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Z Damuni

Publications and source records attributed to Z Damuni.

33 records · Page 2Linked to original sources
PubMed1992

Purification and properties of a protamine kinase from bovine kidney microsomes.

About an eightfold increase in protamine kinase activity was detected following extraction of highly purified microsomes from bovine kidney with 1% Triton X-100. Relative to the soluble fraction, the microsomes contained about 30% protamine kinase activity. The microsomal protamine kinase was purified to apparent homogeneity. The purified enzyme exhibited an apparent M(r) approximately 45,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel permeation chromatography on Sephacryl S-200. Relative to protamine, the purified kinase exhibited about 100% activity with the synthetic peptide RRLSSLRA and about 5, 8, and less than 0.1% activity with casein, histone H2B, and histone H1, respectively. The purified kinase phosphorylated several 40 S ribosome polypeptides. One of these polypeptides was identified as ribosomal protein S6 by N-terminal sequencing. About 2.5 mol of phosphoryl groups was incorporated per mole of ribosomal protein S6 following incubation of the 40 S ribosomes with the purified kinase. Following incubation with protein phosphatase 2A2, purified preparations of the protamine kinase were inactivated. These properties were identical to those of purified preparations of a protamine kinase from extracts of bovine kidney cytosol (Z. Damuni, G.D. Amick, and T.R. Sneed, 1989, J. Biol. Chem. 264, 6412-6418). Near identical peptide patterns were obtained following incubation of purified preparations of the microsomal and cytosolic protamine kinases with Staphylococcus aureus V8 proteinase. The results indicate that a form of the cytosolic protamine kinase is present in microsomes.

Animals↗
PubMed1992

Protamine kinase phosphorylates eukaryotic protein synthesis initiation factor 4E.

Up to 1 mol of phosphoryl groups was incorporated per mol of eukaryotic protein synthesis initiation factor (eIF) 4E following incubation of purified preparations of this factor with purified preparations of a protamine kinase from bovine kidney cytosol. By contrast, purified preparations of two forms of mitogen-activated protein kinase, casein kinase II and two forms of a distinct autophosphorylation-activated protein kinase exhibited little activity, if any, with eIF-4E. Together with previous observations, the results indicate that the protamine kinase could contribute to the insulin-stimulated phosphorylation of eIF-4E.

Animals↗
PubMed1990

Insulin stimulates the activity of a protamine kinase in isolated rat hepatocytes.

Treatment of isolated rat hepatocytes with 10-100 nM insulin for 5-10 min increased by about 2-fold the activity of a protamine kinase which exhibited properties similar to those of a protamine kinase from bovine kidney (Damuni, Z., Amick, G. D., and Sneed, T. R. (1989) J. Biol. Chem. 264, 6412-6416). Half-maximal increase in protamine kinase activity occurred at about 1 nM insulin. This effect of insulin was detected only when 25 mM NaF or 50 mM KPO4 were included in the homogenization buffers and was not prevented by preincubation of the hepatocytes with 10 microM cycloheximide. Insulin stimulation of protamine kinase was maintained following chromatography of extracts on protamine-agarose, DEAE-cellulose, and Sephacryl S-200 gel filtration. The apparent Mr of the protamine kinase from control and insulin-treated hepatocytes was 45,000 as estimated by gel permeation chromatography. Experiments utilizing partially purified protamine kinase from control and insulin-treated hepatocytes indicated that insulin did not affect the apparent Km for protamine, Mg2+, or ATP, but increased the Vmax for the protamine kinase reaction by 1.6-2-fold. Incubation with the catalytic subunit of protein phosphatase 2A completely inactivated the protamine kinase from control and insulin-treated cells. The results indicate that the insulin-stimulated increase in protamine kinase activity may be due to a covalent modification, possibly phosphorylation, of the protamine kinase.

Animals↗
PubMed1990

Inactivation of bovine kidney cytosolic protamine kinase by the catalytic subunit of protein phosphatase 2A.

Incubation of highly purified preparations of the bovine kidney cytosolic protamine kinase in the presence of near homogeneous preparations of the catalytic subunit of protein phosphatase 2A (PrP2Ac) from bovine kidney resulted in time-dependent inactivation of the protamine kinase. By contrast, incubation of bovine kidney cytosolic casein kinase II with PrP2Ac had no effect on the activity of this casein kinase II. In the presence of 10 mM sodium fluoride, 10 mM inorganic orthophosphate, 1 mM pyrophosphate or 0.1 mM ATP, the inactivation of the protamine kinase by PrP2Ac was completely inhibited. Half-maximal inhibition by ATP occurred at about 20 microM. The rate of inactivation of the protamine kinase by PrP2Ac was unaffected by Mg2+, Mn2+, Ca2+, EDTA or EGTA at 1 mM. The results strongly indicate that the activity of the cytosolic protamine kinase is regulated by phosphorylation/dephosphorylation.

Adenosine Triphosphate↗
PubMed1989

Purification and properties of a distinct protamine kinase from the cytosol of bovine kidney cortex.

A protamine kinase has been purified to apparent homogeneity from extracts of the cytosol of bovine kidney cortex. This protamine kinase exhibited an apparent Mr = 43,000 as estimated by gel permeation chromatography on Sephacryl S-200 and an apparent Mr = 45,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The purified protamine kinase exhibited about 5% activity with casein, 8% with histone H2B, and less than 0.1% with histone H1, histone H4, glycogen synthase a from rabbit skeletal muscle, ovalbumin, bovine serum albumin, and phosvitin. The activity of the highly purified protamine kinase was unaffected by cyclic AMP (up to 0.1 mM), cyclic GMP (up to 0.1 mM), the heat-stable protein inhibitor of cyclic AMP-dependent protein kinase (up to 100 micrograms/ml), heparin (up to 100 micrograms/ml), EGTA (up to 1 mM), Ca2+ (up to 1 mM), calmodulin (up to 0.5 microM) in the absence or presence of Ca2+ (0.05 mM), and phosphatidylserine (up to 40 micrograms/ml) and/or diolein (up to 1 microgram/ml) in the absence or presence of Ca2+ (up to 0.5 mM). Experiments in which extracts of kidney cytosol were incubated with [gamma-32P]ATP and MgCl2 revealed that the phosphorylation of numerous polypeptides was markedly increased in the presence of the purified protamine kinase. The results indicate that this protamine kinase of kidney cytosol is a novel protein kinase.

Animals↗
PubMed1988

Purification and properties of a protamine kinase and a type II casein kinase from bovine kidney mitochondria.

Bovine kidney mitochondrial extracts contain an inactive protamine kinase and an inactive casein kinase. The protamine kinase was activated by chromatography on poly(L-lysine)-agarose. Two forms of this soluble mitochondrial protamine kinase were separated by chromatography on protamine-agarose. Both forms were purified about 80,000-fold to apparent homogeneity. Both forms of the protamine kinase consist of a single polypeptide chain with an apparent Mr approximately 45,000. Both enzyme forms underwent autophosphorylation without significant effect on activity, and both forms exhibited identical substrate specificities. The protamine kinase showed little activity toward branched-chain alpha-keto acid dehydrogenase (less than 3%), and it was essentially inactive (less than 0.1%) with pyruvate dehydrogenase, casein, and ovalbumin. The enzyme was active with histone H1 and with bovine serum albumin. Protamine kinase activity was unaffected by heparin (up to 100 micrograms/ml), by the protein inhibitor of cyclic AMP-dependent protein kinase, by Ca2+ and calmodulin, and by monoclonal antibody to the catalytic domain of protein kinase C from rat brain. The casein kinase was activated in the presence of spermine or by chromatography of the extract on DEAE-cellulose or poly(L-lysine)-agarose. The enzyme was purified about 80,000-fold to apparent homogeneity. It exhibited an apparent Mr 130,000 as determined by gel-permeation chromatography on Sephacryl S-300 in the presence of 0.5 M NaCl. Two subunits, with apparent Mr's 36,000 (alpha) and 28,000 (beta) were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The kinase underwent autophosphorylation of its beta-subunit, without significant effect on activity. Casein kinase activity was inhibited 50% by 1.5 micrograms/ml of heparin. Spermine (1.0 mM) stimulated activity of the purified kinase two- to three-fold at 1.5 mM Mg2+. Half-maximal stimulation occurred at 0.1 mM spermine. The kinase utilized both ATP and GTP as substrates. The casein kinase showed little activity (less than 1%) toward pyruvate dehydrogenase and branched-chain alpha-keto acid dehydrogenase from kidney mitochondria, and the kinase was essentially inactive with glycogen synthase a. The properties of this soluble mitochondrial kinase indicate that it is a type II casein kinase.

Animals↗
PubMed1987

Purification and properties of the catalytic subunit of the branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney mitochondria.

The catalytic subunit of the branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase (Damuni, Z., Merryfield, M.L., Humphreys, J.S., and Reed, L.J., (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 4335-4338) has been purified over 50,000-fold from extracts of bovine kidney mitochondria. The apparently homogeneous protein consists of a single polypeptide chain with an apparent Mr = approximately 33,000 as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. BCKDH phosphatase, with an apparent Mr = 460,000, was dissociated to its catalytic subunit with no apparent change in activity, at an early stage in the purification procedure by treatment with 6 M urea. The specific activity of the catalytic subunit was 1,500-2,500 units/mg. The catalytic subunit exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex but was inactive with phosphorylase a and with p-nitrophenyl phosphate. The catalytic subunit, like the Mr = 460,000 species, was inhibited by nanomolar concentrations of BCKDH phosphatase inhibitor protein, was unaffected by protein phosphatase inhibitor 1 and inhibitor 2, and was inhibited by nucleoside tri- and diphosphates but not by nucleoside monophosphates.

Animals↗
PubMed1987

Purification and characterization of a divalent cation-independent, spermine-stimulated protein phosphatase from bovine kidney mitochondria.

A divalent cation-independent and spermine-stimulated phosphatase (protein phosphatase SP) that is active toward the phosphorylated pyruvate dehydrogenase complex has been purified about 15,000-fold to near homogeneity from extracts of bovine kidney mitochondria. Half-maximal stimulation, 1.5- to 3-fold at pH 7.0-7.3, occurred at 0.5 mM spermine. Protein phosphatase SP exhibited an apparent Mr = 140,000-170,000 as estimated by gel-filtration chromatography on Sephacryl S-300. Two major subunits, with apparent Mr = 60,000 and 34,000, were detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Gel-permeation chromatography of protein phosphatase SP on Sephacryl S-200 in the presence of 6 M urea and 1.4 M NaCl increased its activity 3- to 6-fold and was accompanied by conversion to the catalytic subunit with an apparent Mr = approximately 34,000. Protein phosphatase SP was inactive with p-nitrophenyl phosphate and was not inhibited by protein phosphatase inhibitor 1, inhibitor 2, or the protein inhibitor of branched-chain alpha-keto acid dehydrogenase phosphatase. Protein phosphatase SP was inhibited by sheep antibody to the catalytic subunit of protein phosphatase 2A from rabbit skeletal muscle. It appears that protein phosphatase SP is related to protein phosphatase 2A.

Animals↗
PubMed1986

A potent, heat-stable protein inhibitor of [branched-chain alpha-keto acid dehydrogenase]-phosphatase from bovine kidney mitochondria.

A heat- and acid-stable protein inhibitor of the [branched-chain alpha-keto acid dehydrogenase]-phosphatase was purified over 100,000-fold from extracts of bovine kidney mitochondria. The nearly homogeneous protein was recovered with a yield of 4-8%. The apparent molecular weight of the inhibitor is about 36,000. This protein is a noncompetitive inhibitor of the phosphatase, and the inhibitor constant (Ki) is about 0.13 nM. The inhibition was reversed 50% by about 1.3 mM Mg2+ and about 0.1 mM spermine. This protein inhibitor is different from the cytosolic protein phosphatase inhibitors 1 and 2.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗
PubMed1985

Specificity of the heat-stable protein inhibitor of the branched-chain alpha-keto acid dehydrogenase phosphatase.

A potent, heat-stable protein inhibitor of branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase has been identified and purified to near homogeneity from bovine kidney mitochondria (Damuni, Z., Humphreys, J. S., and Reed, L. J., Proc. Natl. Acad. Sci. U.S.A., in press). This protein is a noncompetitive inhibitor of BCKDH phosphatase, with a Ki about 0.13 nM. By contrast, this protein inhibitor did not affect the activity of the cytosolic protein phosphatase-1 and phosphatase-2A or the mitochondrial pyruvate dehydrogenase (PDH) phosphatase at concentrations up to 10 nM. The cytosolic protein phosphatase inhibitor-1 and inhibitor-2 had no effect on the activity of BCKDH phosphatase or PDH phosphatase at concentrations up to 50 and 300 nM respectively. These results, together with previous evidence, demonstrate that BCKDH phosphatase and its inhibitor protein are distinct from the cytosolic protein phosphatase-1 and phosphatase-2A and from protein phosphatase inhibitor-1 and inhibitor-2, respectively.

Animals↗
PubMed1984

Stimulation of pyruvate dehydrogenase phosphatase activity by polyamines.

Pyruvate dehydrogenase phosphatase requires Mg2+ or Mn2+, and its activity in the presence of Mg2+ is markedly stimulated by Ca2+. At saturating Mg2+ and Ca2+ concentrations, the polyamines spermine, spermidine and putrescine stimulated the activity of pyruvate dehydrogenase phosphatase 1.5- to 3-fold. Spermine was the most active of the polyamines. At a physiological concentration of Mg2+ (1 mM) and saturating Ca2+ concentration, the stimulation by 0.5 mM spermine was 4- to 5-fold, and at 0.3 mM Mg2+, the stimulation was 20- to 30-fold. In the absence of Mg2+ or Ca2+, spermine had no effect. These results suggest that a polybasic factor may be involved in the regulation of pyruvate dehydrogenase phosphatase activity.

Animals↗
PubMed1984

Purification and properties of branched-chain alpha-keto acid dehydrogenase phosphatase from bovine kidney.

Branched-chain alpha-keto acid dehydrogenase (BCKDH) phosphatase was purified about 8000-fold from extracts of bovine kidney mitochondria. The highly purified phosphatase exhibited a molecular weight of approximately 460,000, as estimated by gel-permeation chromatography. Another form of the phosphatase, with an apparent molecular weight of approximately 230,000, was also detected under conditions of high dilution. In contrast to pyruvate dehydrogenase phosphatase, BCKDH phosphatase was active in the absence of divalent cations. BCKDH phosphatase was inactive toward 32P-labeled phosphorylase a, but exhibited approximately 10% maximal activity with 32P-labeled pyruvate dehydrogenase complex. BCKDH phosphatase activity was inhibited by GTP, GDP, ATP, ADP, UTP, UDP, CTP, and CDP. Half-maximal inhibition occurred at about 60, 200, 200, 400, 100, 250, 250, and 400 microM, respectively. These inhibitions were reversed completely by 2 mM Mg2+. GTP was replaceable by guanosine 5'-(beta, gamma-imido)triphosphate. GMP, AMP, UMP, CMP, NAD, and NADH showed little effect, if any, on BCKDH phosphatase activity at concentrations up to 1 mM. Heparin showed half-maximal inhibition at 2 micrograms/ml. This inhibition was only partially (30%) reversed by 2 mM Mg2+. CoA and various acyl-CoA compounds exhibited half-maximal inhibition at 150-300 microM. These inhibitions were not reversed by 2 mM Mg2+. BCKDH phosphatase activity was stimulated 1.5- to 3-fold by protamine, poly(L-lysine), and poly(L-arginine) at 3.6 micrograms/ml.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗