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PubMed1970

Mechanism of activation by adenosine 3':5'-cyclic monophosphate of a protein phosphokinase from rabbit reticulocytes.

Two protein phosphokinases (EC 2.7.1.37) were found to be present in rabbit reticulocytes. The two enzymes were separated by DEAE-cellulose chromatography and called kinases I and II. Adenosien 3':5'-cyclic monophosphate stimulated the activity of both enzymes. However, the degree of stimulation was different and depended on the protein acceptor used. In the presence of adenosine 3':5'-cyclic monophosphate, protein kinase I dissociated into two subunits: a subunit binding adenosine 3':5'-cyclic monophosphate, and a catalytic subunit. The component binding the cyclic nucleotide appeared to act as an inhibitory protein, regulating the activity of the catalytic subunit. The mechanism of action of the cyclic nucleotide on kinase II appeared to be different from that of kinase I.

Adenine Nucleotides↗
PubMed1989

In vitro studies of the inhibition of protein kinase C from rat brain by di-(2-ethylhexyl)phthalate.

The environmental contaminant di(2-ethylhexyl)phthalate (DEHP) has been shown to inhibit the phosphorylation of histone by purified protein kinase C (PK-C) from rat brain in a concentration-dependent manner. The inhibition does not involve making the substrate unavailable, although DEHP does bind to some extent to histone. DEHP displaces phorbol dibutyrate from PK-C, indicating that DEHP binds to the regulatory domain of the enzyme. Since DEHP does not affect the PK-C dependent phosphorylation of protamine, DEHP probably does not bind at the catalytic site. DEHP non-competitively blocked activation of PK-C by either phosphatidyl serine or calcium ion. Inhibition of histone phosphorylation by DEHP was enhanced if diglyceride was present, and the enhancement was stereoselective for the isomeric form of the diglyceride. The mechanism of the inhibition is thought to involve interference with the interaction between calcium ion and the regulatory domain of PK-C, and would have significance only for those PK-C substrates that require calcium activation of the enzyme. Thus the presence of DEHP in the high nanomolar concentration range alters the effective substrate specificity of PK-C.

Animals↗
PubMed2006

Thrombin generation during reperfusion after coronary artery bypass surgery associates with postoperative myocardial damage.

BACKGROUND: Cardiopulmonary bypass and coronary artery bypass grafting (CABG) result in significant thrombin generation and activation of fibrinolysis. Thrombin contributes to myocardial ischemia-reperfusion injury in animal studies, but the role of thrombin in myocardial damage after CABG is unknown. OBJECTIVES: We measured thrombin generation and fibrin turnover during reperfusion after CABG to evaluate their associations with postoperative hemodynamic changes and myocardial damage. METHODS: One hundred patients undergoing primary, elective, on-pump CABG were prospectively enrolled. Plasma prothrombin fragment F(1+2) and D-dimer were measured preoperatively and at seven time points thereafter. Mass of the Mb fraction of creatine kinase (Ck-Mbm) and troponin T (TnT) were measured on the first postoperative day. RESULTS: Reperfusion induced an escalation of thrombin generation and fibrin turnover despite full heparinization. F(1+2) during early reperfusion associated with postoperative pulmonary vascular resistance index. F(1+2) at 6 h after protamine administration correlated with Ck-Mbm (r = 0.40, P < 0.001) and TnT (r = 0.44, P < 0.001) at 18 h postoperatively. Patients with evidence of myocardial damage (highest quintiles of plasma Ck-Mbm and TnT) had significantly higher F(1+2) during reperfusion than others (P < 0.002). Logistic regression models identified F(1+2) during reperfusion to independently associate with postoperative myocardial damage (odds ratios 2.5-4.4, 95% confidence intervals 1.04-15.7). CONCLUSIONS: Reperfusion caused a burst in thrombin generation and fibrin turnover despite generous heparinization. Thrombin generation during reperfusion after CABG associated with pulmonary vascular resistance and postoperative myocardial damage.

Adult↗
PubMed1978

Protamine-agarose non-charged alkyl derivatives of agarose in the purification of rat-liver phosphoprotein phosphatases.

1. Protamine-agarose and hydrophobic interaction chromatography were found to be effective in the purification of phosphoprotein phosphatase(s) (phosphoprotein phosphohydrolase, EC 3.1.3.16) of rat-liver. The phosphoprotein phosphatase of rat-liver cytosol were first resolved into three fractions, termed A, B and C, in order of elution from DEAE-cellulose. Whereas all fractions displayed activity towards [32P]phosphoprotamine, only fractions B and C displayed appreciable activity towards [32P]phosphopyruvate kinase. Since fraction B exhibited the most properties and the highest recovery of enzymatic activity towards [32P]phosphoprotamine and [32P]phosphopyruvate kinase, it was selected for further purification. The method developed involves sequential chromatography of fraction B on Sephadex G-200, protamineagarose, histone-agarose and then again on Sephadex G-200 as a final step. A 400-fold enrichment in the phosphoprotamine phosphatase activity of fraction B was obtained. Purified fraction B also displayed substantial phosphatase activity towards [32P]phosphopyruvate kinase and [32P]phosphohistones. An apparent molecular weight of about 250 000 was estimated for purified fraction B on a calibrated Sephadex G-200 column. The present data indicate that rat-liver cytosol contains multiple forms of phosphoprotein phosphatases and suggest a technique which might be applied for the further purification of at least fraction B. 2. In a separate approach, a combination of pentyl-agarose and protamineagarose chromatography was shown to be a conbenient method for the enrichment (up to 20-fold of phosphoprotein phosphatase activity from crude liver extracts.

Alkylation↗
PubMed1992

Multivariate analysis by the minimum spanning tree method of the structural determinants of diphenylethylenes and triphenylacrylonitriles implicated in estrogen receptor binding, protein kinase C activity, and MCF7 cell proliferation.

The response profiles of 36 para-substituted diphenylethylenes (DPEs) and triphenylacrylonitriles (TPEs) have been compared by multivariate analysis. The responses measured were (a) relative binding affinity (RBA) for the cytosol estrogen receptor (ER), (b) ability to promote the growth of the human MCF7 breast cancer cell-line, (c) cytotoxicity in MCF7 cells, and (d) ability to stimulate or inhibit protein kinase C (PKC) III activity under three different conditions of enzyme activation. The prime object of the analysis was to observe the simultaneous influence of diverse combinations of substituents on all these in vitro responses. To do this, the minimum spanning tree (MST) method was used to organize the molecules into a network in which proximate molecules are closely related with regard to their responses whereas remote molecules are distinct. The MST of this population of molecules had four main branches. E2 and its TPE mime were located in a central position within the trunk whereas the tips of the branches tended toward molecules of different specificity, i.e., cytotoxic molecules that bind to ER and interfere with PKC, noncytotoxic molecules that also bind to ER and interfere with PKC but promote cell growth, molecules only active on PKC, and molecules active on all parameters except PKC stimulation. A parallel MST analysis of the relationships among the response parameters themselves confirmed previous conclusions: For this population of molecules, RBAs for ER are fairly closely related to ability to promote MCF7 cell growth and only little to cytotoxicity (Bignon et al. J. Med. Chem. 1989, 32, 2092). Cytotoxicity is much more clearly correlated with inhibition of diacylglycerol-stimulated PKC activity than with RBAs for ER. PKC inhibition differs substantially depending upon whether the substrate is H1 histone or protamine sulfate.

Acrylonitrile↗
PubMed1992

Ca(2+)-dependent protein kinase from the halotolerant green alga Dunaliella tertiolecta: partial purification and Ca(2+)-dependent association of the enzyme to the microsomes.

Ca(2+)-dependent protein kinase (CDPK) was purified 900-fold from the soluble fraction of Dunaliella tertiolecta cells by ammonium sulfate precipitation, DEAE-Toyopearl, phenyl-Sepharose, and hydroxylapatite column chromatography. The CDPK was activated by micromolar concentration of Ca2+ and required neither calmodulin nor phospholipids for its activation. The enzyme phosphorylated casein, myosin light chain, and histone type III-S (histone H-1), but did not phosphorylate protamine and phosvitin. The Km values for ATP and casein were 11 microM and 300 micrograms/ml, respectively. Phosphorylation of casein was inhibited by calmodulin antagonists, calmidazolium, trifluoperazine, and compound 48/80, but not affected by calmodulin. CDPK bound to phenyl-Sepharose in the presence of Ca2+ and was eluted by ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (EGTA). This suggests that hydrophobicity of the enzyme was increased by Ca2+. CDPK was also bound to the microsomes isolated from Dunaliella cells in the presence of micromolar concentration of Ca2+ and released in the presence of EGTA, suggesting the possibility of in vivo Ca(2+)-dependent association of the enzyme. The enzyme phosphorylated many proteins in the microsomes but few in the cytosol, if at all.

Calmodulin↗
PubMed1988

Association of protein kinase C with phospholipid monolayers: two-stage irreversible binding.

The association of protein kinase C (PKC) with phospholipid (PL) monolayers spread at the air-water interface was examined. PKC-PL binding induced surface pressure changes that were dependent on the amount of PKC, the phospholipid composition of the monolayers, the presence of Ca2+, and the initial surface pressure of the monolayer (pi 0). Examination of surface pressure increases induced by PKC as a function of phospholipid surface pressure, pi 0, revealed that PKC-phosphatidylserine (PS) association had a critical pressure of 43 dyn/cm. Above this surface pressure, PKC cannot cause further surface pressure changes. This high critical pressure indicated that PKC should be able to penetrate many biological membranes which appear to have surface pressures of about 30 dyn/cm. PKC-induced surface pressure changes were Ca2+ dependent only for PL monolayers spread at a pi 0 greater than 26 dyn/cm. PKC alone (in the absence of PL) formed a film at the air-water interface with a surface pressure of about 26 dyn/cm. Calcium-dependent binding was studied at the higher surface pressures which effectively excluded PKC from the air-water interface. Subphase depletion measurements suggested that association of PKC with PS monolayers consisted of two stages: a rapid Ca2+-dependent interaction followed by a slower process that resulted in irreversible binding of PKC to the monolayer. The second stage appeared to involve penetration of PKC into the hydrocarbon region of the phospholipid. The commonly used in vitro substrates for PKC, histone and protamine sulfate, also associated with and penetrated PS monolayers with critical pressures of 50 and 60 dyn/cm, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1996

The Candida albicans PKC1 gene encodes a protein kinase C homolog necessary for cellular integrity but not dimorphism.

Using a DNA fragment derived from the Saccharomyces cerevisiae protein kinase C gene (PKC1) as a probe to screen an ordered array library of genomic DNA from the dimorphic pathogenic fungus Candida albicans, the C. albicans PKC1 gene (CaPKC1) was isolated. The CaPKC1 gene is predicted to encode a protein of 1079 amino acids with 51% sequence identity over the entire length with the S. cerevisiae Pkc1 protein and is capable of functionally complementing the growth defects of a S. cerevisiae pkc1 delta mutant strain on hypo-osmotic medium. Deletion of both endogenous copies of the CaPKC1 gene in diploid C. albicans cells resulted in an osmotically remedial cell lysis defect of both the budding and the hyphal growth form and morphologically aberrant cells of the budding form. Despite these abnormalities, the transition between the two growth forms of C. albicans occurred normally in pkc1/pkc1 double disruptants. Capkc1p was modified at its C-terminus with two repeats of the Staphylococcus aureus protein A IgG-binding fragment (ZZ-sequence tag) and partially purified by chromatography on DEAE-Sepharose and IgG-Sepharose. In vitro, Capkc1p preferably phosphorylated the S. cerevisiae Pkc1p pseudosubstrate peptide and myelin basic protein, but not histones, protamine or dephosphorylated casein, and failed to respond to cofactors known to activate several mammalian PKC isozymes.

Amino Acid Sequence↗
PubMed1995

Shedding of tyrosine and serine/threonine ecto-protein kinases from human leukemic cells.

Ecto-protein kinases (ecto-PK), primarily of the serine/threonine kinase type, have been previously described on the surface of various normal, transformed, and tumor cells. We have found that in the presence of ATP and Mg2+, exogenously added substrates such as phosvitin and poly(Glu4-Tyr) are phosphorylated by intact K562 erythroleukemia, HL60 promyelocytic leukemia, and U937 histiocytic leukemia human cells. Phosphoamino acid analysis indicated that phosvitin, histone H2B, casein, and protamine are phosphorylated on serine and threonine residues, whereas poly(Glu4-Tyr) is phosphorylated on tyrosine. We also present evidence showing that the C9 complement protein, a key component of the membranolytic protein complex of the complement system, is exclusively phosphorylated by the K562 cells on serine residues. Phosphorylation of poly(Glu4-Tyr) is markedly enhanced by Mn2+, whereas C9 phosphorylation is rather inhibited by Mn2+. It is concluded that human leukemic cells express on their surface two types of ecto-PK, one phosphorylating serines and threonines and one specific to tyrosines. The ecto-PKs are spontaneously shed from fully viable cells into the medium in a temperature-dependent manner. Upon sedimentation of cell supernatants at 100,000g, the ecto-PKs are found sedimented with small membrane vesicles. Treatment of intact K562 cells or of released membrane vesicles with bacterial phospholipase C, but not with trypsin or pronase, releases the two types of ecto-PK from the cell or vesicle membrane, respectively. This is accompanied by a marked increase in the released phosphorylating activity. It is, therefore, suggested that these ecto-PKs are either covalently linked to phospholipids or strongly attached to lipid-anchored molecules in the cell surface membrane. Several endogenous proteins in the released membranes are phosphorylated by the ecto-PKs on serines and to a lesser extend on threonines. Two proteins (PTP79 and PTP54) are phosphorylated in a manganese-dependent manner on tyrosines.

Adenosine Triphosphate↗
PubMed2000

[Male contraception of triptonide and its function mechanism].

OBJECTIVE: The male contraceptive mechanism in rats of triptonide, isolated from Multiglycosides of Tripterygium wilfordii (GTW), was investigated. METHODS: The dosage of triptonide was 200 micrograms/(kg.d), fed for 8 weeks via gastric intubation. Sperm counts, sperm motility, and sperm silver staining were used to test the antispermatogenic effect of triptonide; the study of morphology was learned from paraffin sections of testis and visceras of triptonide-treated rats; protamine was abstracted from testis by electrophoresis; using antisense mRNA of cyclin D1 and Cdk4 as prodes, we examined the expression of cyclin D1 and Cdk4 through hybridization in situ. RESULTS: Sperm counts (26.43 +/- 3.90) x 10(6)/g and sperm motility(66.0 +/- 6.8)% of triptonide-treated rats were substantially different from those of control, (41.15 +/- 5.51) x 10(6)/g (P < 0.01) and (88.0 +/- 2.0)% (P < 0.001). Paraffin sections of testis of triptonide-treated rats stained by PAS showed that the number of spermatids per 100 sertoli cells (9.15 +/- 0.33) was remarkably decreased compared with control's (10.73 +/- 0.38) (P < 0.001). At the same time, delaying sperm-released process was observed, which affects the nature course of sperm. And the extended vacuoles of vary sizes, the segregation of cells in lumens were also found in the cross-section of testis of rats treated with triptonide. In addition, the contents of protamine (4.6 +/- 2.2)% was extremely decreased, compared with the control's (40.6 +/- 7.2)% (P < 0.001). By using antisense mRNA of cyclin D1 and Cdk4 as prodes, we detected that the expression of the genes was notably increased in spermatids. CONCLUSIONS: The results suggest the antispermatogenic effect of triptonide is very well and it mainly plays its role on spermatids by increasing the expression of cyclin D1 and Cdk4, meanwhile, decreasing the biosynthesis of protamine. The appropriate target region of triptonide makes itself most potency to be a suitable male antifertility drug.

Animals↗
PubMed1982

Properties and purification of the catalytic subunit of cyclic AMP-dependent protein kinase of adipose tissue.

The catalytic subunit of cAMP-dependent protein kinase from rat adipose tissue was purified to apparent homogeneity by making use of the differential binding of the holoenzyme and the free catalytic subunit to CM-Sephadex and by gel chromatography. Stability and yield was improved by inclusion of nonionic detergent in all steps after dissociation of the holoenzyme. Isoelectric focusing separated enzyme species with pI values of 7.8 and 8.6-8.8. The amino acid composition was similar to the enzyme purified from other tissues. Enzyme activity was markedly unstable in dilute solutions (less than 5 micrograms/ml). Additions of nonionic detergent, glycerol, bovine serum albumin and, especially, histones stabilized the enzyme. With protamine, the catalytic subunit had an apparent Km of 60 microM and Vmax of 20 mumol X min-1 X mg-1, corresponding values with mixed histones were 12 microM and 1.2 mumol X min-1 X mg-1. With both protein substrates the apparent Km for ATP was 11 microM. Concentrations of Mg2+ above 10 mM were inhibitory. Histone phosphorylation was inhibited by NaCl (50% at 0.5 M NaCl) while protamine phosphorylation was stimulated (4-fold at 1 M NaCl). Inorganic phosphate inhibited both substrates (histones: 50% at 0.3 M, and protamine: 50% at 0.5 M). pH optimum was around pH 9 with both substrates. The catalytic subunit contained 2.0 (range of three determinations, 1.7-2.3) mol phosphate/mol protein. It was autophosphorylated and incorporated 32Pi from [gamma-32P]ATP in a time-dependent process, reaching saturation when approx. 0.1 mol phosphate/mol catalytic subunit was incorporated.

Adipose Tissue↗
PubMed1989

Modes of inhibition of protein kinase C by triphenylacrylonitrile antiestrogens.

Protein kinase C (PKC) I (gamma), II (beta) and III (alpha) subspecies' activities are inhibited by three triphenylacrylonitrile (TPE) antiestrogens at micromolar concentrations. TPE 1 (having a p-hydroxy and a p-diethylaminoethoxy group on the 3-, and 3'- phenyl rings respectively) and TPE 2 (having a p-diethylaminoethoxy group on both the 3-, and 3'- phenyl rings) are competitive with the mechanism of activation by phosphatidylserine (PS). TPE 3 (having p-hydroxy groups on each of the three phenyl rings) is non-competitive with PS and inhibits the Ca2+- and PS-independent phosphorylation of protamine sulfate by PKC subspecies. This evidence suggests that PKC activity can be inhibited by different routes depending on the TPE structure: diethylaminoethoxy side chain-substituted TPEs (TPE 1 and 2) interact with PS as well as with the regulatory domain, whereas the trihydroxylated derivative (TPE 3) inhibits the enzyme by interacting with the catalytically active site.

Acrylonitrile↗