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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

We have previously reported the isolation of a subclone of the MA-10 mouse Leydig tumor cell line (MA-10 LP) which secretes less than 10% of the steroid synthesized by the parent, accumulates comparable amounts of cAMP and has equivalent cholesterol side-chain cleavage activity as the parent population (Kilgore and Stocco (1989) Endocrinology 124, 1210-1216). In the present study we show that addition of exogenous sterol carrier protein 2 (SCP2) to isolated mitochondria was not able to overcome the deficient steroid response of MA-10 LP. We have also demonstrated that human chorionic gonadotropin (hCG)-stimulated cellular events which activate steroid production by subsequently isolated mitochondria require ongoing protein synthesis, release of intracellular calcium and are mediated through the calcium-calmodulin complex. Additionally, mitochondrial sonicates from hCG-stimulated parent cells were able to stimulate steroid production by intact mitochondria isolated from unstimulated parent cells, whereas sonicates from similarly treated MA-10 LP had no effect on steroid production in these mitochondria. Together these data suggest that hCG induces changes in the mitochondria of the parent stock which are not induced to the same extent in the mitochondria of MA-10 LP.
Mol Cell Endocrinol 1990 Feb 12
PMID:Regulation of steroidogenesis in subclones of the MA-10 mouse Leydig tumor cell line. 169 Nov 16

The effects of divalent metals, metal chelators (EDTA, EGTA) and sodium dodecyl sulfate were investigated on the phosphatase activity of isolated bovine brain calcineurin assayed in the absence (called intrinsic) and presence of calmodulin. Intrinsic phosphatase was increased by Mn2+, was unaffected by Mg2+, Ca2+, and Ba2+, and was markedly inhibited by Ni2+, Fe2+, Zn2+ and Cu2+. When assayed in the presence of calmodulin, many divalent metals (Ni2+, Zn2+, Pb2+, Cd2+), besides Mn2+, increased modestly the phosphatase activity at low concentrations (10-100 microM) and inhibited it markedly at high concentrations. Ca2(+)-calmodulin stimulated phosphatase activity was antagonized by Ni2+, Zn2+, Fe2+, Cu2+, Pb2+, at low concentrations (50 microM), and by Ba2+, Cd2+ at slightly higher concentrations (greater than 100 microM); Mn2+ and Co2+ (50 microM to 1 mM) in fact augmented it. EDTA and EGTA in a concentration and time dependent fashion inhibited the intrinsic phosphatase activity, particularly that of trypsinized calcineurin. SDS in low concentrations (0.005%) augmented the phosphatase activity and inhibited it at high concentrations. Mn2+ (+/- calmodulin) and Ca2+ only with calmodulin present increased the phosphatase activity assayed with low concentrations of SDS. The EDTA dependent inhibition of intrinsic phosphatase was almost abolished in assays containing SDS. Prior exposure of calcineurin to Mn2+ led to a high activity conformation state of calcineurin that was 'long-lived' or 'pseudo-irreversible'. Such Mn2(+)-activated state of calcineurin exhibited no discernible change in the affinity towards myelin basic protein or its inhibition by trifluoperazine. At alkaline pH, Mg2+ supported the intrinsic phosphatase activity, although to a lesser degree than Mn2+. The latter cation, compared to Mg2+ and Ni2+, was also a more powerful stimulator of the calcineurin phosphatase assayed with histone (III-S) and myosin light chain as substrates.
Mol Cell Biochem 1990 Sep 03
PMID:Divalent cation effects on calcineurin phosphatase: differential involvement of hydrophobic and metal binding domains in the regulation of the enzyme activity. 170 Oct 13

B-50 (= GAP-43, F1, and P-57 or neuromodulin) is a nervous tissue-specific, growth-associated protein, localized in the presynaptic membrane. Phosphorylation by protein kinase C at Ser41 appears to play a role in B-50/calmodulin interaction and neurotransmitter release. Previous studies have shown that digestion of the phosphorylated protein with S. aureus V8 protease (SAP) resulted consecutively in 28- and 15-kDa phospho fragments, the latter containing all incorporated phosphate. These proteolytic products of digestion with SAP have frequently been used to identify B-50 in various systems. Therefore we were interested to find out the location of these fragments in the rat B-50 molecule. For this purpose, the rat cDNA for B-50 was used to generate full-length and truncated cRNAs for cell-free translation. B-50 and B-50 peptides were either N-terminally labeled with [35S]methionine (residues 1 and 5) as a tracer, or they were phosphorylated in vitro by protein kinase C. SAP digestion of the immunoprecipitated, 35S-labeled translation products produced similar 28- and 15-kDa fragments as were obtained from 32P-labeled B-50, indicating that these fragments are N-terminal. Relative mobilities of the N-terminal B-50 fragments of known length were used as internal standards for the calculation of the length of SAP and phospho fragments. Comparing the 35S- and 32P-labeled products, four SAP sites at Glu12, Glu28, Glu65, and Glu132 could be deduced. The latter two sites are in accordance with sequence data of C-terminal fragments from the literature. All available data could be fitted into one scheme.
J Mol Neurosci 1991
PMID:Phosphoprotein B-50: localization of proteolytic sites for S. aureus V8 protease using truncated cRNAs for cell-free translation. 172 45

NPC 15437 is a prototype member of a new class of synthetically derived protein kinase C (PKC) inhibitors. PKC activity and binding of phorbol ester to the enzyme were inhibited by NPC 15437, with IC50 values of 19 +/- 2 microM and 23 +/- 4 microM, respectively. No inhibition of cAMP-dependent or calcium/calmodulin-dependent protein kinases was observed at concentrations of NPC 15437 up to 300 microM. To investigate the mechanism by which NPC 15437 exerts its effects, a kinetic analysis of the inhibition with respect to three activators of the enzyme, phosphatidylserine, calcium, and phorbol ester, was performed. NPC 15437 was a competitive inhibitor of the activation of PKC by phorbol ester (Ki = 5 +/- 3 microM). Stimulation of PKC alpha by phosphatidylserine was competitively inhibited by NPC 15437 (Ki = 12 +/- 4 microM). The inhibition was mixed with respect to activation by calcium. These results suggest that NPC 15437 is a selective inhibitor of PKC, interacting at the regulatory region of the enzyme. NPC 15437 inhibited PKC in intact cells, dose-dependently antagonizing the phorbol ester-induced phosphorylation of a 47-kDa protein in human platelets.
Mol Pharmacol 1992 Jan
PMID:2,6-Diamino-N-([1-(1-oxotridecyl)-2-piperidinyl] methyl)hexanamide (NPC 15437): a novel inhibitor of protein kinase C interacting at the regulatory domain. 173 21

Effects of endotoxin administration on the ATP-dependent Ca2+ uptake by canine cardiac sarcoplasmic reticulum (SR) were investigated. Results obtained 4 h after endotoxin administration show that ATP-dependent Ca2+ uptake by cardiac SR was decreased by 27-43% (p less than 0.05). Kinetic analysis indicates that the Vmax values for Ca2+ and for ATP were significantly decreased while the S0.5 and the Hill coefficient values were not affected during endotoxin shock. Magnesium (1-5 mM) stimulated while vanadate (25-250 microM) inhibited the ATP-dependent Ca2+ uptake, but the Mg(2+)-stimulated and the vanadate-inhibited activities remained significantly lower in the endotoxin-treated animals. Phosphorylation of SR by the exogenously added catalytic subunit of the cAMP-dependent protein kinase or by the addition of calmodulin stimulated the ATP-dependent Ca2+ uptake activities both in the control and endotoxin-injected dogs. However, the phosphorylation-stimulated activities remained significantly lower in the endotoxin-injected dogs. Dephosphorylation of SR decreased the ATP-dependent Ca2+ uptake, but the half-time required for the maximal dephosphorylation was reduced by 31% (p less than 0.05) 4 h post-endotoxin. These data indicate that endotoxin administration impairs the ATP-dependent Ca2+ uptake in canine cardiac SR and the endotoxin-induced impairment in the SR calcium transport is associated with a mechanism involving a defective phosphorylation and an accelerated dephosphorylation of SR membrane protein. Since ATP-dependent Ca2+ uptake by cardiac SR plays an important role in the regulation of the homeostatic levels of the contractile calcium, our findings may provide a biochemical explanation for myocardial dysfunction that occurs during endotoxin shock.
Mol Cell Biochem 1991 Nov 13
PMID:Impaired calcium uptake by cardiac sarcoplasmic reticulum and its underlying mechanism in endotoxin shock. 177 Sep 48

Partially purified plasma membrane fractions were prepared from guinea-pig pancreatic acini. These membrane preparations were found to contain an ATP-dependent Ca(2+)-transporter as well as a heterogenous ATP-hydrolytic activity. The Ca(2+)-transporter showed high affinity for Ca2+ (KCa2+ = 0.04 +/- 0.01 microM), an apparent requirement for Mg2+ and high substrate specificity. The major component of ATPase activity could be stimulated by either Ca2+ or Mg2+ but showed a low affinity for these cations. At low concentrations, Mg2+ appeared to inhibit the Ca(2+)-dependent ATPase activity expressed by these membranes. However, in the presence of high Mg2+ concentration (0.5-1 mM), a high affinity Ca(2+)-dependent ATPase activity was observed (KCa2+ = 0.08 +/- 0.02 microM). The hydrolytic activity showed little specificity towards ATP. Neither the Ca(2+)-transport nor high affinity Ca(2+)-ATPase activity were stimulated by calmodulin. The results demonstrate, in addition to a low affinity Ca2+ (or Mg2+)-ATPase activity, the presence of both a high affinity Ca(2+)-pump and high affinity Ca(2+)-dependent ATPase. However, the high affinity Ca(2+)-ATPase activity does not appear to be the biochemical expression of the Ca(2+)-pump.
Mol Cell Biochem 1991 Jul 10
PMID:Relationship between Ca(2+)-transport and ATP hydrolytic activities in guinea-pig pancreatic acinar plasma membranes. 183 23

The plasma membrane Ca2+ ATPase in erythrocytes is vital for the maintenance of intracellular Ca2+ levels. Since the cytoplasmic Ca2+ concentration is elevated in older erythrocytes, the properties of the Ca2+ transport ATPase were examined during cell aging using inside-out vesicles (IOVs) prepared from density-separated, young (less dense, Ey) and old (more dense, Eo) rat and human erythrocytes. The transport of Ca2+ and the coupled hydrolysis of ATP were measured using radiolabeled substrates. The calmodulin-independent Ca2+ transport activity (Ey, 38.8 vs. Eo, 23.3 nmols/min/mg IOV protein) and the Ca2+ dependent ATP phosphohydrolase activity (Ey, 53.5 vs. Eo, 48.8 nmols/min/mg protein) were greater in IOVs prepared from younger (less dense) rat erythrocytes. The calmodulin-independent Ca2+ transport activity in IOVs from human erythrocytes was 12.9 nmols/min/mg IOV protein for Ey and 10.7 nmols/min/mg IOV protein for Eo. Inside-out vesicles from older (more dense) cells exhibited a lower pumping efficiency as determined by the calculated stoichiometry, molecule of Ca2+ transported per molecule of ATP hydrolyzed (rat: Ey, 0.74 vs. Eo, 0.49; human: Ey, 1.22 vs. Eo, 0.77). The enzymatic activity of rat and human Ey IOVs was labile. The Ca2+ transport activity in Ey but not Eo IOVs rapidly declined during cold storage (4 degrees C). The decrease in Ca2+ transport activity during aging may accentuate the age-related decline in several erythrocytic properties.
Mol Cell Biochem 1991 Jul 10
PMID:Ca2+ transport activities of inside-out vesicles prepared from density-separated erythrocytes from rat and human. 183 24

High-affinity Ca(2+)-activated ATPases that do not show any demonstrable dependence on Mg2+ have been reported in the plasma membranes of different trypanosomatids, and it has been suggested [McLaughlin (1985) Mol. Biochem. Parasitol. 15, 189-201; Ghosh, Ray, Sarkar & Bhaduri (1990) J. Biol. Chem. 265, 11345-11351] that these enzymes may have a role in Ca2+ transport by the plasma membrane and in the regulation of intracellular Ca2+ in these parasites. In this report we investigated Ca2+ transport by Trypanosoma cruzi plasma membrane vesicles using Arsenazo III as a Ca2+ indicator. These vesicles accumulated Ca2+ upon addition of ATP only when Mg2+ was present and released it in response to the Ca2+ ionophore A23187, but were insensitive to inositol 1,4,5-trisphosphate. Ca2+ transport was insensitive to antimycin A, oligomycin and carbonyl cyanide p-trifluorophenylhydrazone, ruling out any mitochondrial contamination. Staurosporine and phorbol myristate acetate had no effect on this activity, while low concentrations of vanadate (10 microM) completely inhibited it. In addition, we describe a high-affinity vanadate-sensitive (Ca(2+)-Mg2+)-ATPase in the highly enriched plasma membrane fraction of T. cruzi. Kinetic studies indicated that the apparent Km for free Ca2+ was 0.3 microM. On the other hand, Ca(2+)-ATPase activity and Ca2+ transport were both stimulated by bovine brain calmodulin and by endogenous calmodulin purified from these cells. In addition, trifluoperazine and calmidazolium, at concentrations in the range in which they normally exert anti-calmodulin effects, inhibited the calmodulin-stimulated Ca(2+)-ATPase activity. These observations support the notion that a Mg(2+)-dependent plasma membrane Ca2+ pump is present in these parasites.
 ...
PMID:A calmodulin-activated (Ca(2+)-Mg2+)-ATPase is involved in Ca2+ transport by plasma membrane vesicles from Trypanosoma cruzi. 183 15

Phosphorylation of ribosomal protein S6 of mammals precedes activation of cell growth in numerous biological systems. We have cloned a cDNA for ribosomal protein S6 from T-47D human breast cancer cells by immunoscreening a lambda gt11 expression library with antibody raised against the mitochondrial Ca(2+)-binding ATPase inhibitor protein (CaBI) of bovine heart mitochondria (Yamada & Huzel: J Biol Chem 263: 11498-11503, 1988). Similar clones were obtained by the immunoscreening of a rat heart expression library. In agreement with others, the open reading frames of the cDNAs from the two species coded for the same amino acid sequence. No difference in S6 of the human neoplastic cells compared to that of non-neoplastic cells was found. However, common antigenic determinants in S6 and CaBI were indicated. Accordingly, S6 was purified from rat liver ribosomes and antiserum prepared. Immuno-dot blot and Western blot analyses showed high specific reactivity between S6, the cloned chimeric beta-galactosidase fusion protein from a cDNA clone, and CaBI with anti-S6 and anti-CaBI antibodies. The antibodies also showed a high degree of discrimination for S6 and CaBI. Neither interacted with the other ribosomal proteins nor with another ATPase inhibitor protein from bovine heart mitochondria. Neither interacted with the Ca(2+)-binding proteins, calmodulin, oncomodulin, Protein C, or Factor X. Prothrombin was weakly reactive with anti-CaBI but not with anti-S6. Thus, the results fulfill the specific criteria for the concept and operational definition of common protein epitopes in S6 and CaBI. However, neither prothrombin nor S6 fusion protein inhibited mitochondrial ATPase activity even at 20 times the concentrations at which CaBI gave 97% inhibition.
Mol Cell Biochem 1991 Nov 13
PMID:Antigenic reactivity of ribosomal protein S6 and the calcium-binding ATPase inhibitor protein of mammalian mitochondria. 183 89

The neuronal phosphoprotein B-50/GAP-43 has been implicated in neuritogenesis during developmental stages of the nervous system and in regenerative processes and neuronal plasticity in the adult. The protein appears to be a member of a family of acidic substrates of protein kinase C (PKC) that bind calmodulin at low calcium concentrations. Two of these substrates, B-50 and neurogranin, share the primary sequence coding for the phospho- and calmodulin-binding sites and might exert similar functions in axonal and dendritic processes, respectively. In the adult brain, B-50 is exclusively located at the presynaptic membrane. During neuritogenesis in cell culture, the protein is translocated to the growth cones, i.e., into the filopodia. In view of many positive correlations between B-50 expression and neurite outgrowth and the specific localization of B-50, a role in growth cone function has been proposed. Its phosphorylation state may regulate the local intracellular free calmodulin and calcium concentrations or vice versa. Both views link the B-50 protein to processes of signal transduction and transmitter release.
Mol Neurobiol 1991
PMID:Role of the growth-associated protein B-50/GAP-43 in neuronal plasticity. 184 Apr 22


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