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V Kinzel

Publications and source records attributed to V Kinzel.

At least 55 records · Page 3Linked to original sources
PubMed1992

Accumulation of ester- and ether-linked phosphatidates by HeLa cells in response to ionophore A23187 through activation of phospholipase D.

Phosphatidates seem to play an important role in the control of cell proliferation modified by ligands (M. Kaszkin et al. 1991, Cancer Res. 51, 4328-4335). In this study the potency of calcium ionophore A23187 to alter phosphatidate levels in HeLa cells as a model was studied in detail. HeLa cells prelabeled with [14C]arachidonic acid responded to calcium ionophore A23187 with a rapid accumulation of labeled 1,2-diacylglycerophosphate (acyl-PA) and 2-acyl-1-O-alkylglycerophosphate (alkyl-PA) with a first peak at 5 min and a second increase starting at 20-30 min. In cells prelabeled with [14C]oleic acid the ionophore mobilized relatively more of labeled acyl-PA. The total amount of phosphatidates mobilized was in the order of 0.2 micrograms/10(6) cells, i.e. an almost 10(-4)M concentration. The transphosphatidylation of labeled acyl- and alkyl-PA to 1-butanol in all cases showed that activation of phospholipase D had occurred. The reaction became detectable at 10(-6)M ionophore and was fully expressed at 10(-5)M. Butyl phosphatidate generated during 1 h treatment with ionophore amounted to approx. 0.5 nmol per 10(6) cells (i.e. 10(-4)M conc. within cells) as shown by the use of [14C]butanol. The 3-5-fold rise of the overall phosphatidate level is probably sufficient to alter physically cellular membranes, particularly if the new phosphatidate is restricted to certain compartment(s).

Arachidonic Acid↗
PubMed1991

A proposed common mechanism by which phorbol esters and epidermal growth factor delay the progression from G2 phase to mitosis of HeLa cells through phospholipid metabolites.

The phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA; also called phorbol myristate acetate) and epidermal growth factor both rapidly inhibit the transition of HeLa cells from G2 phase to mitosis in a reversible fashion with characteristics of a receptor-mediated response. It was proposed that an intracellular mediator was responsible for this inhibition. In searching for a common mediator elicited by the action of both ligands, the time course of generation of phospholipid metabolites was compared with the cell cycle response of the G2 cohort monitored by time lapse analysis. The time course and the degree of mobilization of diacylglycerols (DG) effected by TPA and by epidermal growth factor and that of phosphatidic acid correlated to a great extent with the onset and the duration of the G2 inhibition caused by both agonists. Further evidence for the proposed role of DGs and/or phosphatidic acid was obtained by the observation that membrane-penetrating 1,2-DG inhibited HeLa cells in G2 phase directly. The concentration of DG required correlated with that elicited by TPA or epidermal growth factor metabolically. This cellular response to 1,2-DG was also seen in cells after down-regulation of protein kinase C using TPA. The data indicate that cellular DG and/or phosphatidic acid may contribute to a restriction of cells in G2 phase possibly by changing membrane properties.

Arachidonic Acids↗
PubMed1991

Rat C alpha catalytic subunit of the cAMP-dependent protein kinase: cDNA sequence and evidence that it is the only isoform expressed in myoblasts.

A full length cDNA clone encoding the C alpha type catalytic subunit of cAMP-dependent protein kinase was isolated from a cDNA library of differentiated rat myoblast L6 cell line. The 2137 bp clone codes for a protein of 351 amino acid residues having more than 90% sequence identity to C alpha subunits of other mammalians. The C alpha isoform was found to be the only isoform of catalytic subunits expressed in myoblast cells as was determined in Northern blot analysis.

Amino Acid Sequence↗
PubMed1991

A conformational switch is associated with receptor affinity in peptides derived from the CD4-binding domain of gp120 from HIV I.

A 15-residue region within the CD4-binding domain of gp120 from HIV I was identified with use of folding algorithms as conserving the potential for forming a particular secondary structure throughout 11 sequenced HIV strains. The region chosen has a potential for forming both beta-sheet and alpha-helix; the helical form would be amphipathic with the five hydrophobic residues all totally or functionally conserved. Five peptides were synthesized corresponding to this region in strain LAV and the strain most highly divergent from it in primary structure (Z3) plus three additional peptides with critical substitutions in the LAV sequence. The conformation of these five peptides was examined under various conditions with circular dichroism, and the results were compared with the ability of each peptide to bind to a CD4-expressing strain of HeLa cells (HeLa T4). In solution, the unmodified peptides exhibit a bistable structure, existing as beta-sheet in dilute buffer and converting to alpha-helix under more apolar conditions. The transition is reversible and sharp, occurring at a particular point in the polar/apolar gradient with virtually no intermediate state. The ability to undergo this bistable flip is closely associated with binding ability, amino acid substitutions that eliminate binding ability also eliminating the switch, and vice versa. The transition thus may reflect conformational changes occurring in this region of gp120 as it binds to the CD4 receptor.

Algorithms↗
PubMed1991

Isoform C beta 2, an unusual form of the bovine catalytic subunit of cAMP-dependent protein kinase.

The catalytic (C) subunit is the phosphorylating component of the cAMP-dependent protein kinase, a key element in a multitude of hormonally controlled cellular functions. The C-subunit, thought to be a solitary protein until several years ago, is now known to be a group of isoforms comprising as yet C alpha, C beta, and C gamma. We report here the isolation of a full-length cDNA clone coding for a hitherto undiscovered isoform of the bovine C-subunit. The end parts of the 5'-coding region and the 5'-noncoding region of this 3365-base pair clone are unique, whereas the rest of the coding region and the 3'-noncoding region are identical to those of isoform C beta. The clone has therefore been named C beta 2. The deduced amino acid sequence of C beta 2 has a length of 397 amino acid residues and a calculated molecular mass of 46.1 kDa, thus being some 6 kDa higher than that of any known C-subunit. In vitro translation of clone C beta 2 resulted in a single 46-kDa protein. The unique amino-terminal sequence of C beta 2 lacks the usual myristoylation site of C-subunits. It contains a stretch of hydrophobic residues (residues 7-19) and a stretch which may fold into an amphiphilic alpha-helix (residues 16-27) conceivably serving targeting functions. The existence of isoform C beta 2 is confirmed by: (i) the isolation of a second independent C beta 2 clone, (ii) the development of products of expected size and sequence upon amplification from total RNA of various bovine tissues with the polymerase chain reaction using C beta 2-specific primers, and (iii) Northern blots probed with a cDNA fragment containing exclusively C beta 2 sequence. C beta 2 mRNA has a size of 4.4 kilobases and is expressed in various bovine tissues, mainly in heart and brain. Both the size and tissue distribution are indistinguishable from those of C beta mRNA, thus explaining the failure of previous investigations to distinguish it from C beta 2. Southern blotting and polymerase chain reaction with genomic DNA indicate that intron sequence(s) exist at the C beta 2/C beta deviation site (bases 267/268). The deviation site is equivalent to the exon 1/exon 2 splice site of the mouse C-subunit. Since splice sites are highly conserved and since not a single mutation is found downstream of the deviation site, it is tempting to suppose that C beta 2 and C beta are coded by one gene which possesses two alternatively spliced exons 1.

Amino Acid Sequence↗
PubMed1990

Epidermal growth factor inhibits transiently the progression from G2-phase to mitosis: a receptor-mediated phenomenon in various cells.

An immediate effect of epidermal growth factor (EGF) on the cell cycle is described. EGF, when given to replicating cells such as HeLa, A431, and D HER 14, very rapidly inhibits the transition from G2 phase to mitosis (M) in a transient fashion. The influence of EGF (10(-10) to 10(-7) M concentrations) on the G2-M transition of individual cells has been analyzed by time-lapse photography in cell lines carrying intact, mutated, or no EGF receptor. The G2-M transition of cells devoid of EGF receptor or carrying an EGF receptor devoid of most of the cytoplasmic domain was not influenced by EGF. In cells carrying intact EGF receptor, EGF caused a transient and dose-dependent delay in G2 phase which could last for greater than 2 h. Cells were inhibited in G2 within less than 10-20 min prior to prophase. A parasynchronous recovery from G2 inhibition was observed at large EGF concentrations; the G2-M transition rate exceeded that of the controls. The system described may represent a model for the mechanistic analysis of a ligand-induced transient restriction of the cell cycle, particularly at the G2-M border.

Animals↗
PubMed1990

Severe chromosomal damage in mammalian cells induced by pulse labeling with small amounts of [3H]thymidine.

Pulse labeling of cultured mammalian cells with tritiated thymidine ([3H]TdR; different specific activities, 2-47 Ci/mmole) for 30 min at low concentrations of radioactivity (0.2-2 microCi/ml) causes an unusually high degree of chromosomal aberrations which becomes fully evident in the cohort of cells analyzed 8 h later. The effect depends on the total amount of radioactivity. In HeLa cells up to 93% aberrant metaphases with up to 3.6 aberrations per metaphase were observed; in CHO cells the corresponding figures are 62% aberrant metaphases with 1 aberration per metaphase. The results may be valuable (i) for users of the pulse-labeling technique for analysis of cell-cycle kinetics, as this shows the technique as such may delay the labeled cohort of cells and therefore interferes with the matter under study and (ii) for risk assessment.

Animals↗
PubMed1990

Distribution of phorbol ester TPA-induced structural chromosomal aberrations in HeLa cells.

In order to gain further insight into the mechanism of clastogenic action of the phorbol ester 12-O-tetradecanoyl-phorbol-13-acetate (TPA), the localization and distribution of structural chromosomal aberrations on HeLa chromosomes was investigated. We have shown by the G-banding technique that TPA-induced chromosomal aberrations in HeLa cells have practically the same distribution as that occurring spontaneously in control cultures. About two-thirds of the breakpoints are located in areas of known fragile sites; about one-third is located in regions near known oncogenes. The tumor promoter TPA thus appears to enhance pre-existing mechanisms of an 'endogeneous' clastogenesis.

Chromosome Aberrations↗
PubMed1990

Cytogenetic effects of phorbol ester tumor promoters: possible role in multistep tumorigenesis.

The conversion step of multistage tumorigenesis in mouse skin effected by TPA (12-O-tetradecanoylphorbol-13-acetate) but not by RPA (12-O-retinoylphorbol-13-acetate) may be best explained by the clastogenic (and mitogenic) activity of TPA. In mouse keratinocytes in vitro as well as in vivo TPA (but not RPA) was shown earlier to induce a significant degree of chromosomal aberrations. Details of the clastogenic action of TPA were studied in HeLa cells. Only TPA exerted a significant clastogenic activity at non-cytotoxic concentrations (10(-8) to 10(-6) M) measured after 24 and 48 hrs. Values obtained with RPA were close to those obtained in the presence of solvent (acetone 0.2%). The response to TPA was only to some extent correlated with the dose. Chromosomal aberrations including gaps and breaks were predominantly of the chromatid type; they are first measurable after 6-8 hrs, i.e. as soon as the cells recover from TPA-induced G2-inhibition. A 30 min exposure to TPA (10(-7) M) is sufficient to induce aberrations. The data point to an indirect, possibly receptor-mediated action of TPA. Supposing that TPA-induced chromosomal lesions represent a key event required for conversion, it is suggested that the absolute requirement for DNA replication in the conversion step is necessary to "fix" a certain degree of chromosomal damage. Therefore, the clastogenic as well as the mitogenic activity of TPA appear to be necessary for effecting conversion.

Chromosome Aberrations↗
PubMed1989

Conformational analysis of PKI(5-22)amide, the active inhibitory fragment of the inhibitor protein of the cyclic AMP-dependent protein kinase.

Fourier-transform i.r. spectroscopy, 1H-n.m.r. spectroscopy and X-ray scattering were used to study the conformation and shape of the peptide PKI(5-22)amide, which contains the active site of the inhibitor protein of the cyclic AMP-dependent protein kinase [Cheng, Van Pattern, Smith & Walsh (1985) Biochem. J. 231, 655-661]. The X-ray-scattering solution studies show that the peptide has a compact structure with Rg 0.9 nm (9.0 A) and a linear maximum dimension of 2.5 nm (25A). Compatible with this, Fourier-transform i.r. and n.m.r. determinations indicate that the peptide contains approx. 26% alpha-helix located in the N-terminal one-third of the molecule. This region contains the phenylalanine residue that is one essential recognition determinant for high-affinity binding to the protein kinase catalytic site.

Amides↗
PubMed1989

Phosphorylation by cell surface protein kinase of bovine and human fibrinogen and fibrin.

Human and bovine fibrinogen as well as fibrin are shown to be phosphorylated by Co631 (monolayer, hamster) and RPL12 (suspension, chicken) cells by their surface protein kinase of the casein kinase II type. The phosphate label is introduced into the alpha-peptide. The kinase system phosphorylates serine residues and utilizes GTP equally well as ATP. The participation of intact cell surfaces indicates the possibility of phosphorylation of extracellular fibrinogen independently of the site of its biosynthesis.

Animals↗
PubMed1989

Evidence for ecto-protein kinase activity that phosphorylates Kemptide in a cyclic AMP-dependent mode.

The heptapeptide Leu-Arg-Arg-Ala-Ser-Leu-Gly (Kemptide) is a synthetic construct of a substrate for cAMP-dependent protein kinase (PK). In this work we show that Kemptide has all the properties of a cytophilic substrate, i.e. it is a molecule preserving cell membrane intactness when added to cultured cells. Kemptide thus satisfies the prerequisites for employment in assays for cell surface-located ecto-PK activity. Different types of intact cells catalyze the phosphorylation of Kemptide in the presence of extracellular ATP and cAMP with Km values of 3-4 microM for Kemptide. Kemptide phosphorylation was influenced by PKI, the inhibitory protein specific for cAMP-PK. The results of comparative experiments with intact cells and with cell extracts demonstrate the ectoenzyme nature of this cAMP-PK. Further, the possibility was ruled out of a transfer of enzyme activity from damaged cells to the surface of intact cells. The anchorage of the surface cAMP-PK activity to the plasma membrane appears to be relatively stable since (i) cell supernatants, obtained after preincubation of intact cells with cAMP or Kemptide, did not show Kemptide phosphorylation, and (ii) the cAMP-dependent PK activity remained with cells even after five consecutive washes with cAMP or Kemptide. This is in contrast to the ecto-cAMP-independent phosvitin/casein type PK (Kübler, D., Pyerin, W., Burow, E., and Kinzel, V. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4021-4025) which is released from intact cells through the addition of substrate. Data are presented which show that both ectokinase activities are exhibited independently. In conjunction with published evidence for an active export of cAMP from cells as well as for the appearance of extracellular ATP the demonstration of an ecto-cAMP-PK further supports the potential of PK for intercellular regulation. The potential of ecto-cAMP-PK is demonstrated by its ability to phosphorylate biologically active forms of atrial natriuretic peptide, the atrial natriuretic peptide, which possesses the specific sequence for a cAMP-PK-catalyzed phosphorylation.

Amino Acid Sequence↗
PubMed1989

Catalytic subunit of cAMP-dependent protein kinase from bovine heart: several isoforms demonstrated by high resolution focusing in immobilized pH gradient.

The catalytic subunit (C) of cAMP-dependent protein kinase holoenzyme type II from bovine cardiac muscle was separated by isoelectric focusing in Immobiline polyacrylamide gels into 9 protein forms. The major forms (i) appeared at pH 7.1, 7.4, 7.5, and 7.7, (ii) exhibited protein kinase activity and were inhibited by heat and acid stable inhibitor, (iii) represented approx. 30%, 4%, 64%, and 1% of the protein respectively, (iv) refocused in the same position from which they had been eluted from the first gel. Antibodies against C detected additional proteins at approx. pH 7.55, 7.75, and 7.8. Two more bands became detectable at approx. pH 7.3 and 7.45 by application of antibody against C beta (Uhler, M.D. & McKnight G.S. 1987, J.Biol.Chem. 262, 15202-15207). The relation of the different forms of C to the fractions CA and CB (Kinzel V. et al. 1987 Arch. Biochem. Biophys. 253, 341-349) is demonstrated.

Acrylamides↗
PubMed1989

Cytogenetic effects caused by phorbol ester tumor promoters in HeLa cells: mechanistic aspects.

The effect of the convertogenic ('first-stage') tumor promoter TPA (12-O-tetradecanoylphorbol-13-acetate) and the non-convertogenic ('second-stage') tumor promoter RPA (12-O-retinoylphorbol-13-acetate) on chromosomes was investigated in HeLa cells which have previously been shown to exhibit a radiomimetic response to TPA. As in the case of mouse keratinocytes, only TPA had a significant clastogenic activity at non-cytotoxic concentrations ranging from 10(-8) to 10(-6) M measured after 24 and 48 h. The values observed with RPA did not differ significantly from values observed in the presence of the solvent (acetone, 0.2%). The response to TPA was saturable with respect to the dose of TPA. The chromosomal aberrations (mostly gaps and breaks) were predominantly of the chromatid type. Isochromatid aberrations were caused by a 24 or 48 h treatment with 10(-6) M TPA. The aberrations appear as early as 6-8 h after TPA application, i.e. as soon as the cells have recovered from TPA-induced inhibition in G2-phase. Even a 30 min exposure to 10(-7) M TPA gives the same yield of aberrations as longer treatment, i.e. the response to TPA is 'saturable' with respect to time. Both TPA and the non-clastogenic RPA cause a temporal G2-delay thus indicating that the G2-inhibition is not related to induction of chromosomal aberrations by TPA. The data are consistent with the hypothesis that TPA induces chromosomal aberrations via a receptor-mediated pathway.

Carcinogens↗
PubMed1988

The inhibitor protein of the cyclic AMP-dependent protein kinase-catalytic subunit interaction. Composition of multiple complexes.

It has been previously demonstrated that the combination of pure preparations of the inhibitor protein of the cyclic AMP-dependent protein kinase and the catalytic subunit of this enzyme resulted in the formation of multiple complexes [Van Patten, Fletcher & Walsh (1986) J. Biol. Chem. 261, 5514-5523]. In the present study it is demonstrated that these multiple species occur because the bovine heart protein kinase preparation contains multiple forms of catalytic subunit [Kinzel, Hotz, König, Gagelmann, Pyerin, Reed, Köbler, Hofmann, Obst, Gensheimer, Goldblatt & Shaltiel (1987) Arch. Biochem. Biophys. 253, 341-349].

Adenosine Triphosphate↗
PubMed1988

Secondary structure of the Arg-Gly-Asp recognition site in proteins involved in cell-surface adhesion. Evidence for the occurrence of nested beta-bends in the model hexapeptide GRGDSP.

The primary sequence Arg-Gly-Asp has been found in a number of proteins which bind to cell surface receptors. Studies with synthetic peptides have shown that the presence of charged side chains alone is not sufficient to confer binding activity. Application of folding algorithms to proteins and peptides having similar sequences indicates that binding activity is strongly correlated with the presence of two or more closely spaced residues that each have a high probability of initiating a beta-bend. Circular dichroic studies on the hexapeptide GRGDSP, whose sequence is contained in fibronectin and which also shows binding activity, demonstrate that it adopts an unusual conformation in aqueous solution. 1H-NMR spectra of the peptide in aqueous solution show that the two amide hydrogens of Asp4 and Ser5 exchange very slowly. Computer-assisted modeling using restrained molecular dynamics and energy minimization results in conformations that include two beta-bends of type III-III or III-I (hydrogen bonds 4----1 and 5----2), fully consistent with constraints imposed by 1H- and 13C-NMR data. It is suggested that this unusual secondary structure provides an additional specificity determinant.

Amino Acid Sequence↗
PubMed1988

Dual effect of the phorbol ester TPA on arachidonic acid release from HeLa cells.

The tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) induces release of arachidonic acid (AA) from HeLa cells with a maximum at 2-3 h. Subsequently the extracellular level of AA decreases. Cycloheximide (CH, 10(-5) M does not influence the release of AA, however, it causes the AA level to remain elevated. In the presence of TPA and CH (i) re-uptake of AA is not altered, (ii) re-incorporation of AA into phosphatidylinositol (and phosphatidylethanolamine) is largely increased, and (iii) the level of lysophosphatidylinositol is elevated. The latter two phenomena can be prevented by fluocinolone acetonide (10(-8) M), i.e. by inhibition of phospholipase A2 (PLA2). These data point to a continuously elevated PLA2 activity in the presence of TPA and CH. The phorbol ester appears to induce a proteinaceous principle which diminishes PLA2 activity.

Arachidonic Acid↗