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At least 775 records · Page 43Linked to original sources
PubMed1995

The heat-stable protein kinase C (PKC) stimulatory factor in the rat ovary may allow PKC to be active independent of cell membrane lipids.

A protein kinase C (PKC) stimulatory factor in heat-treated ovarian cytosol appears to be protein in nature as it was susceptible to proteinase K digestion and strong acid, and its lipid-substituting properties were retained after extraction of lipids. Phosphorylation of substrate by PKC stimulated by the factor from heat-treated ovarian cytosol in the absence of lipids was subject to dephosphorylation by the ovarian phosphatase. The factor stimulated purified PKC obtained from a commercial source as well as PKC partially purified from rat brain. Heat-treated ovarian cytosol stimulated PKC phosphorylation of myelin basic protein, but not protamine sulphate, in a calcium-dependent manner.

Animals↗
PubMed1997

Protein kinase C from bat brain: the enzyme from a hibernating mammal.

Protein kinase C (PKC) from brain of euthermic and hibernating bats (Myotis lucifugus) showed only one form as determined by hydroxylapatite chromatography, compared with three forms found in rat brain. Cross-reaction with antibodies to rabbit alpha, beta, and gamma isozymes showed that bat brain contained only PKC(gamma). During hibernation the activity of PKC in bat brain decreased to 63% of the euthermic value but the percentage that was membrane-associated did not change. Bat and rat brain PKC(gamma) were purified to homogeneity. Both enzymes phosphorylated all three of the substrates tested (FKKSFKL-NH2 peptide substrate, histone H1, protamine), the bat enzyme having significantly higher K(m) values than rat PKC for both peptide and histone. Both enzymes required phospholipids and Ca2+ for activation with rat brain PKC depending almost exclusively on phosphatidylserine. Bat PKC, however, made use of other phospholipids and showed relative activities of 100:81:33:42 for euthermic PKC and 100:91:45:35 for hibernator PKC with phosphatidylserine, phosphatidylinositol, phosphatidylcholine, and phosphatidylethanolamine (each at 50 microM), respectively. Activation of bat PKC by phosphatidylserine was temperature sensitive, being 3.5-fold at 4 degrees C (hibernating body temperature) compared with 14-18-fold at 33 degrees C (near euthermic body temperature). Arrhenius plots for bat brain PKC showed a sharp break below 10 degrees C; activation energies below this temperature were 11.5- and 5.2-fold greater than at higher temperatures for the enzyme from hibernating versus euthermic animals. By contrast, plots for the rat enzyme were linear over the range 0-42 degrees C. The data suggest that a sharp suppression of PKC activity by several mechanisms (reduced total activity, low temperature effects on activity and sensitivity to phospholipids) may be important to overall metabolic rate suppression during hibernation.

Animals↗
PubMed1976

Phosphorylation of cardiac regulatory proteins by cyclic AMP-dependent protein kinase.

Cardiac myofibrils were purified from canine myocardium, and the regulatory proteins (troponin + tropomyosin) were extracted and shown to contain endogenous cyclic AMP-dependent protein kinase activity. Other cyclic nucleotide stimulated the protein kinase activity but only at higher concentrations. The enzyme was able to catalyze phosphorylation of conventional substrates such as histones and casein as well as a component of the regulatory protein fraction with a molecular weight of 28,000 daltons. Endogenous phosphorylation required the presence of Mg2+ and was inhibited by Ca2+. A protein kinase inhibitor obtained from skeletal muscle inhibited the cyclicAMP-dependent phosphorylation. Escherichia coli alkaline phosphatase dephosphorylated the endogenous substrates. The level of phosphorylation found is severalfold higher than we have previously reported. A protein kinase, with its close association with the regulatory proteins, seems to be well suited to transmitting the message from the cyclic AMP to the regulatory proteins, a phenomenon that may influence the cardiac contractility via the troponin phosphorylation. The inhibitory effect of troponin on actomyosin might be changed by its state of phosphorylation.

Actins↗
PubMed1987

In vitro inhibition of rat brain protein kinase C by rhodamine 6G. Profound effects of the lipid cofactor on the inhibition of the enzyme.

Rhodamine 6G inhibited protein kinase C (PKC) when the enzyme was activated by Ca2+ plus phosphatidylserine, 12-O-tetradecanoyl-phorbol-13-acetate (TPA) or mezerein plus phosphatidylserine, Ca2+ plus arachidonic acid, or arachidonic acid alone. Rhodamine 6G did not affect protein kinase C activity in the absence of lipid cofactor and, thus, does not appear to inhibit the enzyme through direct interactions with the active site. The inhibitory potency of the drug was affected dramatically by the nature of the lipid cofactor. Thus, 50 microM rhodamine 6G inhibited the Ca2+ plus arachidonic acid dependent protein kinase activity approximately 50%, whereas 800 microM rhodamine 6G was required to cause 50% inhibition of the Ca2+ plus phosphatidylserine dependent protein kinase activity. These results, along with studies demonstrating a reversal of inhibition by high lipid concentrations, provide evidence that rhodamine 6G exerts its inhibitory effect on PKC through drug-lipid interactions. The dramatic effect of the lipid cofactor on the potency of rhodamine 6G as a PKC inhibitor suggests that the lipid environment of the cell may profoundly affect the abilities of rhodamine 6G and related cationic lipophilic drugs to inhibit PKC in vivo.

Animals↗
PubMed1981

Regulation by human chorionic gonadotropin of protein kinases in rabbit endometrium.

The effect of human chorionic gonadotropin (CG) on protein kinase levels has been studied in rabbit endometrium. A good correlation was observed between alterations of total protein kinase activity and DNA content in the tissue. The level of type I cyclic AMP-dependent protein kinase had slightly increased by 6 h after human CG treatment, and then decreased later. In contrast, the type II enzyme gradually increased after 3 days. The increase of the activity at 7 days was mainly due to that of type II enzyme, and the ratio of type II to type I enzyme increased. The activity pattern at the later stage closely resembled that of treatment with progesterone.

Animals↗
PubMed1987

The regulatory and basal phosphorylation sites of hormone-sensitive lipase are dephosphorylated by protein phosphatase-1, 2A and 2C but not by protein phosphatase-2B.

The activity of hormone-sensitive lipase, the rate-limiting enzyme in adipose tissue lipolysis, is controlled by cAMP-mediated phosphorylation at a specific regulatory phosphorylation site. The lipase is also phosphorylated at a site, termed basal, without any effects on its activity [Strålfors et al. (1984) Proc. Natl Acad. Sci. USA 81, 3317-3321]. The capacity of protein phosphatase-1, 2A, 2B and 2C to dephosphorylate the lipase, selectively phosphorylated by glycogen synthase kinase-4 and cAMP-dependent protein kinase at the basal and regulatory phosphorylation sites, was compared with that towards glycogen phosphorylase and phosphorylase kinase (alpha subunit). Protein phosphatase-1, 2A and 2C were found to dephosphorylate both phosphorylation sites of hormone-sensitive lipase, while protein phosphatase-2B had no measureable activity towards any of the sites. When the activities of protein phosphatase-1, 2A and 2C were normalized with respect to the reference substrates, they were found to dephosphorylate the lipase regulatory site in the approximate relations of 1:4:3 and the basal site in the approximate relations of 1:6:4. Protein phosphatase-1 showed 20% higher and protein phosphatase-2A and 2C 80% higher activity towards the basal site compared to the regulatory site. The two phosphorylation sites of the lipase were comparable to good substrates for protein phosphatase-2A and 2C, but relatively poor substrates for protein phosphatase-1. Protein phosphatase-2C activity towards the lipase was completely dependent on Mg2+ with a half-maximal effect at 3 mM. Protamine increased the lipase dephosphorylation by protein phosphatase-1 3-5-fold with half-maximal effect at 0.6 microgram/ml, and by protein phosphatase-2A about 2-fold with half-maximal effect at 3-5 micrograms/ml, thus illustrating the potential for control of these lipase phosphatase activities.

Adipose Tissue↗
PubMed1989

Effect of basic polycations and proteins on purified insulin receptor. Insulin-independent activation of the receptor tyrosine-specific protein kinase by poly(L-lysine).

Since the studies on tyrosine phosphorylation of calmodulin by the insulin receptor kinase in vitro suggested that protamine and poly(L-lysine) may activate phosphorylation of the receptor beta subunit [Sacks & McDonald (1988) J. Biol. Chem. 263, 2377-2383], we examined the effects of a variety of basic polycations/proteins and polyamines on insulin receptor kinase activity. The insulin receptor purified from human placental membranes was incubated with each basic polycation/protein or polyamine and assayed for tyrosine-specific protein kinase activity by measuring 32P incorporation into the src-related peptide. At a concentration of 1 microM, poly(L-lysine) and poly(L-ornithine) markedly stimulated kinase activity, whereas poly(L-arginine) and histones H1 and H2B inhibited insulin receptor kinase. In contrast, at a concentration of 1 mM, three polyamines (spermine, spermidine and putrescine) did not alter kinase activity. Poly(L-lysine) and poly(L-ornithine) stimulated the insulin receptor kinase by 5-10-fold at concentrations of 0.1-1 microM. Protamine sulphate also showed a significant stimulatory effect at a concentration of 100 microM. Preincubation of the receptor with poly(L-lysine) or poly(L-ornithine) for 20-60 min resulted in maximal kinase activation. Poly(L-lysine), the most effective activator of the receptor kinase, was used to characterize further the mechanisms of the kinase activation. Poly(L-lysine) activates the insulin receptor kinase by increasing the Vmax. without changing the Km. Poly(L-lysine) markedly stimulates the kinase activity of insulin receptor preparations that have lost both basal kinase activity and the ability to be stimulated by insulin. Insulin and poly(L-lysine) also differed in their ability to stimulate the kinase activity of prephosphorylated receptors. Prephosphorylation of the receptors did not affect the stimulation of the kinase by insulin. In contrast, prephosphorylation of receptors resulted in a markedly enhanced ability of poly(L-lysine) to stimulate kinase activity. These studies suggest that the mechanisms by which poly(L-lysine) and insulin activate the kinase are different. In conjunction with other additional evidence, it is suggested that poly(L-lysine) interacts directly with the beta-subunit of the receptor, thereby activating the receptor kinase.

Amino Acid Sequence↗
PubMed1998

Inhibition of membrane lipid-independent protein kinase Calpha activity by phorbol esters, diacylglycerols, and bryostatin-1.

The activity of membrane-associated protein kinase C (PKC) has previously been shown to be regulated by two discrete high and low affinity binding regions for diacylglycerols and phorbol esters (Slater, S. J., Ho, C., Kelly, M. B., Larkin, J. D., Taddeo, F. J., Yeager, M. D., and Stubbs, C. D. (1996) J. Biol. Chem. 271, 4627-4631). PKC is also known to interact with both cytoskeletal and nuclear proteins; however, less is known concerning the mode of activation of this non-membrane form of PKC. By using the fluorescent phorbol ester, sapintoxin D (SAPD), PKCalpha, alone, was found to possess both low and high affinity phorbol ester-binding sites, showing that interaction with these sites does not require association with the membrane. Importantly, a fusion protein containing the isolated C1A/C1B (C1) domain of PKCalpha also bound SAPD with low and high affinity, indicating that the sites may be confined to this domain rather than residing elsewhere on the enzyme molecule. Both high and low affinity interactions with native PKCalpha were enhanced by protamine sulfate, which activates the enzyme without requiring Ca2+ or membrane lipids. However, this "non-membrane" PKC activity was inhibited by the phorbol ester 4beta-12-O-tetradecanoylphorbol-13-acetate (TPA) and also by the fluorescent analog, SAPD, opposite to its effect on membrane-associated PKCalpha. Bryostatin-1 and the soluble diacylglycerol, 1-oleoyl-2-acetylglycerol, both potent activators of membrane-associated PKC, also competed for both low and high affinity SAPD binding and inhibited protamine sulfate-induced activity. Furthermore, the inactive phorbol ester analog 4alpha-TPA (4alpha-12-O-tetradecanoylphorbol-13-acetate) also inhibited non-membrane-associated PKC. In keeping with these observations, although TPA could displace high affinity SAPD binding from both forms of the enzyme, 4alpha-TPA was only effective at displacing high affinity SAPD binding from non-membrane-associated PKC. 4alpha-TPA also displaced SAPD from the isolated C1 domain. These results show that although high and low affinity phorbol ester-binding sites are found on non-membrane-associated PKC, the phorbol ester binding properties change significantly upon association with membranes.

Allosteric Regulation↗
PubMed1976

Purification and general properties of guanosine 3':5'-monophosphate-dependent protein kinase from guinea pig fetal lung.

Guanosine 3':5'-monophosphate (cyclic GMP)-dependent protein kinase was purified from the guinea pig fetal lung, a tissue shown to be the richest in this enzyme in all mammalian sources examined, and its general properties studied. The enzyme was purified 150-fold from crude extract by steps of pH 5.4 isoelectric precipitation, Sephadex G-200 filtration, hydroxylapatite treatment and DEAE-cellulose chromatography. The purified enzyme, free from contamination with adenosine 3':5'-monophosphate (cyclic AMP)-dependent protein kinase, had a specific activity at least equivalent to 600-fold purification of the enzyme from the adult lung. The pulmonary enzyme exhibited an absolute requirement of protein kinase modulator (prepared from various mammalian tissues with an exception of skeletal muscle) for its activity. Inhibitor protein of cyclic AMP-dependent protein kinase purified from rabbit skeletal muscle could not stimulate nor inhibit the cyclic GMP target enzyme, indicating the factors from mammalian sources regulating the two classes of protein kinases may not be the same. The enzyme had Ka values of 1.3 times 10(-8) and 3.3 times 10(-8) M for 8-bromo cyclic GMP and cyclic GMP, respectively, compared to 3.0 times 10(-6) M for cyclic AMP. Cyclic GMP lowered the Km of the enzyme for ATP from 6.3 times 10(-5) M in its absence to 2.1 times 10(-5) M in its presence, accompanied by an approximate doubling of the Vmax. The molecular weight of the enzyme (assayed by its catalytic and cyclic GMP-binding abilities) was estimated to be 123,000, corresponding to a sedimendation coefficient of 7.06 S, by means of sucrose density gradient ultracentrifugation. The cyclic GMP-dependent enzyme required Mg2+ and Co2+ for its activity with optimal concentrations of about 30 and 0.7 mM, respectively. The maximal activity seen in the presence of Mg2+, however, was nearly twice as high as that seen in the presence of Co2+. Histones were generally effective substrates for the enzyme, whereas protamine, casein, phosvitin, phosphorylase kinase, and activator protein of phosphodiesterase were not. The cyclic GMP-dependent enzyme exhibited a greater affinity for histones than did the cyclic AMP-dependent enzyme in the presence of Mg2+.

Animals↗
PubMed1987

Substrate-specific stimulation of protein kinase C by polyvalent anion.

The activity of protein kinase C (PKC) toward arginine-rich substrates was greatly stimulated by sulfate and phosphate, but not by monovalent anions. This stimulation did not require phospholipid, calcium, or diacylglycerol, and appeared to mimic the stimulation by phospholipid. Anionic proteins such as bovine serum albumin also promoted PKC activity toward certain substrates that were characterized by either high arginine or high lysine content. The mechanism of both of these stimulations appeared to be related to formation of a substrate-PKC complex which is essential to phosphorylation by PKC. Polyvalent anions bind the cationic substrate and, together with PKC, form an aggregate which allows phosphorylation. Potential physiological relevance of this stimulation is discussed.

Animals↗
PubMed1999

Resveratrol preferentially inhibits protein kinase C-catalyzed phosphorylation of a cofactor-independent, arginine-rich protein substrate by a novel mechanism.

Resveratrol, a polyphenolic natural product abundantly present in grape skins, is a candidate cancer chemopreventive agent that antagonizes each stage of carcinogenesis and inhibits protein kinase C (PKC), a key mediator of tumor promotion. While resveratrol has been shown to antagonize both isolated and cellular forms of PKC, the weak inhibitory potency observed against isolated PKC cannot account for the reported efficacy of the polyphenol against PKC in cells. In this report, we analyze the mechanism of PKC inhibition by resveratrol. Our results indicate that resveratrol has a broad range of inhibitory potencies against purified PKC that depend on the nature of the substrate and the cofactor dependence of the phosphotransferase reaction. Resveratrol weakly inhibited the Ca2+/phosphatidylserine-stimulated activity of a purified rat brain PKC isozyme mixture (IC(50) = 90 microM) by competition with ATP (K(i) = 55 microM). Consistent with the kinetic evidence for a catalytic domain-directed mechanism, resveratrol inhibited the lipid-dependent activity of PKC isozymes with divergent regulatory domains similarly, and it was even more effective in inhibiting a cofactor-independent catalytic domain fragment (CDF) of PKC generated by limited proteolysis. This suggested that regulatory features of PKC might impede resveratrol inhibition of the enzyme. To explore this, we examined the effects of resveratrol on PKC-catalyzed phosphorylation of the cofactor-independent substrate protamine sulfate, which is a polybasic protein that activates PKC by a novel mechanism. Resveratrol potently inhibited protamine sulfate phosphorylation (IC(50) = 10 microM) by a mechanism that entailed antagonism of the activation of PKC by protamine sulfate and did not involve competition with either substrate. On the basis of the presence of PKC isozymes at subcellular sites rich in polybasic proteins, it has been proposed that certain endogenous polybasic PKC substrates may activate PKC in cells by the same mechanism as protamine sulfate. Our results suggest that antagonism by resveratrol of the phosphorylation of cellular PKC substrates that resemble protamine sulfate in their interactions with PKC may contribute to the efficacy of resveratrol against PKC in cells.

Animals↗
PubMed1975

Phosphorylation of purified thyroid plasma membranes incubated with [32-P]ATP.

Plasma membranes have been prepared from porcine thyroid glands using sucrose gradients. The fractions having a density in sucrose of 1.18 g/ml mainly contained plasma membranes and were moderately contaminated with other subcellular components as shown by marker enzyme data. Purified plasma membranes incubated in the presence of [32-P]gamma ATP incorporated 32-P. Kinetics of incorporation of 32-P into endogenous substrates studied in various buffers and with increasing ATP concentration suggest a phosphodephosphorylating system related to cAMP-dependent protein kinase and phosphoprotein phosphatase activities. The two enzymatic activities associated with plasma membranes have been demonstrated using exogenous substrates. cAMP increases and fluoride ions decrease the extent of membrane phosphorylation. The specific activity of protein kinase was 10-12 times higher than in the initial homogenate and was only slightly enhanced in the presence of 0.5% Nonidet as compared to microsomal fraction. cAMP binding to membrane proteins was 3 times higher than to the other particulate fractions. TSH present in the incubating medium or added after 5 min of 32-P labelling induced a rapid stimulation of endogenous phosphorylation followed by a rapid decrease. Phosphorylated membrane substrates were analyzed: high voltage paper electrophoresis after partial hydrolysis indicated that [32-P]phosphate is incorporated into serine and threonine residues as o-phosphate derivatives. SDS-polyacrylamide gel electrophoresis showed several 32--labelled fractions. When enhanced by cAMP, no specific phosphorylation of protein components was observed.

Adenosine Triphosphate↗
PubMed1977

Protein kinase and phosphoproteins of avian myeloblastosis virus.

A protein kinase associated with purified virions of avian myeloblastosis virus, BAI strain A, was highly purified by ion-exchange chromatography and gel filtration. On the basis of molecular sieving on Sephadex G-200, the enzyme protein appeared to have a molecular weight of about 50,000 to 60,000; disc gel electrophoresis in sodium dodecyl sulfate-acrylamide gels revealed the presence of at least two polypeptide chains; and isoelectric focusing on acrylamide gels revealed two protein bands with activity. Of the nonviral proteins used as phosphate acceptors, the greatest rate of phosphorylation was obtained with alpha-casein. Potential physiological substrates for this activity included specific virion polypeptide of avian myeloblastosis virus. One of the virion polypeptides found in association with reverse transcriptase activity from avian myeloblastosis virus accepted more phosphate than any of nonviral or viral polypeptides examined on the basis of nanomoles of 32P incorporated per milligram of protein.

Avian Leukosis Virus↗
PubMed2003

Inhibition of protein kinase C catalytic activity by additional regions within the human protein kinase Calpha-regulatory domain lying outside of the pseudosubstrate sequence.

The N-terminal pseudosubstrate site within the protein kinase Calpha (PKCalpha)-regulatory domain has long been regarded as the major determinant for autoinhibition of catalytic domain activity. Previously, we observed that the PKC-inhibitory capacity of the human PKCalpha-regulatory domain was only reduced partially on removal of the pseudosubstrate sequence [Parissenti, Kirwan, Kim, Colantonio and Schimmer (1998) J. Biol. Chem. 273, 8940-8945]. This finding suggested that one or more additional region(s) contributes to the inhibition of catalytic domain activity. To assess this hypothesis, we first examined the PKC-inhibitory capacity of a smaller fragment of the PKCalpha-regulatory domain consisting of the C1a, C1b and V2 regions [GST-Ralpha(39-177): this protein contained the full regulatory domain of human PKCalpha fused to glutathione S-transferase (GST), but lacked amino acids 1-38 (including the pseudosubstrate sequence) and amino acids 178-270 (including the C2 region)]. GST-Ralpha(39-177) significantly inhibited PKC in a phorbol-independent manner and could not bind the peptide substrate used in our assays. These results suggested that a region within C1/V2 directly inhibits catalytic domain activity. Providing further in vivo support for this hypothesis, we found that expression of N-terminally truncated pseudosubstrate-less bovine PKCalpha holoenzymes in yeast was capable of inhibiting cell growth in a phorbol-dependent manner. This suggested that additional autoinhibitory force(s) remained within the truncated holoenzymes that could be relieved by phorbol ester. Using tandem PCR-mediated mutagenesis, we observed that mutation of amino acids 33-86 within GST-Ralpha(39-177) dramatically reduced its PKC-inhibitory capacity when protamine was used as substrate. Mutagenesis of a broad range of sequences within C2 (amino acids 159-242) also significantly reduced PKC-inhibitory capacity. Taken together, these observations support strongly the existence of multiple regions within the PKCalpha-regulatory domain that play a direct role in the inhibition of catalytic domain activity.

Animals↗
PubMed2004

Activation of protein kinase C by 1-beta-D-arabinofuranosylcytosine conjugates of phospholipid.

1-beta-D-arabinofuranosylcytosine (ara-C) conjugates of phospholipid were shown to be highly antineoplastic against various tumor cells. In this study, we report that these conjugates are potent activators of protein kinase C (PKC, EC) in vitro. Although required Ca2+, PKC activation by the conjugates occurred even in the absence of phospholipid and diacylglycerol. Among the conjugates, 1-beta-D-arabino-furanosylcytosine 5'-diphosphate-rac-1-O-octadecyl-2-O-palmitoylglycerol [(ara-CDP-DL-PBA); ara-C conjugate of ether phospholipid], was employed to investigate its mode of activation, since ether phospholipid has been reported to be a regulator of the PKC. When PKC was activated by ara-CDP-DL-PBA, diacylglycerol enhanced its activity with 3-fold reduction of an apparent Ka value for ara-CDP-DL-PBA and no change in the Vmax. During the PKC activation by phosphatidylserine, ara-CDP-DL-PBA exhibited a synergistic effect on the activation. Studies on the relationship between the structures of ara-CDP-DL-PBA and their effects on PKC activity showed that phosphate group of ether lipid was important for its activation of PKC, and that conjugation of ara-C and ether lipid further enhanced the enzyme activity. These results suggest that the ara-C conjugate of phospholipid activates PKC in a co-operative manner with diacylglycerol and/or phosphatidylserine, however, the exact mechanism of the antineoplastic effect of ara-CDP-DL-PBA through PKC activation still remains speculative.

Animals↗
PubMed1976

Nucleoside-dependent protein kinase from Trypanosoma gambiense.

A nucleoside-dependent protein kinase (EC 2.7.1.37) was partially purified from Trypanosoma gambiense, the pathogenic agent of sleeping sickness. This enzyme catalyzes the phosphorylation of histone and protamine. Various nucleosides at the concentration of 10(-4) M stimulated the histone kinase activity about two-fold, whereas cyclic AMP and cyclic GMP were without effect. The pH-optimum for histone phosphorylation was at about pH 7.0. The enzyme activity absolutely depends on Mg2+, Mn2+ or Co2+. The apparent Km-value for histone was 0.3 mg/ml and those for ATP were 2 - 10(-4) M and 6 - 10(-5) M in the absence or presence of 10(-4) M adenosine respectively. IDP and ADP complete with ATP. The inhibition constants were calculated to be 2 - 10(-4) M and 2.5 - 10(-4) M, respectively. The molecular weight of the histone kinase was found to be 95 000 by gel filtration and 88 000 by sedimentation in a sucrose gradient.

Animals↗
PubMed1985

Pyrimidine nucleoside monophosphate kinase from rat bone marrow cells: purification to high specific activity by a two-step affinity chromatography procedure.

This report describes a two-column scheme for purifying a pyrimidine nucleoside monophosphate kinase from rat bone marrow cells. Purification was achieved by affinity chromatography on Blue Sepharose and cellulose phosphate, with selective elution of the enzyme by substrates (UMP, ATP). The enzyme preparation appeared to be about 90% pure upon polyacrylamide gel electrophoresis, exhibited an exceptionally high specific activity (greater than 600 mumol/min/mg protein), and was obtained with 30-36% recovery of enzyme activity. It was concluded that UMP, dUMP, and CMP serve as phosphate acceptors for the enzyme, based on the parallel behavior displayed by enzyme activity with these substrates both during the purification process and during other procedures. The purified enzyme preparation did not display dTMP kinase activity. This report also describes a simplified radiotracer assay for pyrimidine nucleoside monophosphate kinases. Thin-layer chromatography on polyethyleneimine-cellulose is used to resolve residual substrates and products. Because both nucleoside di- and triphosphates remain at the origin, the assay is insensitive to the action of nucleoside diphosphate kinases and does not require the use of marker compounds. A variety of radiolabeled substrates can be used with this assay, including UMP, dUMP, CMP, and dTMP.

Animals↗