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1.
Ca2+ regulation of thyroid NADPH-dependent H2O2 generation 总被引:1,自引:0,他引:1
A thyroid particulate fraction contains an NADPH-dependent H2O2-generating enzyme which requires Ca2+ for activity. A Chaps solubilized extract of the thyroid particulate fraction partially purified by DEAE chromatography did not show a dependence on Ca2+ for activity. Preincubation of the particulate fraction with Ca2+ yielded a preparation insensitive to Ca2+. The non-particulate fraction obtained after incubation of the particles in the presence of Ca2+ was able to inhibit, in the presence of EGTA, the Ca2+-desensitized particulate fraction and the enzyme isolated on DEAE. It is concluded that the reversible Ca2+ activation of the NADPH-dependent H2O2 generation was modulated in porcine thyroid tissue by (a) calcium-releasable inhibitor protein(s). 相似文献
2.
The release of plasma-membrane-bound enzymes by phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis was investigated. Among the ectoenzymes of plasma membrane tested, alkaline phosphodiesterase I was released markedly from rat kidney cortex slices, in addition to alkaline phosphatase and 5'-nucleotidase. Other membrane-bound enzymes; alanine aminopeptidase, leucine aminopeptidase, dipeptidyl peptidase, leucine aminopeptidase, dipeptidyl peptidase IV, esterase and gamma-glutamyl transpeptidase could not be liberated from the treated slices. Alkaline phosphodiesterase I was released linearly from rat kidney slices with the concentration of phosphatidylinositol-specific phospholipase C, but little enzyme was released from rat liver slices. Alkaline phosphodiesterase I separated from kidney tissue with n-butanol still retained phosphatidylinositol and was transformed into a lower molecular weight form by phosphatidylinositol-specific phospholipase C. This suggests an important function for phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membrane of rat kidney cells. The alkaline phosphodiesterase I released from rat kidney had a molecular weight of about 240,000 and an isoelectric point (pI) of 5.4. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9 and had a Km value of 0.3 mM. The enzyme was activated by Mg2+ and Ca2+, but was inhibited by EDTA. Strong inhibition took place on the addition of adenosine 5'-phosphosulfate or the nucleotide pyrophosphates, i.e., UDP-galactose and alpha, beta-methylene ATP. 相似文献
3.
The effect of thyroid hormone on the high affinity Ca2+-ATPase activity in rat liver plasma membrane was studied. The high affinity Ca2+-ATPase activity in plasma membrane was activated by 10(-7)-10(-5) M of Ca2+ and was inhibited by 70 microM trifluoperazine. Thyroidectomy of rats was associated with an increase in the activity of high affinity Ca2+-ATPase. The increased enzyme activity was normalized by T4 administration to the animals. On the other hand, Na+-K+-ATPase activity in the membrane was decreased by thyroidectomy and the decreased enzyme activity was normalized by T4 administration. The results suggest that thyroid hormone inhibits the Ca2+ extrusion system by inhibiting calmodulin-independent high affinity Ca2+-ATPase in liver plasma membrane. 相似文献
4.
Y Banno S Nakashima T Tohmatsu Y Nozawa E G Lapetina 《Biochemical and biophysical research communications》1986,140(2):728-734
The hydrolysis of [3H]phosphatidylinositol 4,5-bisphosphate (PIP2) by cytosolic phospholipase C from human platelets was determined. Cytosolic fractions were prepared from platelets that had or had not been preactivated with thrombin. Thrombin pretreatment did not affect cytosolic phospholipase C activity. In both cytosolic fractions, phospholipase C was activated by GTP and GTP gamma S. This action is observed in the presence of 2 mM EGTA. GDP was as effective as GTP in stimulating cytosolic phospholipase C in the presence of Ca2+ or EGTA. Partially purified phospholipase C obtained from platelet cytosol is activated by GTP, but not by GTP gamma S, in the presence of 2 mM EGTA. However, in the presence of 6 microM Ca2+, both GTP and GTP gamma S stimulated the partially purified phospholipase C. Our present information indicates that GTP and GDP have a direct effect on the cytosolic phospholipase C. 相似文献
5.
From various rat organs, alkaline phosphodiesterase I was liberated by the action of phosphatidylinositol-specific phospholipase C obtained from Bacillus thuringiensis. Especially, a large amount of alkaline phosphodiesterase I was released from slices of small intestine, testis, lung, and kidney, but not from pancreas and liver. The release of the enzyme induced by phospholipase C was dependent on, or proportional to, the reaction time and the concentrations of the phospholipase C and the weight of the slices of small intestine or testis. Furthermore, little enzyme was released from the homogenate of pancreas. These results suggest an important role of phosphatidylinositol in the binding of alkaline phosphodiesterase I to the plasma membranes of rat small intestine and pancreas. The alkaline phosphodiesterase I released from slices of rat small intestine and testis had a molecular weight of about 240,000, and was activated by Mg2+ and Ca2+ but inhibited by EDTA. The enzyme hydrolyzed the phosphodiester linkage of p-nitrophenyl-thymidine 5'-monophosphate at pH 8.9, having the Km values of 0.36 mM (small intestine) and 0.25 mM (testis). The intestinal enzyme differed from the testis enzyme in pI values, thermostability, and Arrhenius plot having a single breakpoint. 相似文献
6.
Detergent Effects on the Phosphatidylinositol-Specific Phospholipase C in Rat Brain Synaptosomes 总被引:2,自引:1,他引:1
In the presence of Ca2+ (2.5 mM) and using [14C]arachidonoyl phosphatidylinositol (PI) membrane as substrate, phosphatidylinositol-specific phospholipase C (PI-PLC) (EC 3.1.4.10) in rat brain synaptosomes was activated by deoxycholate but not taurocholate. Calcium stimulated enzymic hydrolysis by both detergents, but the stimulatory effect of taurocholate was less than that of deoxycholate. Peak stimulation for deoxycholate was observed at 1 mg/ml, whereas that for taurocholate was 4 mg/ml. When 1 mM EDTA was added to the taurocholate (4 mg/ml) and Ca2+ (3.5 mM) system, synaptosomal PI-PLC activity was greatly stimulated, to almost the same level as the deoxycholate + Ca2+ system. This system required the presence of all three factors, and EGTA could not effectively replace EDTA in the stimulatory action. The detergent-induced hydrolysis of synaptosomal PI by the deoxycholate + Ca2+ and the taurocholate + Ca2+ + EDTA systems was strongly inhibited by divalent metal ions such as Zn2+, Cu2+, Pb2+, and Fe2+, whereas Mg2+ and Ca2+ were ineffective. Nevertheless, only the deoxycholate + Ca2+ system was responsive to enzyme inhibition by membrane-perturbing agents such as lysophospholipids and free fatty acids. The specific requirement for EDTA in the taurocholate system may be due to the release of a pool of inhibitory divalent metal ions from the membranes. 相似文献
7.
Compound 48/80 is a potent inhibitor of phospholipase C and a dual modulator of phospholipase A2 from human platelet 总被引:2,自引:0,他引:2
C Bronner C Wiggins D Monté F M?rki A Capron Y Landry R C Franson 《Biochimica et biophysica acta》1987,920(3):301-305
Compound 48/80 inhibited phosphatidylinositol-specific phospholipase C activity from human platelets. Whereas 1 microgram/ml of compound 48/80 slightly stimulated Ca2+-dependent phospholipase A2, higher concentrations led to dose-dependent inhibition of this platelet enzyme. This biphasic effect was confirmed with phospholipases A2 purified from rat liver and human synovial fluid. The aggregation of human platelets induced by ADP and PAF-acether was inhibited by compound 48/80, whereas the aggregation induced by ionophore A23187 was not modified by this compound. These results demonstrate that the inhibition of platelet aggregation by compound 48/80 is not due solely to effects on calmodulin as previously reported, but that inhibition of phospholipases and probably arachidonate mobilization may also be involved. 相似文献
8.
The selective release of phospholipase A2 by resident mouse peritoneal macrophages. 总被引:3,自引:0,他引:3
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Resident mouse peritoneal macrophages have three phospholipase activities: a phospholipase A2 active at pH 4.5, a Ca2+-dependent phospholipase A2 active at pH 8.5 and a phosphatidylinositol-specific phospholipase C activity. When macrophages are exposed to zymosan in culture, the cellular activity of pH-4.5 phospholipase A2 is diminished in a manner dependent on zymosan concentration and time of exposure, whereas the cellular activities of pH-8.5 phospholipase A2 and phospholipase C remain unchanged. The depletion of pH-4.5 phospholipase A2 activity from the cell is paralleled by a quantitative recovery of this activity in the culture medium in a manner similar to the cellular depletion and extracellular recovery of two lysosomal enzymes. This release is specifically elicited by an inflammatory substance such as zymosan, since macrophages incubated with 6 micrometer latex spheres retain pH-4.5 phospholipase A2 activity and lysosomal enzyme activities intracellularly. 相似文献
9.
Calcium-independent phospholipase A2 in rat tissue cytosols 总被引:3,自引:0,他引:3
A J Pierik J G Nijssen A J Aarsman H Van den Bosch 《Biochimica et biophysica acta》1988,962(3):345-353
Cytosols (105,000 X g supernatant) from seven rat tissues were assayed for Ca2+-independent phospholipase A2 activity with either 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine, 1-acyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphoethanolamine or 1-O-hexadecyl-2-[9,10-3H2]oleoyl-sn-glycero-3-phosphocholine as substrate. Low but consistent activities ranging from 10-120 pmol/min per mg protein were found in all tissues. The highest activities were present in liver, lung and brain. Total activities in mU/g wet weight were rather constant, ranging from 0.43 (heart) to 1.36 (liver). The soluble enzyme from rat lung cytosol was further investigated and was found to be capable of hydrolyzing microsomal membrane-associated substrates without exhibiting much selectivity for phosphatidylcholine species. Comparative gel filtration experiments of cytosol prepared from non-perfused and perfused lungs indicated that part of the Ca2+-independent phospholipase A2 originated from blood cells, but most of it was derived from lung cells. Lung cytosol also contained Ca2+-dependent phospholipase A2 activity, a small part of which originated from blood cells, presumably platelets. The major amount of Ca2+-dependent phospholipase A2 activity, however, came from lung cells. Neither this enzyme nor the Ca2+-independent phospholipase A2 from lung tissue showed immunological cross-reactivity with monoclonal antibodies against Ca2+-dependent phospholipase A2 isolated from rat liver mitochondria. 相似文献
10.
A calcium-dependent mechanism for associating a soluble arachidonoyl-hydrolyzing phospholipase A2 with membrane in the macrophage cell line RAW 264.7 总被引:14,自引:0,他引:14
Arachidonoyl-hydrolyzing phospholipase A2 plays a central role in providing substrate for the synthesis of the potent lipid mediators of inflammation, the eicosanoids, and platelet-activating factor. Although Ca2+ is required for arachidonic acid release in vivo and most phospholipase A2 enzymes require Ca2+ for activity in vitro, the role of Ca2+ in phospholipase A2 activation is not understood. We have found that an arachidonoyl-hydrolyzing phospholipase A2 from the macrophage-like cell line, RAW 264.7, exhibits Ca2(+)-dependent association with membrane. The intracellular distribution of the enzyme was studied as a function of the Ca2+ concentration present in homogenization buffer. The enzyme was found almost completely in the 100,000 x g soluble fraction when cells were homogenized in the presence of Ca2+ chelators and there was a slight decrease in soluble fraction activity when cells were homogenized at the level of Ca2+ in an unstimulated cell (80 nM). When cells were homogenized at Ca2+ concentrations expected in stimulated cells (230-450 nM), 60-70% of the phospholipase A2 activity was lost from the soluble fraction and became associated with the particulate fraction in a manner that was partly reversible with EGTA. Membrane-associated phospholipase A2 activity was demonstrated by [3H]arachidonic acid release both from exogenous liposomes and from radiolabeled membranes. With radiolabeled particulate fraction as substrate, this enzyme hydrolyzed arachidonic acid but not oleic acid from membrane phospholipid, and [3H]arachidonic acid was derived from phosphatidylcholine, phosphatidylethanolamine, and phosphatidylinositol/phosphatidylserine. We suggest a mechanism in which the activity of phospholipase A2 is regulated by Ca2+: in an unstimulated cell phospholipase A2 is found in the cytosol; upon receptor ligation the cytosolic Ca2+ concentration increases, and the enzyme becomes membrane-associated which facilitates arachidonic acid hydrolysis. 相似文献
11.
Incubation of human platelets with C. perfringens phospholipase C caused an increase in soluble protein kinase activity assayed in the presence of EGTA, and a decrease in Ca2+/phospholipid-dependent protein kinase activity. Fractionation of extracts on DEAE-cellulose columns showed that phospholipase C treatment resulted in a new peak of protein kinase active in the presence of EGTA. On Sephadex G-100 chromatography this enzyme eluted as a single peak of protein kinase activity of MW about 50,000. An extract from untreated platelets eluted as a single peak of Ca2+/phospholipid-dependent protein kinase of MW about 77,000. It was concluded that phospholipase C treatment resulted in the proteolysis of this latter enzyme to the lower MW form. 相似文献
12.
Calcium-requirement and its pH dependency of lysophosphoinositide-specific phospholipase C in porcine platelet membranes 总被引:1,自引:0,他引:1
The lysophosphoinositide-specific phospholipase C (lysoPI-PLase C) in porcine platelet membranes had an optimal pH of 9.2 and the activity at a physiological pH of 7.3 was 20% of the maximum in the absence of added divalent metals (Murase, S. et al. (1985) J. Biol. Chem. 260, 262). The activity was completely inhibited by 1 mM EGTA in the assay mixture but was restored by addition of excess Ca2+ or Mn2+, indicating that this is a metalloenzyme. However, membranes pretreated with 1 mM EGTA and washed with buffer retained full activity at a free Ca2+ concentration of 5 nM and no stimulation was observed by added Ca2+ at pH 9.2. In contrast to the results obtained at pH 9.2, addition of Ca2+ stimulated lysoPI-PLase C activity severalfold at pH 7.3, apparently by shifting down the optimal pH and broadening the pH profile. The effect of Ca2+ at pH 7.3 was to enhance Vmax with no significant change in Km value. The stimulatory effect of Ca2+ at pH 7.3 alone did not appear to be of physiological significance since millimolar concentrations of Ca2+ were necessary to reach the maximum activity. However, a shift in pH had a profound effect on the Ca2+-dependency of the activity. A rise in 2 pH units increased the apparent affinity for Ca2+ 10,000-fold. These results indicate that the alkalinization and the rise in free Ca2+ concentration known to occur in stimulated platelets could synergistically provide conditions under which the lysoPI-PLase C exerts its activity when the substrate lysoPI is generated by phospholipase A. 相似文献
13.
The present study examined (a) the source of arachidonic acid for Ca2+-stimulated renal inner medullary prostaglandin synthesis, (b) the Ca2+-dependence of enzymes of the phospholipase A2 and C pathways, and (c) the role of calmodulin in these Ca2+ actions. Ca2+ plus the ionophore A23187 stimulated (2-4-fold) release of labeled arachidonate, diglyceride, prostaglandin E2 or F2 alpha from inner medullary slices with a concomitant fall in labeled phosphatidylcholine, phosphatidylinositol, and phosphatidylethanolamine. The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide hydrochloride (W-7) (10-100 microM) abolished or suppressed Ca++-stimulated immunoreactive prostaglandin E, labeled arachidonate and prostaglandin release, and the fall in labeled phospholipids but did not suppress labeled diglyceride or inositol accumulation. Studies in subcellular fractions demonstrated a particulate phospholipase A2 activity and a phosphatidylinositol-specific phospholipase C activity which was predominantly soluble (80%). W-7 or trifluoperazine (25 microM) abolished Ca2+-stimulated phospholipase A2 activity and particulate phospholipase C activity but were without effect on soluble phospholipase C. W-7 (100 microM) was without effect on Ca2+-stimulated diglyceride lipase and phosphatidic acid-specific phospholipase A2 activities. Hypertonic urea at concentrations that pertain in the inner medulla of hydropenic rats in vivo inhibited Ca2+-induced increases in labeled arachidonate release and immunoreactive prostaglandin E in slice incubates and Ca2+-responsive phospholipase C and A2. The results are consistent with the involvement of phospholipase A2, C, or both in the Ca2+ (+A23187)-stimulated release of free arachidonate for prostaglandin synthesis and support a role for calmodulin in Ca2+ activation of phospholipase A2 and particulate phospholipase C. 相似文献
14.
Y Takeuchi 《Biochimica et biophysica acta》1988,960(2):157-163
The interaction between Penicillium notatum phospholipase B and divalent cations such as Ca2+ and Mg2+ was studied. When the purified enzyme, present at concentrations of submicrogram to microgram per ml, was incubated with submillimolar to millimolar concentrations of CaCl2 or MgCl2, the enzymatic activity was remarkably decreased (to no more than 30% of original activity, when the enzyme was incubated with 2 mM CaCl2 for 15 min). The inhibitory effect of divalent cations was reversible, since dialysis against a metal chelator, such as EDTA or EGTA, substantially restored the enzymatic activity. Atomic absorption analysis showed the purified enzyme molecule to be present in a complex with Ca2+ at a ratio approaching 1:1, and this Ca2+ binding was shown to be extremely tight, since repeated dialyses of the enzyme molecules against EDTA or EGTA could remove the divalent cations only in a gradual manner. During this process, the enzyme activity increased also gradually. The remnant fraction of tightly bound Ca2+ was released from the enzyme molecule after the denaturation of the enzyme by treatment with guanidine hydrochloride, and the apoenzyme recovered its substantial activity after removal of the denaturing agent by dialysis. On the other hand, the content of Mg2+ in the purified enzyme molecule was lower than that of Ca2+, and the association of Mg2+ with the enzyme was much weaker in comparison to that of Ca2+. Atomic absorption analysis of the enzyme exposed to exogenous Ca2+ showed a fast removal, by dialysis, of unbound and weakly bound divalent cation, followed by a gradual removal of endogenous Ca2+ and a concomitant increase of enzymatic activity, which are similar to data obtained for the purified enzyme. Results shown in this report suggest some regulatory roles of divalent cations, especially of Ca2+, in the enzymatic function of P. notatum phospholipse B. 相似文献
15.
When rat liver mitochondria are allowed to accumulate Ca2+, treated with ruthenium red to inhibit reverse activity of the Ca2+ uniporter, and then treated with an uncoupler, they release Ca2+ and endogenous Mg2+ and undergo large amplitude swelling with ultrastructural expansion of the matrix space. These effects are not produced by Ca2+ plus uncoupler alone. Like other "Ca2+-releasing agents" (i.e. N-ethylmaleimide, t-butylhydroperoxide, oxalacetate, etc.), the development of nonspecific permeability produced by ruthenium red plus uncoupler requires accumulated Ca2+ specifically and is antagonized by inhibitors of phospholipase A2. The permeability responses are also antagonized by ionophore A23187, indicating that a rapid pathway for Ca2+ efflux from deenergized mitochondria is necessary to prevent the development of nonspecific permeability. EGTA can be substituted for ruthenium red to produce the nonspecific permeability change in Ca2+-loaded, uncoupler-treated mitochondria. The permeability responses to EGTA plus uncoupler again require accumulated Ca2+ specifically and are antagonized by inhibitors of phospholipase A2 and by ionophore A23187. The equivalent effects of ruthenium red and EGTA on uncoupled, Ca2+-containing mitochondria indicate that reducing the extramitochondrial Ca2+ concentration to the subnanomolar range produces inhibition of reverse uniport activity. It is proposed that inhibition reflect regulation of the uniporter by a Ca2+ binding site which is available from the cytoplasmic side of the inner membrane. EDTA cannot substitute for EGTA to induce nonspecific permeability in Ca2+-loaded, uncoupled mitochondria. Furthermore, EDTA inhibits the response to EGTA with an I50 value of approximately 10 microM. These data suggest that the uniporter regulatory site also binds Mg2+. The data suggest further that Mg2+ binding to the regulatory site is necessary to inhibit reverse uniport activity, even when the site is not occupied by Ca2+. 相似文献
16.
M. Quik 《Journal of neurochemistry》1987,48(1):217-224
Superior cervical ganglion phospholipase A2 activity was characterized using 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine as a substrate. The enzyme activity exhibited linearity with interval of incubation and tissue concentration; there appeared to be two pH optima of the enzyme, at pH 6.0 and 9.0. A Lineweaver-Burk plot of the reciprocal of activity versus substrate concentration yielded an apparent Km of 0.53 mM and a Vmax of 5.3 nmol/h/mg of protein. The enzyme exhibited a partial Ca2+ dependence; in the absence of Ca2+ and presence of EGTA, activity was reduced by 40%. The phospholipase A2 activity was heat sensitive and was completely inactivated after treatment at 100 degrees C for 30 min. For determination of whether the enzyme had a preference for hydrolysis of specific fatty acid substituents in the 2 position of phosphatidylcholine, several different substrates were tested. The order of preference for hydrolysis by the ganglionic enzyme was 1-palmitoyl-2-[1-14C]arachidonoyl-sn-glycero-3-phosphocholine = 1-palmitoyl-2-[1-14C]linoleoyl-sn-glycero-3-phosphocholine greater than 1-palmitoyl-2-[1-14C]palmitoyl-sn-glycero-3-phosphocholine. For determination of the localization of the phospholipase A2 enzyme in sympathetic ganglia, two approaches were used. Guanethidine, which results in destruction of adrenergic cell bodies in sympathetic ganglia, was administered to rats; an approximately 50% decline in phospholipase A2 activity was observed after this treatment. In other experiments, the preganglionic nerve to the ganglion was sectioned in rats; after 2 weeks of denervation, no significant change in ganglionic phospholipase A2 activity was seen.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
We have previously demonstrated that 5'-nucleotidase, known as a plasma membrane enzyme, is also distributed both in rat liver tritosomal membranes and contents (J. Biochem. 101, 1077-1085, 1987). When the lysosomal membranes isolated from rat livers were incubated with phosphatidylinositol-specific phospholipase C purified from B. thuringiensis, about 70% of 5'-nucleotidase activity was released from the membranes. Judging from the result by phase separation with Triton X-114, the enzyme solubilized by the phospholipase C digestion showed a hydrophilic nature such as that of the tritosomal contents. Immunoblot analysis showed that the molecular weight of 5'-nucleotidase released from the lysosomal membranes by the phospholipase C digestion was almost identical with that of the enzymes from the Tritosomal contents. The above results showed that the phosphatidylinositol-specific phospholipase C-like enzyme in the lysosomes may be responsible for the conversion of the lysosomal membrane-bound 5'-nucleotidase to the soluble form present in the lysosomal matrix. 相似文献
18.
An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine. 相似文献
19.
H Krause P Dieter A Schulze-Specking A Ballhorn K Decker 《European journal of biochemistry》1991,199(2):355-359
In cell-free extracts of rat liver macrophages (Kupffer cells) phospholipase A2 was found to be rapidly associated with the particulate fraction in a Ca(2+)-dependent manner at Ca2+ concentrations of 0.1-1.0 microM. This is also the range of the levels of intracellular Ca2+ reported for basal and various stimulated conditions. After translocation, phospholipase A2 could be released from the membranes in the presence of Ca2+ chelators, increasing the specific activity of phospholipase A2 in the supernatant fraction. These findings support the view that translocation is a regulatory mechanism of phospholipase A2 by bringing the enzyme to its substrate. Unlike the situation with protein kinase C, Mg2+ exerted little effect on phospholipase A2 translocation, indicating that this process is regulated in vivo mainly by fluctuations of the intracellular Ca2+ content. 相似文献
20.
S N Orlov N I Pokudin V I Bo?tsov A V Sitozhevski? P V Gulak 《Biokhimii?a (Moscow, Russia)》1985,50(6):883-890
The activity of Ca-pump in inside-out oriented vesicles obtained from erythrocyte membranes after their 30 min treatment with EGTA at 20 degrees C (membranes A) and 37 degrees C (membranes B) was investigated. It was shown that in membranes A placed into an incubation medium containing 0.1 mM EGTA (pH 7.4) the overall effect of exogenous calmodulin is due to the increase in the maximal activity of the enzyme, its affinity for Ca2+ being unaffected thereby. In membranes B placed into the same medium (pH 6.75) the activation of the Ca-pump by calmodulin is due to the increased affinity for Ca2+ at a constant maximal activity of the enzyme. The dependencies of the value of the calmodulin-stimulated component of membranes A and the Ca2+-binding capacity of calmodulin measured by the intensity of N-phenyl-1-naphthylamine fluorescence on the concentration of free Ca2+ are coincident. In the case of membranes B, the stimulation of Ca-pump by calmodulin occurs at much lower Ca2+ concentrations than the Ca2+ binding-induced conformational shifts in calmodulin. The experimental results suggest that the affinity of the Ca-pump for Ca2+ may affect calmodulin existing in a Ca2+-independent state. The hydrophobic interactions between the Ca-calmodulin complex and the Ca-ATPase molecule are apparently essential for the regulation of the maximal enzyme activity. 相似文献