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W Pyerin

Publications and source records attributed to W Pyerin.

At least 73 records · Page 4Linked to original sources
PubMed1987

Chromatographic separation of two heterogeneous forms of the catalytic subunit of cyclic AMP-dependent protein kinase holoenzyme type I and type II from striated muscle of different mammalian species.

Electrophoretically homogeneous preparations of catalytic subunit (C) of cAMP-dependent protein kinase isolated according to two different procedures from holoenzyme type I and type II from rabbit and from holoenzyme type II from rat skeletal muscle and from bovine cardiac muscle can be separated on carboxymethyl cellulose or on a Mono S column (Pharmacia) by salt gradient elution into two enzymatically active peaks called A and B, which do not interconvert on rechromatography. Cochromatography of peak A fractions or of peak B fractions derived from both holoenzymes respectively yields single enzyme peaks in each case, thus indicating that both represent different entities, which were named CA and CB. The separate character of both enzyme forms is supported by the fact that CB under all conditions is degraded faster by the C-specific protease (E. Alhanaty et al. (1981) Proc. Natl. Acad. Sci. USA 78, 3492-3495) than CA, a phenomenon which is enhanced in both enzyme forms by substrate (Kemptide). The separation of both subtypes from each other is probably based on differences in isoelectric values (delta pH less than or equal to 0.5 units). The reason for the charge difference is not presently known. CA and CB do not differ significantly in their phosphate content. No differences between CA and CB have been detectable so far with respect to their migration in SDS gels, kinetic behavior regarding both substrates and cosubstrate, pH dependence, inhibition by regulatory subunits of holoenzyme type I (rabbit skeletal muscle) and of type II (bovine cardiac muscle), and inhibition by specific-heat and acid-stable inhibitor-modulator. The peptide pattern of both forms after limited proteolysis exhibits small differences.

Animals↗
PubMed1987

Catalytic and molecular properties of highly purified phosvitin/casein kinase type II from human epithelial cells in culture (HeLa) and relation to ecto protein kinase.

Phosvitin/casein type II kinase was purified from HeLa cell extracts to homogeneity and characterized. The kinase prefers phosvitin over casein (Vmax phosvitin greater than Vmax casein; apparent Km 0.5 microM phosvitin and 3.3 microM casein) and utilizes as cosubstrate ATP (apparent Km 3-4 microM), GTP (apparent Km 4-5 microM) and other purine nucleoside triphosphates, including dATP and dGTP but not pyrimidine nucleoside triphosphates. Enzyme reaction is optimal at pH 6-8 and at 10-25 mM Mg2+.Mg2+ cannot be replaced by, but is antagonized by other divalent metal ions. The kinase is stimulated by polycations (spermine) and monovalent cations (Na+,K+), and is inhibited by fluoride, 2,3-diphosphoglycerate, and low levels of heparin (50% inhibition at 0.1 microgram/ml). The HeLa enzyme is composed of three subunits with Mr of approximately 43,000 (alpha), 38,000 (alpha'), and 28,000 (beta) forming alpha alpha'beta 2 and alpha'2 beta 2 structures with obvious sequence homology of alpha with alpha' but not with beta. Photoaffinity labeling with [alpha-32P]- and [gamma-32P]8-azido-ATP revealed high affinity binding sites on subunits alpha and alpha' but not on subunit beta. The kinase autophosphorylates subunit beta and, much weaker, subunits alpha and alpha'. Ecto protein kinase, detectable only by its enzyme activity but not yet as a protein (J. Biol. Chem. 257, 322-329), was characterized in cell-bound form and in released form, and the released form both with and without prior separation from phosvitin which was employed to induce the kinase release from intact HeLa cells (Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025). Ratios of phosvitin/casein phosphorylation (greater than 2) and of ATP/GTP utilization (1.5-2.1), inhibition by heparin (50% inhibition at 0.1 microgram/ml), and amino-acid side chains phosphorylated in phosvitin and casein (serine, threonine) are comparable for cell-bound and released form. These properties resemble those of type II kinase as does Mr of released ecto kinase (120-150,000). Consistently, a protein with Mr 125,000 in calf serum and a protein (possibly two) with Mr greater than 300,000 in calf plasma which are selectively phosphorylated by the ecto kinase are also substrates of the type II kinase. Thus, nearly all properties examined of the ecto kinase are characteristic for a type II kinase.

Anions↗
PubMed1987

Substrates for the cell surface protein kinase in blood: a calf serum protein and its precursor in plasma.

A protein in calf serum with molecular mass of 125,000 is selectively phosphorylated by the surface kinase activity of intact tissue culture cells and erythrocytes. The protein, termed pp125, is phosphorylated at serine and threonine residues to a ratio of greater than 1 mol P/mol. The pp125 is an acidic protein (pI 4.4) which also serves as substrate for purified phosvitin/casein kinases but not for cyclic AMP-dependent protein kinases. About 80-fold purification of pp125 was achieved by ion exchange and affinity chromatography. Gel filtration under non-reducing conditions showed that pp125 is part of a complex (Mr 535,000). The pp125 obviously originates from a large plasma protein: the incubation of calf plasma with intact cells in the presence of [gamma-32P]ATP resulted in the labeling of a protein with Mr greater than 300,000 (pp greater than 300). The relationship between pp greater than 300 in plasma and pp125 in serum was demonstrated by cyanogen bromide peptide patterns, and the use of specific anti-serum raised against pp125. Furthermore, it was shown that pp125 is derived from pp greater than 300 during blood clotting.

Animals↗
PubMed1986

Phosphorylation of microsome-bound cytochrome P-450 LM2.

The phosphorylation of a microsomal protein of rabbit liver by catalytic subunit of cyclic AMP-dependent protein kinase was shown, and the protein was identified as cytochrome P-450 LM2 on basis of comparative peptide-mapping. Acid hydrolysis of microsome-bound phosphorylated cytochrome P-450 revealed that phosphorylation occurred exclusively on serine residues. This serine residue was identified as the same residue phosphorylated in purified, soluble P-450, that is, serine in position 128.

Animals↗
PubMed1986

Phosphatase affects microsomal monooxygenase mainly via reductase.

Treatment with alkaline phosphatase of hepatic microsomes prepared from rabbit, rat, and mouse caused a marked decrease of their specific monooxygenase activity (7-ethoxycoumarin-deethylation). This decrease occurred without a significant change in the microsomal content of cytochrome P-450, but with an equally marked decrease of NADPH-cytochrome P-450 reductase (cytochrome c reduction). Thus the phosphatase effect on monooxygenase is mainly due to the inactivation of the reductase.

7-Alkoxycoumarin O-Dealkylase↗
PubMed1985

Protein kinase activities in mammalian blood fluid.

Two protein kinase activities, one specific for phosvitin and another specific for histone, were detected in serum and plasma of calf as well as of human blood after precipitation with ammonium sulfate (40%) and chromatography on DEAE-Sephacel. The enzymes were separated by chromatography on phosphocellulose. The histone kinase is not related to the cyclic AMP-dependent protein kinase; it may derive at least partly from damaged cells. The phosvitin kinase activity carries characteristics of the so called casein kinase type II similar to that present at the surface of cells including blood cells.

Adenosine Triphosphate↗
PubMed1985

Conversion of hepatic microsomal cytochrome P-450 to P-420 upon phosphorylation by cyclic AMP dependent protein kinase.

Cytochrome P-450, purified from liver microsomes of phenobarbital-induced rabbits, was phosphorylated by the catalytic subunit of cyclic AMP-dependent protein kinase. Upon phosphorylation P-450 was found to be converted to its denatured form, P-420, as verified spectroscopically from the CO-bound form of the reduced cytochrome. The conversion was dependent on both kinase and ATP. Thus, cyclic AMP may regulate the biotransformation system through the control of the degradation rate of microsomal P-450 in vivo.

Adenosine Triphosphate↗
PubMed1984

Phosphorylation of rabbit liver cytochrome P-450 LM2 and its effect on monooxygenase activity.

The phosphorylation of rabbit liver microsomal cytochrome P-450 LM2 by catalytic subunit of cyclic AMP-dependent protein kinase (W. Pyerin et al. (1983) Carcinogenesis 4, 573) has now been studied in detail with purified soluble form of cytochrome P-450 as well as with the purified protein incorporated into model membranes. The apparent Km values for P-450 of the phosphorylation reaction in all experimental systems were in a range of 2-8 microM, while the Vmax values were dependent on the state of P-450. Upon phosphorylation, the reconstituted enzyme activities with benzphetamine (N-demethylation) and 7-ethoxycoumarin (O-deethylation) as substrates were reduced to 30-40% of control.

Animals↗
PubMed1983

Substrate-effected release of surface-located protein kinase from intact cells.

Protein kinase activity that is independent of cAMP has been reported to exist on the surface of intact HeLa cells. Here we report that the protein kinase activity can be released by the use of casein or phosvitin within a short period of time. The discharge of the enzyme occurs from intact cells since (i) the cells do not release intracellular material and (ii) the cultures continue to grow within any morphological alteration. As shown with phosvitin, the release of protein kinase depends on substrate concentration, incubation time, and temperature. The degree of inducible release or surface protein kinase is inversely related to cell density. Four incubations with phosvitin (1 mg/ml) are sufficient to liberate most of the enzyme, thus greatly reducing the capacity of the cells to phosphorylate cellular substrates at the surface. Within approximately 24 hr after protein kinase removal, cultures have restored their surface protein kinase. Cultured cells of different origin (rat liver, mouse cerebellum, and human lung) exhibited phosvitin-induced protein kinase release from intact cells. The possible significance of these observations with respect to extracellular protein phosphorylation is discussed.

Caseins↗
PubMed1983

Phosphorylation of cytochrome-P-450-dependent monooxygenase components.

Most chemical carcinogens require activation by polysubstrate monooxygenase. The phosphorylation of essential components of this cytochrome P-450 monooxygenase system, isolated from rabbit liver microsomes, cytochrome P-450 (LM2) and cytochrome reductase, was tested using two different protein kinases. One of the kinases, a cyclic AMP-independent phosvitin kinase (kinase P), was inactive in all systems tested. However, the catalytic subunit of a cyclic AMP-dependent protein kinase (kinase C) catalyzed phosphoryl group transfer to both proteins, but to different extents. Cytochrome P-450 was phosphorylated when added as sole component and also when in the presence of P-450 reductase and phosphatidylcholine. In contrast, the weak phosphorylation of P-450 reductase was reduced considerably in a complete reconstituted system containing P-450 and phosphatidylcholine. The inclusion of kinase P did not alter these results which excludes the possibility that these kinases participate in a sequential phosphorylation mechanism. The monooxygenase constituents themselves were without kinase activity. When hepatic microsomes were isolated in presence of the phosphatase inhibitor sodium fluoride no significant change in monooxygenase (7-ethoxycoumarin O-deethylation) activity was observed, whilst after preincubation with either acid or alkaline phosphatase a significant reduction in monooxygenase activity was measured. Thus, cytochrome P-450 (LM2) is phosphorylatable by protein kinase C and the catalytic activity of polysubstrate monooxygenase decreases after preincubation of microsomes with phosphatases.

Acid Phosphatase↗
PubMed1982

Protein kinase activity and substrates at the surface of intact HeLa cells.

Evidence is presented for the location at the surface of HeLa cells of a protein kinase capable of phosphorylating surface as well as extracellular (foreign) proteins. The reaction products have been found to be proteins containing phosphoryl groups as monoesters of seryl and threonyl residues (but not of tyrosine). The enzyme is of the cyclic AMP-independent type, since neither cyclic AMP nor the heat- and acid-stable inhibitor protein (specific for cyclic AMP-dependent protein kinases) influenced its activity. Further, co-substrate ATP could in part be substituted by GTP, and the spectrum of proteins phosphorylated by the ecto-enzyme differed from that phosphorylated by cyclic AMP-dependent protein kinases. Evidence for the ecto-enzymic nature of this protein kinase includes (a) utilization of co-substrate and location of products at the surface of cells carefully controlled as being in an intact state and (b) phosphorylation of exogenous protein (phosvitin; specific serum proteins) by intact cells. Conclusive proof was gained by qualitative and quantitative comparative studies of phosphorylation in cultures with varying degrees of damaged cells either as a whole or after separation into groups of intact and damaged cells by electronic cell sorting. The results of experiments with cell sonicates excluded the possibility that either enzyme or substrates released from damaged cells were simply adsorbing to the cell surface.

Amino Acids↗
PubMed1982

Assays of cell surface protein kinase: importance of selecting cytophilic substrates.

The ability of phosvitin and histone to serve as substrates for possible protein kinase(s) at the cell surface was examined with regard to their suitability as indicators of such activity. While phosvitin did not interfere with the membrane barrier of HeLa cells, 3T3 and SV 3T3 cells, histone caused severe damage as indicated by both uptake of viability stains Trypan Blue and diamidino-phenylindol and by release of intracellular compounds such as lactate dehydrogenase, cAMP-dependent protein kinase(s), and metabolically prelabelled proteins. Where intactness of the cell membrane is prerequisite for verification of ecto-protein kinase, histone cannot be used as substrate. In contrast, we found that phosvitin is suitable for assays of cell surface located protein kinase activity.

Animals↗
PubMed1980

Evidence for a "mute" catalytic subunit of cyclic AMP-dependent protein kinase from rat muscle and its mode of activation.

An isoenzyme of the catalytic subunit of type II cyclic AMP-dependent protein kinase from rat muscle is reported which coelutes with the classical catalytic subunit but differs from it in isoelectric point (pI 8.7 vs pI 9.1) and is enzymmatically inactive. After reaction with a heat- and acid-stable component of the protein kinase modulator fraction from the same tissue, the "mute" isoenzyme displays a high activity when assayed on isoelectric focusing gels. This activation process does not occur through proteolytic degradation and is not characteristic of a turnover-type reaction. The data imply direct interaction between the isoenzyme and a modulating protein which may subsequently be separated from the enzyme without reversal of the activation. The modulator protein thus appears to act as a template, inducing a conformational change. The implications of such a mute isoenzyme and its control through small modulator proteins are discussed.

Animals↗
PubMed1980

Generation of pyrophosphate from extracellular ATP at the surface of HeLa cells.

On incubation of intact HeLa cells with micromolar concentrations of [gamma 32 P]ATP under physiological conditions, the generation of inorganic pyrophophate in the supernatant has been observed. The reaction depends on incubation time and cell number employed; the enzymatic activity is not shed into the supernatant. Isotope dilution data support that the responsible enzyme(s) is located at the cell surface.

Adenosine Triphosphatases↗
PubMed1979

Isolation of the catalytic subunits of cyclic AMP-dependent protein kinases from different mammalian tissues on the basis of charge differences of their subunits.

Both, the experimental data and a literature survey presented reveal common charge differences in the subunit composition of cAMP-dependent protein kinases from a variety of mammalian tissues. In general, the holoenzymes (type I and II) focus at pH 4.5-5.5, the cAMP-binding regulatory subunit below pH 4, whereas catalytic subunits are found at pH 6.7-9.1. For the purification of the catalytic subunits, the anionic holoenzymes of a variety of rat and rabbit organs as well as mouse and human-derived tissue culture cells were adsorbed to DEAE-cellulose. The catalytic subunit from the particular isoenzyme were selectively eluted at an appropriate ionic strength depending on the isoenzyme elution pattern by small amounts of cAMP. Extraction of tissues with Triton X-100 increased enzyme yield. Improved elution of the enzyme was accomplished by 8-(4-aminobutyl)amino-cAMP instead of cyclic AMP. Carboxymethyl-cellulose chromatography leads to both, concentration and, if necessary, to further purification of the enzyme.

Animals↗