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C L Carpenter

Publications and source records attributed to C L Carpenter.

At least 19 recordsLinked to original sources
PubMed1992

Purification and characterization of human erythrocyte phosphatidylinositol 4-kinase. Phosphatidylinositol 4-kinase and phosphatidylinositol 3-monophosphate 4-kinase are distinct enzymes.

PtdIns 4-kinase has been purified 83,000-fold from human erythrocyte membranes. The major protein detected by SDS/PAGE is of molecular mass 56 kDa, and enzymic activity can be renatured from this band of the gel. The characteristics of this enzyme are similar to other type II PtdIns kinases previously described: PtdIns presented in Triton X-100 micelles is preferred as a substrate over PtdIns vesicles, the enzyme possesses a relatively low Km for ATP (20 microM), and adenosine is an effective inhibitor. A monoclonal antibody raised against bovine brain type II PtdIns 4-kinase is an effective inhibitor of the purified enzyme. PtdIns(4,5)P2 inhibits by approx. 50% when added in equimolar amounts with PtdIns; PtdIns4P has little effect on activity. A PtdIns3P 4-kinase activity has also been detected in erythrocyte lysates. Approximately two-thirds of this activity is in the cytosolic fraction and one-third in the membrane fraction. No PtdIns3P 4-kinase activity could be detected in the purified type II PtdIns 4-kinase preparation, nor could this activity be detected in a bovine brain type III PtdIns 4-kinase preparation. The monoclonal antibody that inhibits the type II PtdIns 4-kinase does not affect the PtdIns3P 4-kinase activity in the membrane fraction. The cytosolic PtdIns3P 4-kinase can be efficiently recovered from a 60%-satd.-(NH4)2SO4 precipitate that is virtually free of PtdIns 4-kinase activity. We conclude that PtdIns3P 4-kinase is a new enzyme distinct from previously characterized PtdIns 4-kinases, and that this enzyme prefers PtdIns3P over PtdIns as a substrate.

1-Phosphatidylinositol 4-Kinase↗
PubMed1992

Polyoma virus middle T antigen-pp60c-src complex associates with purified phosphatidylinositol 3-kinase in vitro.

Reconstitution of the polyoma virus middle T antigen (mT)-pp60-src complex and phosphatidylinositol 3-kinase (PtdIns 3-kinase) has been accomplished in vitro with immunopurified baculovirus-expressed mT-pp60c-src and PtdIns 3-kinase purified from rat liver. Both the 110- and 85-kDa subunits of the PtdIns 3-kinase associated with the mT-pp60c-src complex. The association of PtdIns 3-kinase with the mT-pp60c-src complex was dependent on the protein-tyrosine kinase activity of pp60c-src as a kinase-inactive mutant (pp60(295c-src)) still complexed with mT, but the mT-pp60(295c-src)) complex was unable to bind PtdIns 3-kinase. The mT-pp60c-src complex phosphorylated both subunits of PtdIns 3-kinase on tyrosine residues. The immunopurified mT-pp60c-src complex also associated with PtdIns 3-kinase activity from whole cell lysates, and this association was dependent upon the protein-tyrosine kinase activity of pp60c-src. Comparison of 35S-labeled proteins from whole cell lysates which associated with immunopurified mT-pp60c-src and mT-pp60(295c-src) revealed proteins of 110 and 85 kDa as the major peptides dependent on protein-tyrosine kinase activity for association with the complex. In addition, a synthetic phosphopeptide (13-mer) containing sequences conserved between the major tyrosine phosphorylation site of murine polyoma virus mT, hamster polyoma virus mT, and the insulin receptor substrate (IRS-1) specifically blocked the association of the 85- and 110-kDa polypeptides with the mT-pp60c-src complex. The ability to block the association was dependent on the tyrosine phosphorylation of the peptide. Association of PtdIns 3-kinase activity was blocked concurrently. This is the first demonstration that the 110-kDa subunit of PtdIns 3-kinase can associate with mT-pp60c-src. This association in vitro is a step toward understanding protein-protein interactions important in the signal transduction pathway of oncogenic proteins.

3T3 Cells↗
PubMed1992

Interactions of polyomavirus middle T with the SH2 domains of the pp85 subunit of phosphatidylinositol-3-kinase.

The binding of phosphatidylinositol-3-kinase to the polyomavirus middle T antigen is facilitated by tyrosine phosphorylation of middle T on residue 315. The pp85 subunit of phosphatidylinositol-3-kinase contains two SH2 domains, one in the middle of the molecule and one at the C terminus. When assayed by blotting with phosphorylated middle T, the more N-terminal SH2 domain is responsible for binding to middle T. When assayed in solution with glutathione S transferase fusions, both SH2s are capable of binding phosphorylated middle T. While both SH2 fusions can compete with intact pp85 for binding to middle T, the C-terminal SH2 is the more efficient of the two. Interaction between pp85 or its SH2 domains and middle T can be blocked by a synthetic peptide comprising the tyrosine phosphorylation sequence around middle T residue 315. Despite the fact that middle T can interact with both SH2s, these domains are not equivalent. Only the C-terminal SH2-middle T interaction was blocked by anti-SH2 antibody; the two SH2 fusions also interact with different cellular proteins.

1-Phosphatidylinositol 4-Kinase↗
PubMed1990

Purification and characterization of phosphoinositide 3-kinase from rat liver.

Phosphoinositide 3-kinase was purified 27,000-fold from rat liver. The enzyme was purified by acid precipitation of the cytosol followed by chromatography on DEAE-Sepharose, S-Sepharose, hydroxylapatite, Mono-Q, and Mono-S columns. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified phosphoinositide 3-kinase preparation contained an 85-kDa protein and a protein doublet of approximately 110 kDa. The 85- and 110-kDa proteins focus together on native isoelectric focusing gels and are cross-linked by dithiobis(succinylamide propionate), showing that the 110- and 85-kDa proteins are a complex. The apparent size of the native enzyme, as determined by gel filtration, is 190 kDa. The 85-kDa subunit is the same protein previously shown to associate with polyoma virus middle T antigen and the platelet-derived growth factor receptor (Kaplan, D. R., Whitman, M., Schaffhausen, B., Pallas, D. C., White, M., Cantley, L., and Roberts, T. M. (1987) Cell 50, 1021-1029). The two proteins co-migrate on two-dimensional gels; and, using a Western blotting procedure, 32P-labeled middle T antigen specifically blots the 85-kDa protein. The purified enzyme phosphorylates phosphatidylinositol, phosphatidylinositol 4-phosphate, and phosphatidylinositol 4,5-bisphosphate. The apparent Km values for ATP were found to be 60 microM with phosphatidylinositol 4-phosphate or phosphatidylinositol 4,5-bisphosphate as the substrate. The apparent Km for phosphatidyinositol is 60 microM, for phosphatidylinositol 4-phosphate is 9 microM, and for phosphatidylinositol 4,5-bisphosphate is 4 microM. The maximum specific activity using phosphatidylinositol as the substrate is 0.8 mumol/mg/min. The enzyme requires Mg2+ with an optimum of 5 mM. Substitution of Mn2+ for Mg2+ results in only approximately 10% of the Mg2(+)-dependent activity. Physiological calcium concentrations have no effect on the enzyme activity. Phosphoinositide 3-kinase has a broad pH optimum around 7.

Animals↗
PubMed1990

Aerobic biodegradation of vinyl chloride in groundwater samples.

Studies were conducted to examine the biodegradation of 14C-labeled vinyl chloride in samples taken from a shallow aquifer. Under aerobic conditions, vinyl chloride was readily degraded, with greater than 99% of the labeled material being degraded after 108 days and approximately 65% being mineralized to 14CO2.

Aerobiosis↗
PubMed1989

Phosphatidylinositol 3-kinase and its novel product, phosphatidylinositol 3-phosphate, are present in Saccharomyces cerevisiae.

The metabolism of polyphosphoinositides has been shown to be an important factor in controlling the proliferation of Saccharomyces cerevisiae. The monophosphate form of phosphatidylinositol has been assumed to be phosphatidylinositol 4-phosphate (PI-4-P). Recent evidence from our laboratory has established that a phosphatidylinositol (PI) kinase, which phosphorylates the D-3 position of the inositol ring (PI 3-kinase), is associated with many activated protein-tyrosine kinases and may play an important role in the signaling of cell proliferation (Auger, K. R., Serunian, L. A., Soltoff, S. P., Libby, P., and Cantley, L. C. (1989) Cell 57, 167-175). To determine the evolutionary conservation of this enzymatic activity, we investigated its presence in yeast. In vitro PI kinase assays of yeast cell homogenates demonstrated that PI 3-kinase activity was present. Preliminary biochemical characterization of the activity suggested that it was quite different from the mammalian enzyme yet catalyzed the same reaction, i.e. phosphorylating the D-3 hydroxyl position of the inositol ring of phosphatidyl-myo-inositol. [3H]Inositol labeling of intact yeast cells with the subsequent extraction, deacylation, and high performance liquid chromatography analysis of the lipids demonstrated that PI-3-P was as abundant as the PI-4-P isomer. The conservation of the enzymatic activity from yeast to man suggests that it has an important functional role in the cell cycle.

Inositol↗
PubMed1989

Calcium channel antagonist receptors in cerebral cortex from alcoholic patients.

Exposure to ethanol for days to weeks enhances the expression of voltage-dependent Ca2+ channels in the brains of experimental animals and in cultured neural cell lines. To determine if similar changes occur in the brains of alcoholic patients, we measured the binding of (+)-[3H]PN 200-110 to cerebral cortex samples obtained at autopsy from alcoholic and non-alcoholic patients. No difference in (+)-[3H]PN 200-110 binding was observed, suggesting that ethanol-induced changes in Ca2+ channel expression are more likely to be related to acute withdrawal than to chronic ethanol exposure alone.

Aged↗
PubMed1989

Comparative effects of chronic exposure to ethanol and calcium channel antagonists on calcium channel antagonist receptors in cultured neural (PC12) cells.

Treatment with 200 mM ethanol for 6 days increased binding of the Ca2+ channel antagonist, (+)-[3H]PN 200-110, to intact PC12 cells in culture. Enhancement of binding by ethanol was due to an increase in binding site number without appreciable change in binding affinity. Long-term exposure to Ca2+ channel antagonist drugs (nifedipine, verapamil, or diltiazem), which, like ethanol, acutely inhibit Ca2+ flux, failed to alter (+)-[3H]PN 200-110 binding to PC12 membranes. Cotreatment of ethanol-containing cultures with the Ca2+ channel agonist, Bay K 8644, did not attenuate the response to ethanol; instead, chronic exposure to Bay K 8644 alone increased (+)-[3H]PN 200-110 binding. These results suggest that chronic exposure to ethanol increases Ca2+ channel antagonist receptor density in living neural cells, but that acute inhibition of Ca2+ flux by ethanol is unlikely to trigger this response.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1988

Dextromethorphan and dextrorphan as calcium channel antagonists.

Dextromethorphan and dextrorphan, which reduce excitatory amino acid-induced neurotoxicity, decreased K+ depolarization-evoked 45Ca2+ uptake into brain synaptosomes and cultured neural (PC12) cells. Half-maximal inhibition of synaptosomal 45Ca2+ uptake occurred with 48 microM dextromethorphan or 200 microM dextrorphan, which are similar to concentrations associated with protection from excitotoxicity. The ability to decrease Ca2+ flux through N-type (synaptosomal) and L-type (PC12) voltage-gated Ca2+ channels may therefore contribute to the neuroprotective effects of these compounds.

Animals↗
PubMed1987

Ethanol-induced component of 45Ca2+ uptake in PC12 cells is sensitive to Ca2+ channel modulating drugs.

We compared the properties of depolarization-dependent 45Ca2+ uptake in PC12 cells grown with and without 200 mM ethanol for 6 days. Ethanol exposure increased 45Ca2+ uptake by 54%, but the ethanol-induced component of uptake retained properties of Ca2+ flux through voltage-dependent Ca2+ channels, including sensitivity to Ca2+ channel modulating drugs. Such drugs may therefore have a role in counteracting ionic events underlying ethanol dependence and withdrawal.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1987

Interaction of calmodulin inhibitors and protein kinase C inhibitors with voltage-dependent calcium channels.

We compared the relative abilities of a series of calmodulin inhibitors and protein kinase C inhibitors to influence 45Ca2+ influx through voltage-dependent Ca2+ channels in PC12, a clonal neural cell line. K+-depolarization-dependent 45Ca2+ uptake was reduced by the calmodulin inhibitors calmidazolium, trifluoperazine, W-7, W-13, and W-5 at concentrations comparable to those that affect calmodulin, while the protein kinase C inhibitors polymyxin B and H-7 were weak or ineffective. The Ca2+ channel antagonist properties of calmodulin inhibitors should be considered in interpreting their effects on Ca2+-dependent cellular events.

Animals↗
PubMed1987

Lectin-induced enhancement of voltage-dependent calcium flux and calcium channel antagonist binding.

Concanavalin A (Con A), a tetravalent lectin with preferential affinity for mannosyl and glucosyl residues of membrane glycoconjugates, increased K+ depolarization-evoked uptake of 45Ca2+ in the PC12 neural cell line. Enhancement of uptake by Con A was concentration dependent, with maximal (24%) stimulation at 100 micrograms/ml of Con A, and was preferentially inhibited by mannoside and glucoside. Succinyl-Con A, a divalent analog with reduced biological potency, increased uptake by only 7%. The effect of Con A on 45Ca2+ uptake was dependent on membrane depolarization, was abolished by ionic Ca2+ channel blockers and organic Ca2+ channel antagonists, and was accompanied by an equivalent increase in Ca2+ channel 3H-labeled antagonist binding, observations suggesting that the voltage-dependent Ca2+ channel was the site of Ca2+ entry. The mechanism for enhancement of 45Ca2+ uptake by Con A appeared to be separate from that used by the Ca2+ channel agonist BAY K 8644 and independent of that involved in Ca2+ channel regulation by phorbol esters. These findings suggest that voltage-dependent Ca2+ channels may link cell surface carbohydrate interactions with intracellular effector processes.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1986

Ethanol regulates calcium channels in clonal neural cells.

The acute and long-term effects of ethanol on voltage-dependent Ca channel function were studied in PC12, a clonal cell line of neural crest origin. Acute exposure to ethanol produced a concentration-dependent decrease in depolarization-evoked 45Ca2+ uptake, while prolonged (2-10 days) exposure led to a reciprocal increase in 45Ca2+ uptake and in the number of Ca-channel binding sites labeled by the dihydropyridine Ca-channel antagonist [3H]nitrendipine. Uptake was restored to control levels following withdrawal of ethanol from cultures. These findings indicate that cellular adaptation to ethanol may involve enhanced expression of dihydropyridine-sensitive, voltage-dependent Ca channels.

Animals↗
PubMed1986

Depolarization-dependent binding of the calcium channel antagonist, (+)-[3H]PN200-110, to intact cultured PC12 cells.

Voltage-dependent Ca++ channels of excitable cell membranes are coupled to drug recognition sites that influence Ca++ channel gating behavior. In cardiac tissue, these sites are themselves regulated by membrane potential, which may explain the apparent dependence of drug effects on Ca++ channel conductance state. Whether a similar relationship pertains in other (e.g., neural) cells is unknown. To examine this issue, we investigated the effect of K+-depolarization on binding of the dihydropyridine Ca++ channel antagonist, (+)-[3H]PN200-110, to intact PC12 cells in culture. Specific (nifedipine-sensitive) binding of 50 pM (+)-[3H]PN200-110 to intact PC12 cells was increased approximately 3-fold by K+-depolarization. Binding was also increased when membrane potential was abolished by treatment with digitonin; elevated [K+] had no additional effect under these conditions. Enhancement of binding by K+-depolarization was reversible upon repolarization and resulted from an increase in binding affinity (decrease in KD from 274 to 55 pM in equilibrium saturation experiments and from 625 to 44 pM in kinetic studies), without an increase in binding site number. These findings are in accord with a modulated receptor model of Ca++ channel function in which affinity for dihydropyridine Ca++ channel antagonists is enhanced by depolarization, and provide evidence that this form of Ca++ channel regulation occurs in neural, as well as muscle, cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1985

Inactivation of 45Ca2+ uptake by prior depolarization of PC12 cells.

45Ca2+ uptake evoked by depolarization of PC12 pheochromocytoma cells with K+ was reduced approximately 90% by prior depolarization in Ca2+-containing medium. Prior depolarization without added Ca2+ reduced 45Ca2+ uptake by only about 20%. The Ca2+ channel agonists, BAY K 8644 and CGP 28392, had no effect on inactivation of 45Ca2+ uptake. These findings suggest that (1) voltage-gated Ca2+ channels of PC12 cells undergo inactivation, (2) inactivation is Ca2+-dependent rather than voltage-dependent, and (3) Ca2+ channel agonists do do not promote Ca2+ flux by inhibiting Ca2+ channel inactivation.

Animals↗
PubMed1985

Stimulation of calcium uptake in PC12 cells by the dihydropyridine agonist BAY K 8644.

Methyl 1,4-dihydro-2, 6-dimethyl-3-nitro-4-(2-trifluoromethylphenyl)-pyridine-5-carboxylate (BAY K 8644), an analog of dihydropyridine calcium channel antagonists, stimulated 45Ca uptake into PC12 pheochromocytoma cells. Half-maximal stimulation occurred at 80 nM BAY K 8644. Enhancement of uptake was inhibited by cationic and organic calcium channel blockers, but not by tetrodotoxin, which is consistent with an effect on voltage-dependent calcium channels. Stimulation of 45Ca uptake by BAY K 8644 occurred only at elevated concentrations of extracellular K+, suggesting that BAY K 8644 may interact with calcium channels in the open (activated) state.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗