development and in response to androgenic manipulation: this suggests that in addition to proteinases which are secreted, others may be involved in intracellular functions or in mediating tissue organization changes.     

A secretory protease inhibitor requires androgens for its expression in male sex accessory tissues but is expressed constitutively in pancreas.

A full length cDNA clone encoding a mouse prostatic secretory glycoprotein (p12) whose synthesis is dependent upon testicular androgens has been cloned and characterized. The predicted amino acid sequence of p12 shares extensive homology with several members of the Kazal family of secretory protease inhibitors, in particular the pancreatic secretory trypsin inhibitors. In agreement with sequence data, prostatic secretory p12, purified from mouse ventral prostate secretion, exhibits anti-trypsin activity. Steady-state levels of protease inhibitor mRNA in ventral prostate are reduced from approximately 0.06% in normal mice to undetectable after androgen withdrawal but are inducible within 4 h by re-administration of testosterone. Androgen-dependent expression of the secretory protease inhibitor mRNA was also observed in coagulating gland and seminal vesicle. In seminal vesicle, a tissue of different embryonic origin to the prostate, the kinetics of secretory protease inhibitor mRNA loss after castration are not as rapid as in the ventral prostate and coagulating gland. Low-level androgen independent expression was also observed in the pancreas. There appears to be a single gene for this secretory protease inhibitor and yet expression is markedly stimulated by testosterone in the sex accessory tissues and unaffected by this hormone in the pancreas.     

Ca2+ transport and Ca2+-dependent ATP hydrolysis by Golgi vesicles from lactating rat mammary glands.

Ca2+ transport across mammary-gland Golgi membranes was measured after centrifugation of the membrane vesicles through silicone oil. In the presence of 2.3 microM free Ca2+ the vesicles accumulated 5.8 nmol of Ca2+/mg of protein without added ATP, and this uptake was complete within 0.5 min. In the presence of 1 mM-ATP, Ca2+ was accumulated at a linear rate for 10 min after the precipitation of intravesicular Ca2+ with 10 mM-potassium oxalate. ATP-dependent Ca2+ uptake exhibited a Km of 0.14 microM for Ca2+ and a Vmax. of 3.1 nmol of Ca2+/min per mg of protein. Ca2+-dependent ATP hydrolysis exhibited a Km of 0.16 microM for Ca2+ and a Vmax. of 10.1 nmol of Pi/min per mg of protein. The stoichiometry between ATP-dependent Ca2+ uptake and Ca2+-stimulated ATPase varied between 0.3 and 0.7 over the range 0.03-8.6 microM-Ca2+. Both Ca2+ uptake and Ca2+-stimulated ATPase were strongly inhibited by orthovanadate, which suggests that the major mechanism by which Golgi vesicles accumulate Ca2+ is through the action of the Ca2+-stimulated ATPase. However, Ca2+ uptake was also decreased by the protonophore CCCP (carbonyl cyanide m-chlorophenylhydrazone), indicating that it may occur by other mechanisms too. The effect of CCCP may be related to the existence of transmembrane pH gradients (delta pH) in these vesicles: the addition of 30 microM-CCCP reduced delta pH from a control value of 1.06 to 0.73 pH unit. Golgi vesicles also possess a Ca2+-efflux pathway which operated at an initial rate of 0.5-0.57 nmol/min per mg of protein.     

Fortilin binds Ca2+ and blocks Ca2+-dependent apoptosis in vivo

Fortilin, a 172-amino-acid polypeptide present both in the cytosol and nucleus, possesses potent anti-apoptotic activity. Although fortilin is known to bind Ca2+, the biochemistry and biological significance of such an interaction remains unknown. In the present study we report that fortilin must bind Ca2+ in order to protect cells against Ca2+-dependent apoptosis. Using a standard Ca2+-overlay assay, we first validated that full-length fortilin binds Ca2+ and showed that the N-terminus (amino acids 1-72) is required for its Ca2+-binding. We then used flow dialysis and CD spectropolarimetry assays to demonstrate that fortilin binds Ca2+ with a dissociation constant (Kd) of approx. 10 mM and that the binding of fortilin to Ca2+ induces a significant change in the secondary structure of fortilin. In order to evaluate the impact of the binding of fortilin to Ca2+ in vivo, we measured intracellular Ca2+ levels upon thapsigargin challenge and found that the lack of fortilin in the cell results in the exaggerated elevation of intracellular Ca2+ in the cell. We then tested various point mutants of fortilin for their Ca2+ binding and identified fortilin(E58A/E60A) to be a double-point mutant of fortilin lacking the ability of Ca2+-binding. We then found that wild-type fortilin, but not fortilin(E58A/E60A), protected cells against thapsigargin-induced apoptosis, suggesting that the binding of fortilin to Ca2+ is required for fortilin to protect cells against Ca2+-dependent apoptosis. Together, these results suggest that fortilin is an intracellular Ca2+ scavenger, protecting cells against Ca2+-dependent apoptosis by binding and sequestering Ca2+ from the downstream Ca2+-dependent apoptotic pathways.     

Ca2+-dependent ATPases in the basolateral membrane of rat kidney cortex

The basolateral segment of the rat renal tubular plasma membrane possesses Ca2+-dependent ATPase activity which was independent of Mg2+. Two kinetic forms were found: one, was a high affinity (apparent Km for free Ca2+ of 172 nM) low capacity (Vmax of 144 nmol of Pi X min-1 mg-1 protein) type; the other, had low affinity (apparent Km of 25 microM) and high capacity (896 nmol of Pi X min-1 X mg-1 protein). Mg2+ inhibited both Ca2+-ATPases. The high affinity enzyme exhibited positive cooperativity with respect to ATP, with a n value of 1.6. Ca2+-ATPase activity was not affected by calmodulin and was not inhibited by vanadate. On the other hand, both high and low affinity Ca2+-ATPase activities were increased when 1,25-dihydroxycholecalciferol was given to vitamin D-deficient rats. Kinetically, the enhanced activities were due to an increase in the Vmax values; the apparent affinities for free Ca2+ were not changed. The physiological function of the vitamin D-sensitive, Mg+-independent, Ca2+-ATPase activities remains to be established.     

Ca2+-dependent conformational changes in bovine GCAP-2.

GCAP-2, a mammalian photoreceptor-specific protein, is a Ca2+-dependent regulator of the retinal membrane guanylyl cyclases (Ret-GCs). Sensing the fall in intracellular free Ca2+ after photo-excitation, GCAP-2 stimulates the activity of Ret-GC leading to cGMP production. Like other members of the recoverin superfamily, GCAP-2 is a small N-myristoylated protein containing four EF-hand consensus motifs. In this study, we demonstrate that like recoverin and neurocalcin, GCAP-2 alters its conformation in response to Ca2+-binding as measured by a Ca2+-dependent change in its far UV CD spectrum. Differences in the conformation of the Ca2+-bound and Ca2+-free forms of GCAP-2 were also observed by examining their relative susceptibility to V8 protease. In contrast to recoverin, we do not observe proteolytic cleavage of the myristoylated N-terminus of Ca2+-bound GCAP-2. NMR spectra also show that, in contrast to recoverin, the chemical environment of the N-terminus of GCAP-2 is not dramatically altered by Ca2+ binding. Despite the similarity of GCAP-2 and recoverin, the structural consequences of Ca2+-binding for these two proteins are significantly dissimilar.