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O H Martini

Publications and source records attributed to O H Martini.

17 recordsLinked to original sources
PubMed1989

Mitogen-responsive S6 kinase.

Many cell lines respond to mitogenic stimuli (serum, growth factors) with rapid phosphorylation of the ribosomal protein S6 at several serine sites. We have tried to identify the protein kinase(s) mediating this effect of growth stimuli. Examining post-DEAE chromatography fractions of S49 kin- cell extracts, we could detect a highly active effector-independent S6 kinase with specificity for serine residues. The study was extended to the presumably homologous human enzyme, using HeLa S3 cells as model system. Activity yields increased up to sevenfold when exhausted HeLa cells were supplied with fresh medium plus serum. The enzyme uses ATP, not GTP, as cosubstrate, 40-S or 80-S (reassociated from subunits) ribosomal particles being substrate. The optimal K+ concentration, measured at 3 mM Mg2+, is 35 mM. Under optimized assay conditions S6 phosphorylation proceeded faster in vitro than it appeared to do in vivo. The apparent Mr of the enzyme, as estimated by gel filtration on Sephadex G-100, is 56,000 (determination in the presence of 200 mM KCl in 25 mM phosphate buffer). Tighter binding to DEAE-Sephacel and higher specificity for S6 distinguishes this enzyme from the following S6-phosphorylating protein kinases: protein kinase C, protease-activated kinase II, histone-4 phosphotransferase and an enzyme with the properties of casein kinase I. In published summaries of observations shown here and in a follow-up study with chick embryo fibroblasts, the enzyme(s) has been referred to as mitogen-responsive S6 kinase(s) [Martini, O. H. W. and Lawen, A. (1985) in Hormones and cell regulation (Dumont, J. E., Hamprecht, B. and Nunez, J., eds) vol. 9, pp. 411-412, Elsevier Company, North-Holland, Amsterdam; Lawen, A. and Martini, O. H. W. (1985) FEBS Lett. 185, 272-276].

Adenosine Triphosphate↗
PubMed1989

Insulin-induced S6 kinase activation in HeLa cells and its reversal by hyperthermic stress.

Insulin treatment of HeLa S3 cells activates an S6-phosphorylating protein kinase. Although this enzyme has chromatographic properties resembling those of described proteolytic fragments of other protein kinases, namely protein kinase C, protease-activated kinase II and histone-4 protein kinase, and although insulin has been proposed by others to cause S6 phosphorylation via proteolytic protein kinase activation, the insulin-induced increase in S6-kinase activity described here is probably not due to proteolysis. Rather, the activity indicates the existence, in HeLa cells, of an interconvertible S6 kinase, since the insulin-induced activity increase was rapidly reversed under hyperthermic stress, and since this effect of hyperthermia was itself reversible. The S6-kinase activities from serum- and from insulin-stimulated HeLa cells resemble each other closely and are likely to represent the same enzyme. The enzyme may therefore mediate both signals delivered by mitogens and the insulin signal. Analysed at an in vitro transfer of 1 mol phosphate/mol S6, this S6 kinase activity does not phosphorylate the (principal) S6 site recognized by the cAMP-dependent protein kinase.

Binding Sites↗
PubMed1988

Occurrence of 40 S.polysomal complexes in polysome profiles of reticulocyte lysates.

In most reticulocyte lysates 40 S.polysomal complexes have such a short lifetime that they will not show up in the polysome profile. Here we describe a reticulocyte lysate where these 40 S.polysomal complexes apparently have a highly increased lifetime and therefore these complexes can be seen in the polysome profile as shoulders on the di-, tri- and tetrasome peak. The presence of these complexes in lysates probably signals an increased speed in the association of 40 S subunits with mRNA since similar alterations as described above show up in the polysome profile of normal lysates to which native ribosomal 40 S subunits were added.

Animals↗
PubMed1987

Regulation of ribosomal protein S6 phosphorylation in heat-shocked HeLa cells.

Decreases in energy charge, ribosomal protein phosphorylation and rate of protein synthesis are well-documented facets of the cellular response to hyperthermia in non-vertebrates. We have tried to reproduce this response pattern in 32P-labelled HeLa cells in order to investigate the hypothetical causal relationship between these effects. In HeLa cells shifted from 36 degrees C to 42 degrees C, dephosphorylation of S6 and inhibition of protein synthesis, owing to a decreased initiation rate, were observed, but could not have been mediated by changes in the cells' general energy charge since the ATP and GTP levels were not reduced. In addition, we found that the hyperthermic translation block developed faster than the overall dephosphorylation of S6, showing that S6 dephosphorylation cannot be responsible for the translation block unless site-specific effects play a critical role.

Adenosine Triphosphate↗
PubMed1987

Are highly phosphorylated 40-S subunits preferentially utilized during protein synthesis in a cell-free system from HeLa cells?

It has been concluded from circumstantial evidence obtained with HeLa cells in vivo that the phosphorylation of ribosomal protein S6 increases the affinity of 40S particles for mRNP [Duncan, R. and McConkey, E. H. (1982) Eur. J. Biochem. 123, 535-538; Thomas, G., Martin-Pérez, J., Siegmann, M. and Otto, A.M. (1982) Cell 30, 235-242]. This conclusion needs to be tested in vitro in a reinitiating cell-free translation system from growth-competent cells. We have prepared such a system from HeLa cells and have compared the capacity of homologous 40S subunits of various degrees of phosphorylation to enter the existing polysome pool. The 40S subunits' degree of phosphorylation was manipulated by exposing aliquots of growth-stimulated HeLa cells to hyperthermia (see accompanying paper). 40S subunits from heat-shocked and control cells, despite differences in S6 phosphorylation level as verified by two-dimensional electrophoresis, did not differ with respect to their recruitment into the existing polysome fraction. Owing to the reinitiation activity of the translation system, assay times could be kept sufficiently short, to avoid any serious interference by the S6 phosphatase activities of the system. Our results suggest that increased S6 phosphorylation by itself is not sufficient to accelerate the participation of 40S subunits in protein synthesis.

Cell-Free System↗
PubMed1986

Effects of addition of derived 40 S subunits on translation rate and polysome profile of the reticulocyte lysate.

We have investigated the way in which the addition of exogenous 40 S subunits to a reinitiating cell-free translation system, prepared from reticulocytes, may affect translational parameters of the system. The disturbance of the system's subunit stoichiometry resulted in the following changes in the ribosome profile: (1) rapid exhaustion of the pool of native 60 S subunits; (2) appearance of humps on the peaks of the polysome profile, which probably represent unusually long-lived [40 S. polysomal] complexes; (3) at higher doses of exogenous particles, the amount of polysomes decreased. This latter effect reflected a corresponding decrease in the overall translation (i.e. initiation) rate. The phenomena are interpreted as follows: exogenous 40 S subunits combine with 60 S subunits, forming idle 80 S ribosomes. The shortage of 60 S subunits delays the utilization of [40 S. polysomal] complexes, which is compensated for by a pool increase of these complexes. At high 40 S subunit doses this compensatory mechanism fails, and the 60 S shortage begins to determine the overall translation rate. The observations underline that the various translational parameters of the lysate function in an optimally concerted manner, so that only small amounts of derived 40 S subunits are tolerated by the system for analysis.

Animals↗
PubMed1985

A chick embryo fibroblast protein kinase recognizing ribosomal protein S6. Activity increase after serum stimulation.

We sought protein kinase(ser) activity in DEAE-Sephacel chromatography fractions of the 10000 X g supernatants of chick embryo fibroblasts using 40 S ribosomal subunits as kinase substrate, and detected a new S6-recognizing kinase activity. There was one order of magnitude more enzyme activity in chromatography fractions derived from serum-stimulated than from serum-deprived cells. Known protein kinase regulators and a low dose tryptic treatment did not increase the enzyme's activity.

Animals↗
PubMed1983

Inhibition of the elongation step of protein synthesis by vaccinia virus.

Vaccinia cores inhibit translation in cell-free protein synthesis systems at two stages: initiation; and, as shown here, elongation. The former effect tends to obscure the latter. Elongation control could, however, be revealed as follows: when, in a reticulocyte of L-cell lysate, initiation was blocked by a drug (edein), the residual [35S]methionine incorporation was severely reduced by the subsequent addition of vaccinia cores. The elongation block could also be demonstrated by analysis of ribosome profiles: treatment with edein alone permitted ribosomal run-off; treatment with either the elongation inhibition anisomycin or with cores preserved the polyribosomes.

Animals↗
PubMed1976

Characterisation of eukaryotic ribosomal proteins.

A simple method of two-dimensional polyacrylamide gel electrophoresis is described which affords: (1) high resolution of eukaryotic ribosomal proteins; (2) good recovery of protein in the transfer from first to second dimension; and (3) characterisation of the separated proteins in terms of molecular weights and other electrophoretic properties. Using this method, we have characterised 70 proteins in rabbit reticulocyte ribosomes, 30 from the small subunit and 40 from the large subunit. The molecular weight distribution is compared with those obtained by other authors after fractionation of the proteins in two dimensions.

Animals↗
PubMed1976

Molecular weight distribution of ribosomal proteins from several vertebrate species.

Two-dimensional polyacrylamide gel electrophoresis of proteins from the separated ribosomal subunits of rabbit reticulocytes, rabbit liver, mouse liver, rat liver, chicken liver, and toad liver was performed using the "pH 4.5/SDS" system previously described (Martini and Gould, 1975), with internal standards to measure the molecular weight distributions. With few exceptions, the patterns were remarkably similar, indicating a high degree of conservation during evolution of both net charge (largely determining mobility in the first dimension) and size (determining mobility in the second dimension). The aggregate mass (sum of molecular weights) of both small and large subunit proteins, about 0.65 X 10(6) and 0.95 X 10(6) daltons respectively, were invariant. These figures are significantly smaller than the hydrodynamically determined mass of protein in the subunits. The implications of this discrepancy, which is opposite that found in the prokaryotes, is discussed.

Animals↗