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PubMed1978

Human erythrocyte proteins associated with adenosine 3',5'-cyclic monophosphate action.

Two adenosine 3',5'-cyclic monophosphate (cyclic-AMP)-binding protein factors (molecular weight 230,000) have been partially purified from human erythrocytes. One of these proteins seems to be different from the cyclic-AMP-binding component of the cyclic-AMP-dependent protein kinases. These protein factors are also capable of binding adenosine. We present data also on two forms of cyclic-AMP-dependent protein kinases (ATP: protein phosphotransferase, EC 2.7.1.37) partially purified from the cytosol of normal human erythrocytes. Kinase I has been classified as type I enzyme on the basis of its activation when preincubated with protamine, histone or NaCl. The substrate specificities of the two kinases and many of their kinetic parameters are rather similar. Their subunit structure is reminiscent of that of kinases obtained from other sources. The catalytic subunit of both enzymes reversibly cross-react with the regulatory subunit of kinase I from the rabbit red blood cell.

Adenosine↗
PubMed1984

Immunological and molecular characterization of the cAMP-dependent protein kinases in AtT20 cells.

The properties of the cAMP-dependent protein kinases in AtT20 mouse pituitary tumor cells were characterized by a combination of immunological and biochemical techniques. Ninety per cent of the total cAMP-dependent protein kinase was in the 40,000 X g supernatant fraction. Protein kinases I and II were immunoprecipitated with specific antisera directed against their regulatory subunits. The immunoprecipitated kinases bound [3H]cAMP and were catalytically active when incubated with [gamma-32P]ATP-Mg and protamine or histone H2B. Immunoprecipitated protein kinases I and II bound [3H]cAMP with apparent Kb values of 1.5 and 15 nM, respectively. Regulatory subunit concentrations in AtT20 cells were measured by immunoprecipitation of [3H]cAMP-R complexes. R-I and R-II levels were 2.7 and 3.0 pmol of [3H]cAMP binding activity per mg of cytosolic protein, respectively, however, the ratio of protein kinase II to protein kinase I was 2.5 indicating the presence of a significant amount of free R-I. This was confirmed by DEAE-cellulose chromatography and the isolation of immunoreactive R-I devoid of protein kinase activity. A significant amount of R-I also coeluted with protein kinase II when AtT20 cell extracts were subjected to DEAE-cellulose chromatography. In quantitative immunoprecipitation experiments, 0.1 microliter of anti-brain R-II serum complexed up to 0.5 pmol of the [3H]cAMP-binding activity of protein kinase II prepared from bovine and rat brain, and AtT20 cells while 2 microliter of anti-brain R-II serum was required to precipitate an equal amount of protein kinase II from bovine skeletal muscle showing that the protein kinase II in AtT20 cells contained the neural-specific R-II subunit.

Affinity Labels↗
PubMed1981

Nuclear protein kinases of GH3 cells.

Three distinct cAMP-independent nuclear protein kinase activities (PCV, PC-II and PC-III) have been separated by phosphocellulose chromatography of GH3 nuclear NaCl extracts. The kinase activities exhibit distinct substrate specificities and preference for either the basic substrates protamine sulfate and histones H1 and H3 (kinase activity PCV) or the acidic protein substrates casein (PC-II) and phosvitin (PC-III). The three nuclear protein kinases further differ in their estimated molecular weights, apparent Km values and in their sensitivity toward NaCl. All three kinases require Mg2+ for optimal activity.

Animals↗
PubMed1984

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity.

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.

Animals↗
PubMedDate not supplied

Purification and characterization of a protein kinase from goat sperm plasma membrane.

A protein kinase that causes phosphorylation of serine and threonine residues of casein has been partially purified from goat cauda-epididymal sperm plasma membrane and characterized. The kinase, solubilized from the membrane with 1.0% Triton X-100, was purified to 480-fold by using DEAE-cellulose and casein-Sepharose affinity chromatographic techniques. The kinase is a strongly basic protein with pI of 9.5. The enzyme has a molecular mass of 310 kilodaltons as estimated by Sephacryl S-300 gel exclusion. The kinase showed affinity for protein substrates in the order membrane proteins > casein > phosvitin > histone > protamine. The apparent Km values of the kinase for casein and membrane proteins were 1 and 0.15 mg/mL, respectively. The synthetic peptides Kemptide and poly(Glu80Tyr20) did not serve as substrates of the enzyme. ATP, rather than GTP or PP(i), is the donor of phosphate for the phosphorylation reaction. Cyclic AMP and GMP, NaCl (0.25 M), KCl (0.25 M), Ca2+, calmodulin, phosphatidylserine, and muscle protein kinase inhibitor had no appreciable effect on the kinase activity. Heparin (0.5 microgram/mL) showed high affinity for inhibiting only 40% of the kinase activity, whereas polyamines at a relatively high concentration (5 mM) inhibited 40-50% of the enzymic activity. The kinase appears to be distinct from other protein kinases including casein kinases. The activity of the kinase derived from the purified sperm plasma membrane was markedly (approximately 90%) lost when the intact spermatozoa were pretreated with diazonium salt of sulfanilic acid, a membrane nonpenetrating surface probe.(ABSTRACT TRUNCATED AT 250 WORDS)

4-Chloromercuribenzenesulfonate↗
PubMed1985

Basic polyaminoacids and protamine stimulate enzyme release from frog muscles.

Polylysines, polyornithine, and protamine increased the release of creatine kinase (CK) and lactate dehydrogenase (LDH) from frog muscles. With 5 micrograms/ml (50 nM) of a polylysine of molecular weight 100,000, enzyme release reached peak values (ten-fold above control) within 30 minutes; subsequent removal of the polylysine did not reduce significantly the release rates. The effectiveness of the polycations increased with the degree of polymerization. Their effects were prevented by pretreatment of the muscles with polyanions (heparin or polyglutamic acids) and were reversed when heparin was applied within 30 minutes of exposure to polylysine. The initial stimulation of enzyme release by polylysine was attenuated by Ca-free solutions and by low temperature (4 degrees C). It is suggested that, while fiber damage can explain the increased loss of sarcoplasmic enzymes after prolonged treatment of frog muscles with polycations, other mechanisms (perhaps exocytosis) are involved in the stimulation of CK and LDH release during brief exposures to nanomolar concentrations of polylysines or polyornithine.

Animals↗
PubMed1984

Protein kinase and its endogenous substrates in coated vesicles.

Coated vesicles prepared from bovine brains contained a protein kinase activity which catalyzed the phosphorylation of endogenous structural proteins, Mr 150 000, 120 000, 48 000 and 32 000. An endogenous protein, Mr 48 000 was most strongly phosphorylated by this kinase. This protein kinase also phosphorylated exogenous proteins, phosvitin intensely and casein slightly but not histone or protamine. The enzyme activity was independent of cyclic nucleotides or Ca2+/calmodulin. Mg2+ stimulated the kinase activity. Some divalent cations were substituted for Mg2+; the potency decreased in the order Mn2+, Mg2+, Co2+, Ca2+, Zn2+. Two separate subfractions, the outer coat and the inner vesicle (core), were prepared from coated vesicles by a urea treatment followed by sucrose density gradient centrifugation and dialysis. The kinase activity was found predominantly in the coat subfraction.

Animals↗
PubMed1993

Molecular cloning of two new interferon-induced, highly related nuclear phosphoproteins.

During the molecular cloning of the human dsRNA activated-p68 kinase (PKR), polyclonal antibodies against PKR selected, in addition to cDNAs corresponding to PKR, another cDNA presenting only slight homology with PKR cDNA. This cDNA recognized an mRNA species of 2 kilobases induced by both alpha- and gamma-interferons. Its transcription did not require protein synthesis. On further library screening, it selected two highly related cDNAs, referred to as 75 and 41, displaying perfect homology over 612 base pairs and divergent at both ends. In addition, cDNA 75 presents an insertion of 150 base pairs highly homologous to a region common to both sequences. The 75 and 41 peptidic sequences are very hydrophilic, rich in basic amino acid residues, and contain several potential phosphorylation sites for different serine/threonine kinases. Furthermore, they present two protamine- and histone-like nuclear targeting sequences as well as some homology with helix-loop-helix motifs of some DNA-binding proteins. The 75-encoded product, which resolved as a 52-kDa protein after in vitro expression in rabbit reticulocyte lysates, was found to migrate as a 65-67-kDa protein after in vivo expression in insect cells. In accord with sequence data, this 65-67-kDa protein was found to be phosphorylated in vivo in the insect cells and was recovered from the membrane/nuclear pellet. In contrast, the 41-encoded product (30-kDa protein in reticulocyte lysates) could not be expressed in vivo, as it provoked a rapid and severe shut-off of protein synthesis in insect cells. The function of the 75 and 41 proteins and their relation to PKR remains to be determined. However, the presence of nuclear targeting sequences, phosphorylation sites, and helix-loop-helix motif is consistent with a role of these proteins in the mechanism of transduction of the interferon action.

Amino Acid Sequence↗
PubMed1991

The substrate specificity of protein kinases which phosphorylate the alpha subunit of eukaryotic initiation factor 2.

The alpha subunit of eukaryotic protein synthesis initiation factor (eIF-2 alpha) is phosphorylated at a single serine residue (Ser51) by two distinct and well-characterized protein kinase, the haem-controlled repressor (HCR) and the double-stranded RNA-activated inhibitor (dsI). The sequence adjacent to Ser51 is rich in basic residues (Ser51-Arg-Arg-Arg-Ile-Arg) suggesting that they may be important in the substrate specificity of the two kinases, as is the case for several other protein kinases. A number of proteins and synthetic peptides containing clusters of basic residues were tested as substrates for HCR and dsI. Both kinases were able to phosphorylate histones and protamines ar multiple sites as judged by two-dimensional mapping of the tryptic phosphopeptides. These data also showed that the specificities of the two kinases were different from one another and from the specificities of two other protein kinases which recognise basic residues, cAMP-dependent protein kinase and protein kinase C. In histones, HCR phosphorylated only serine residues while dsI phosphorylated serine and threonine. Based on phosphoamino acid analyses and gel filtration of tryptic fragments, dsI was capable of phosphorylating both 'sites' in clupeine Y1 and salmine A1, whereas HCR acted only on the N-terminal cluster of serines in these protamines. The specificities of HCR and dsI were further studied using synthetic peptides with differing configurations of basic residues. Both kinases phosphorylated peptides containing C-terminal clusters of arginines on the 'target' serine residue, provided that they were present at positions +3 and/or +4 relative to Ser51. However, peptides containing only N-terminal basic residues were poor and very poor substrates for dsI and HCR, respectively. These findings are consistent with the disposition of basic residues near the phosphorylation site in eIF-2 alpha and show that the specificities of HCR and dsI differ from other protein kinases whose specificities have been studied.

Amino Acid Sequence↗
PubMed1991

Structure and organization of the mouse acrosin gene.

The genomic region carrying the mouse acrosin gene, including the 5'-flanking sequence, has been isolated and characterized. The acrosin gene consists of five exons separated by four introns. Organization of this gene is very similar to those of the genes for other typical serine proteases, except for the phase class of the first intron. Riboprobe mapping and primer extension analyses showed that the start site of transcription initiation in the acrosin gene is heterogeneous, including three major sites. Thus, the structure and organization of the mouse acrosin gene are different from those of the human gene [Keime, S., Adham, I.M., & Engel, W. (1990) Eur. J. Biochem. 190, 195-200] in two respects: the number of transcription initiation sites and the phase class of the third intron. The putative promoter regions of the mouse and human acrosin genes lack typical sequences of TATA, CAAT, and GC boxes, but contain a consensus sequence, GGGTGGG, known to be specific for the phosphoglycerate kinase-2 gene, and the protamine-1 and 2 genes that are uniquely expressed during spermatogenesis.

Acrosin↗
PubMed1986

Specificity of protein phosphatases in the dephosphorylation of protein kinase C.

Protein kinase C can autophosphorylate in vitro and has also been shown to be phosphorylated in vivo. In order to investigate the factors that may determine the phosphorylation state of protein kinase C in vivo, we determined the ability of the ATP + Mg2+-dependent phosphatase and the polycation-stimulated (PCS) phosphatases to dephosphorylate protein kinase C in vitro. These studies show that all the oligomeric forms of the PCS phosphatases (PCSH1, PCSH2, PCSM and PCSL phosphatases) are effective in the dephosphorylation of protein kinase C, showing 34-82% of the activity displayed with phosphorylase a as substrate. In contrast both the catalytic subunit of the PCS phosphatase and that of the ATP+Mg2+-dependent phosphatase showed only weak activity with protein kinase C as substrate. All these phosphatases, however, were activated by protamine (Ka 14-16 micrograms/ml) through what appears to be a substrate-directed effect. The relative role of these phosphatases in the control of protein kinase C is discussed.

Adenosine Triphosphate↗
PubMedDate not supplied

[Study of mechanisms of regulation of disulphide reductase in mouse liver].

An increase in activity of disulphide reductase system (DRS) in supernatant of liver tissue was caused by 3',5'-AMP, ATP, GTP, UTP, Mg2+, Ca2+, EDTA, protamine, noradrenaline and F-. The effect was connected with arsenite resistant fraction of DRS. After rapid homogenization the effect of noradrenaline disappeared and the effects of ATP, GTP, UTP and Ca2+ were distinctly decreased. Treatment with adsorbents prevented the effects of 3',5,-AMP, ATP and EDTA and markedly decreased the effects of protamine and Mg2+. A protein inhibitor of protein kinase prevented completely the activation of DRS with 3',5'-AMP, ATP, GTP, UTP and noradrenaline and distinctly decreased the effect of protamine, Mg2+ and Ca2+ but did not alter the influence of EDTA. Addition of 2',3'-AMP blocked the effect of 3',5-AMP, ATP and Mg2+ but did not influence the effect of protamine and EDTA. The data obtained suggest that protein kinase participated in activation of DRS by most of the regulators.

Adenine Nucleotides↗
PubMed1985

The protein phosphatases involved in cellular regulation. 1. Modulation of protein phosphatases-1 and 2A by histone H1, protamine, polylysine and heparin.

The phosphorylase phosphatases in rat and rabbit liver cytosol that are markedly stimulated by histone H1, protamine and polylysine were identified as protein phosphatases-2A0, 2A1 and 2A2 by anion-exchange chromatography, gel-filtration and immunotitration experiments. Histone H1 and protamine also stimulated the dephosphorylation of phosphorylase kinase, glycogen synthase, fructose-1,6-bisphosphatase, pyruvate kinase, acetyl-CoA carboxylase and phenylalanine hydroxylase by phosphatases-2A1 and 2A2, and with several of these substrates activation was even more striking (20-100-fold) than that observed with phosphorylase (approximately 5-fold). Activation by basic polypeptides did not involve dissociation of these phosphatases to the free catalytic subunit. The dephosphorylation of phosphorylase by protein phosphatase-1 was suppressed by basic polypeptides, protamine and polylysine being the most potent inhibitors. However, the dephosphorylation of glycogen synthase, pyruvate kinase and acetyl-CoA carboxylase were markedly stimulated by histone H1 and protamine (2-13-fold). Consequently, with the appropriate substrates, protein phosphatase-1 can also be regarded as a basic-polypeptide-activated protein phosphatase. Heparin stimulated (1.5-2-fold) the dephosphorylation of phosphorylase by phosphatases-2A0 and 2A1, provided that Mn2+ was present, but phosphatase-2A2 and the free catalytic subunit of phosphatase-2A were unaffected. Heparin, in conjunction with Mn2+, also stimulated (1.5-fold) the dephosphorylation of glycogen synthase (labelled in sites 3 abc), phosphorylase kinase and phenylalanine hydroxylase by phosphatase-2A1, but not by phosphatase-2A2. By contrast, the dephosphorylation of phosphorylase and phosphorylase kinase by protein phosphatase-1 was inhibited by heparin. However, dephosphorylation of glycogen synthase and pyruvate kinase by phosphatase-1 was stimulated by this mucopolysaccharide. The studies demonstrate that basic proteins can be used to distinguish protein phosphatase-1 from protein phosphatase-2A, but only if phosphorylase is employed as substrate. Optimal differentiation of the two phosphatases is observed at 30 micrograms/ml protamine or at heparin concentrations greater than 150 microM.

Animals↗
PubMed1992

Alteration of the kinetic properties of the epidermal growth factor receptor tyrosine kinase by basic proteins.

Previous studies have shown that lysine- and arginine-rich proteins can enhance the activity of tyrosine and serine/threonine protein kinases. However, the kinetics and mechanism of this activation are not fully understood. Therefore we investigated the ability of poly(amino acids) and the arginine-rich protein, protamine, to alter the kinetic properties of epidermal growth factor (EGF) receptor protein-tyrosine kinase activity using immunoaffinity-purified receptor isolated from human epidermoid carcinoma (A431) cells. Poly(L-lysine), poly(L-arginine) and protamine stimulated EGF receptor kinase activity by 3-5-fold at non-saturating doses of ATP and peptide substrate, while poly(L-glutamate) had no effect. Initial kinetic studies demonstrated an increase in the maximum velocity and a decrease in the apparent Km for the peptide substrate angiotensin II in the presence of the basic effectors. Further analysis of the kinetic mechanism by product inhibition revealed that protamine altered the pattern of ADP inhibition towards the peptide substrate but not towards ATP. The change was indicative of the receptor's ability to form an enzyme-angiotensin II-ADP ternary complex in the presence of protamine but not in its absence. In addition, the basic effectors had a substantially decreased influence on the kinase activity of a C-terminally truncated form of the EGF receptor. Thus the changes in kinase activity may be partially mediated by the C-terminal region of the receptor, which contains the sites of receptor self-phosphorylation. These results suggest that the basic domains of proteins can interact with the EGF receptor to induce changes in its kinetic properties, especially with regard to reactant recognition and binding.

Amino Acid Sequence↗
PubMed1990

Control of src kinase activity by activators, inhibitors, and substrate chaperones.

The activities of src tyrosine kinases are greatly influenced by substrate modulators (chaperones). In the presence of bovine serum albumin, the phosphorylation of a random polymer of glutamic acid, alanine, and tyrosine (1:1:1) by src kinases is stimulated 20- to 100-fold, but there is little stimulation with a polymer of glutamic acid and tyrosine (4:1) as substrate. This suggests that serum albumin interacts with the substrates rather than with the enzyme. groEL and several other heat shock proteins also stimulate the phosphorylation of a random polymer of glutamic acid, alanine, and tyrosine (1:1:1). In the absence of substrate modulators, the phosphorylation of calmodulin and of several ras proteins by src kinase is barely detectable. In the presence of polylysine or protamine, marked phosphorylation is observed. Another type of control of src kinase activities appears to be directed toward the enzyme rather than the substrate. Triton X-100 extracts of plasma membranes of bovine brain contain a heat-stable factor that stimulates c-src kinase activity with any of the polymers as substrate. The same extract contains a heat-labile factor that preferentially inhibits c-src kinase activity. The two factors are separated by DEAE-Sephacel and phosphocellulose chromatography. The presence of the activator enhances the potency of the inhibitor.

Animals↗
PubMed1985

Partial purification and characterization of a new phosphoprotein kinase from cells infected with pseudorabies virus.

Cytoplasmic fractions from normal baby hamster kidney fibroblasts and from fibroblasts infected with pseudorabies virus were fractionated by DEAE-cellulose chromatography and fractions assayed for protein kinase activity. In preparations from uninfected and infected cells protein kinase activities identified as casein kinase I and II, the two isoforms of the cyclic-AMP-dependent protein kinase, protein kinase C, and a presumed proteolytic fragment of protein kinase C were present in comparable amounts. However in infected cells a new protein kinase activity was detected, appearing about 4 h after infection and increasing during the following 6 h at least. This new protein kinase was purified 100-fold by high-performance gel-permeation and ion-exchange chromatography, and characterized. It has an apparent relative molecular mass of 68 000 on the basis of gel-permeation chromatography, and a sedimentation coefficient of 4.3 S. It catalysed the phosphorylation of serine residues of basic proteins in vitro, with protamine a better substrate than mixed histones; and used ATP (apparent Km = 60 microM), but not GTP, as phosphoryl donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, Ca2+ + calmodulin, Ca2+ + phospholipid, double-stranded RNA, and heparin) did not significantly alter the activity of this enzyme. A distinguishing characteristic of the protein kinase was a high KCl concentration optimum with the persistence of activity up to 800 mM KCl, at least.

Animals↗
PubMed2000

Expression pattern of germ cell-specific genes in the testis of patients with nonobstructive azoospermia: usefulness as a molecular marker to predict the presence of testicular sperm.

OBJECTIVE: To analyze the expression pattern of testis-specific genes of patients with various spermatogenic defects and their usefulness as a molecular marker to predict the presence of testicular spermatozoa in patients with nonobstructive azoospermia undergoing IVF. DESIGN: Prospective, controlled study. SETTING: Hospital-based infertility research laboratory. PATIENT(S): Fifty-eight men with azoospermia or severe oligozoospermia. INTERVENTION(S): Testicular biopsy was done in the patients with obstructive or nonobstructive azoospermia, including Sertoli cell-only syndrome, maturation arrest, severe hypospermatogenesis, and normal spermatogenesis. MAIN OUTCOME MEASURE(S): Reverse transcriptase polymerase chain reaction was performed using 1 microgram of total RNA extracted from testicular tissues. Three pairs of primers were used for amplification of male germ cell-specific genes (DAZ, transcribed in male germ cells; PGK2, in late spermatocytes and spermatids; protamine-2, in spermatids) as molecular markers. Testicular sperm was obtained by multiple testicular sperm extraction. RESULT(S): The DAZ, PGK2, and protamine-2 genes were expressed in 38, 30, and 21 of the 43 patients with nonobstructive azoospermia, respectively. Testicular spermatozoa were successfully extracted in 4 of 43 patients with nonobstructive azoospermia with the use of multiple testicular sperm extraction. Detection of protamine-2 transcripts predicted the presence or absence of spermatozoa in the testicular tissue in 39 of 43 patients (91%). CONCLUSION(S): Expression of the protamine-2 gene may be a useful molecular marker to predict the presence of testicular sperm in patients with nonobstructive azoospermia.

Adult↗