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K L Manchester

Publications and source records attributed to K L Manchester.

At least 55 records · Page 3Linked to original sources
PubMed1987

Kinetic constants in the functioning of eIF-2 and eIF-2B.

Minimum rate constants for reactions catalysed by the eukaryotic initiation factor eIF-2B in promoting formation of the ternary complex eIF-2.GTP.met-tRNAi from eIF-2.GDP are estimated from published data. The most plausible sequence of reactions in vivo is when eIF-2B remains bound to eIF-2.GTP.met-tRNA. Rate constants for reaction of eIF-2B and eIF-2.GDP are too large for protein:protein interactions at cellular concentrations in free solution. This finding suggests some form of sequestration of eIF-2 and eIF-2B in the cell to facilitate interaction, which may result in only a portion of cellular eIF-2 being actively engaged in initiation.

Eukaryotic Initiation Factor-2↗
PubMed1985

Evaluation and significance of kinetic parameters governing function of protein synthesis initiation factors eIF-2 and eIF-2B.

Published data dealing with the formation of the ternary complex eIF-2 X GTP X met-tRNAi involved in eukaryotic initiation have been evaluated to calculate the expected inhibition by GDP and the role of eIF-2B in limiting this inhibition. It is concluded that cellular levels of GDP are unlikely seriously to inhibit ternary complex formation if the reaction can proceed to equilibrium. However, derivation of 'on' and 'off' rates for the interaction of GTP and GDP with eIF-2 demonstrates that these are too slow in the absence of eIF-2B to support active protein synthesis, particularly if eIF-2 is released from ribosomes as eIF-2 X GDP. Whilst eIF-2 X GDP and eIF-2 X GTP appear to dissociate equally slowly, it is concluded that GDP binds to eIF-2 100-times faster than GTP. Addition of eIF-2B has the effect of raising k-1 for both GDP and GTP several hundred-fold and k+1 50- and 7000-fold, respectively. Thus, a kinetic block can be relieved even if there is no change in the thermodynamic state. Phosphorylation of the alpha-subunit of eIF-2 appears to affect only those parameters influenced by eIF-2B. The reported rescue of inhibited lysates by addition of 1 mM GTP is not by mass action but by some other mechanism. Consideration of the kinetic parameters favours the formation of a ternary complex of eIF-2 X eIF-2B X GDP en route to eIF-2 X GTP as opposed to displacement of GDP from eIF-2 X GDP by eIF-2B.

Eukaryotic Initiation Factor-2↗
PubMed1985

Optimisation of protein synthesis rates in reticulocyte lysates and reconstituted systems.

Means of increasing the very low activity of a reconstituted protein synthesising system from rabbit reticulocytes were investigated. Increasing the concentration of labelled amino acid, addition of polyamines, use of Sepharose-filtered as opposed to centrifuged ribosomes, use of untreated as opposed to gel-filtered cytosol and an increase in ratio of cytosol to ribosomes all contributed to the increase in activity of the system to the point where activity was clearly consistent with initiation taking place. Similar activities could not be attained with rat liver cytosol though rat liver ribosomes incorporated well in reticulocyte cytosol. Incorporation by lysates was also found to be dependent on the concentration of the labelled amino acid added.

Amino Acids↗
PubMed1985

Control of cell-free protein synthesis by amino acids: effects on tRNA charging.

In order to resolve the observation that addition of glutamine and glutamate appears to be of particular importance in enhancing the activity of a cell-free protein synthesis system derived from rat liver (Manchester and Tyobeka, 1980), we have measured the KM of the aminoacyl-tRNA synthetases towards amino acids and the extent of aminoacylation of tRNA under the conditions of our earlier experiments. During incubation of the cell-free system in the presence of an amino acid mixture the extent of acylation to tRNA of 15 amino acids studied showed no clear change from initial time values. When incubation took place in the absence of added amino acids, however, the levels of glutamate and glutamine bound to their appropriate tRNAs dropped more rapidly and to lower levels than for other amino acids except tryptophan. The pronounced drop for these two amino acids does not seem to result from an abnormally high KM value for the synthetases towards the respective amino acids, nor an abnormally low Vmax, but probably from the fact that the amounts of glutamyl and glutaminyl-tRNA in the cell-free system are comparatively low.

Amino Acids↗
PubMed1984

Protein and fat metabolism of hepatocytes from hyperosmolar non-ketotic diabetic rats.

The secretion of newly synthesized proteins and urea, triacylglycerol and beta-hydroxybutyrate by hepatocytes in cultures from normal and hyperosmolar non-ketotic diabetic rats has been compared. Secretion of protein and of urea was very similar in the two preparations. Secretion of protein was enhanced by addition of insulin to the culture medium, that of urea was decreased. Output of triacylglycerol was substantially greater and output of beta-hydroxybutyrate much reduced in hepatocytes from the diabetic rats in comparison with controls during the 1st day of culture.

Animals↗
PubMed1984

Effects of preparative procedures on the sedimentation behaviour of 40-S ribosomal subunits.

The sedimentation characteristics on sucrose gradients of derived 40-S subunits from rat liver depend on the ionic conditions used in their preparation. Following the procedure of Wettenhall, R.E.H., Wool, I.G. and Sherton, C.C. (Biochemistry 12 (1973) 2403-2411) in which 12.5 mM Mg2+ is present throughout the preparation of the subunits, sucrose gradients of the 40-S fraction show two peaks consistent with the presence of a mixed and non-interchanging population of monomers and dimers. By contrast, when ribosomes and subunits are prepared in 2 mM Mg2+ it is shown here that 40-S subunits sediment as a single peak at a rate intermediate between that of monomers and dimers as established by prior fixation with glutaraldehyde or formaldehyde - a phenomenon observed by Spirin, A.S., Belitsina, N.V. and Lishnevskaya, E.B. (FEBS Lett. 24 (1972) 219-224) for the Escherichia coli 30-S--50-S couple but not previously shown for subunits of eukaryotic origin. Under conditions of low [Mg2+] it is suggested that the subunits are of a uniform population capable of dimerization and that equilibrium can continue during sedimentation. In the present experiments it was not possible to find tRNA in 40-S subunits as shown by Wettenhall et al. By fixation before sedimentation the relative proportions of monomers and dimers of 40 S incubated under a variety of conditions was determined. Warming from 0 to 37 degrees C increased the proportion of monomers markedly, but variation of [Mg2+] had little effect. Incubation with albumin or tRNA or spermine also had little effect, but addition of ribosomal salt-wash increased the proportion of monomers. It seems most likely that many of the 40-S subunits prepared in 12.5 mM Mg2+ have initiation factors or other proteins bound to them which prevent dimerization, but that subunits prepared at any stage with buffer containing only 2 mM Mg2+ have lost these proteins and therefore are able to dimerize to a degree depending on concentration. Different relative proportions of monomers and dimers were observed depending on whether glutaraldehyde or formaldehyde was used as fixative. Reasons are suggested for believing that formaldehyde is to be preferred to glutaraldehyde for fixation in experiments of this nature.

Animals↗
PubMed1984

The influence of hyperosmolar non-ketotic diabetes on protein synthesis in rat tissues.

We have compared the effects of acute ketotic diabetes on the capacity of isolated ribosomes to carry out protein synthesis with the effects of the non-ketotic but hyperosmolar state developed in rats as a model of the human condition by the procedure described by Joffe et al. These workers concluded that the hyperosmolar non-ketotic condition was consistent with the liver receiving adequate insulin but with the periphery being insulin-deficient. In support of this hypothesis we find that there is no impairment of hepatic protein synthesis as judged by the activity of isolated ribosomes, nor of the proportion of ribosomes in polysomes in the hyperosmolar non-ketotic condition, this being in contrast with the ketotic state, where protein synthesis and polysome levels are markedly reduced. However, the hyperosmolar non-ketotic condition was characterized by a reduced capacity for protein synthesis by ribosomes isolated from kidney and cardiac and skeletal muscle.

Animals↗
PubMed1983

Histochemical and biochemical characteristics of the transient hypertrophy of the denervated rat hemidiaphragm.

A systematic study of the different fiber types of rat diaphragm muscle in the first 10 days after unilateral denervation showed approximately a 10% decrease in diameter of the white fibers, 25% increase in that of intermediate fibers, and 35% increase in that of red fibers together with diminished differential staining properties both for myosin ATPase and Sudan black. There was also a doubling of the amount of connective tissue. A reduction in total lipid concentration of the tissue included decreases in both triacylglycerol and phospholipid, but not cholesterol. Magnesium concentration in the tissue also declined, as did activity of Ca2+-activated, though not Mg2+-activated, myosin ATPase.

Adenosine Triphosphatases↗
PubMed1982

The phosphorylation of ribosomal protein S6 in rat tissues following cycloheximide injection, in diabetes, and after denervation of diaphragm. A simple immunological determination of the extent of S6 phosphorylation on protein blots.

The extent of phosphorylation of S6 was measured in a variety of cells, organs, and tissues using a simplified procedure. Ribosomal proteins were separated by electrophoresis on a one-dimensional urea-sodium dodecyl sulfate gel. They were transferred electrophoretically to nitrocellulose, and S6 was specifically stained by means of its reaction with a monoclonal antibody. The mobility of the stained protein was shown to be a measure of the extent of its phosphorylation. Using this method, we found that cycloheximide injection of rats gives rise to the phosphorylation of S6 in a variety of organs and tissues: diaphragm, heart and skeletal muscle liver and kidney, but not brain. Diabetes induced with alloxan did not result in the phosphorylation of S6 in any of the tissues examined, and subsequent insulin injection resulted in a slight increase in the phosphorylation of S6 in liver. Insulin injection in normal rats also caused the phosphorylation of liver S6. Unilateral denervation of rat diaphragm resulted in phosphorylation of S6 in the denervated portion but not in the innervated portion of the diaphragm.

Animals↗
PubMed1981

Cyclic nucleotides in the denervated rat diaphragm and the effect of cyclic AMP on ornithine and adenosylmethionine decarboxylases.

The concentrations of cyclic AMP and cyclic GMP were measured in the denervated rat diaphragm at various times following unilateral phrenicectomy. Cyclic AMP concentration was raised by the second day after operation, reached a peak by the third day, followed by another increase at around 10 days. By contrast, cyclic GMP concentration was decreased within a day after denervation and remained below control levels at all subsequent times studied. Epinephrine in vitro produced a comparable increase in the concentration of cyclic AMP in both normal and denervated tissue. The concentration of adenosine appeared unchanged in the denervated diaphragm by comparison with its innervated control. Activity of ornithine decarboxylase was elevated in the diaphragms of rats treated with dibutyryl cyclic AMP, but this effect could also be achieved with sodium butyrate alone. Adenosylmethionine decarboxylase activity, was unaffected after treatment with either compound. These observations and others discussed are taken to indicate a lack of direct relationship between cyclic AMP concentrations and the activity of the rate-limiting enzymes of polyamine biosynthesis in the rat diaphragm.

Adenosylmethionine Decarboxylase↗
PubMed1981

Influence of serum and amino acids on the accumulation of aminoisobutyrate by rat hepatoma cells. A dedifferentiation of transport routes?

Rat hepatoma cells accumulate considerably less 2-aminoisobutyrate after cultivating in the absence of serum--the change in rate of aminoisobutyrate uptake takes place within 1 h of serum starvation. Starvation of amino acids by contrast raises aminoisobutyrate uptake in the presence or absence of serum, but the cells are much less responsive to amino acid supply than to availability of serum. Phosphate (10 mM) reduced aminoisobutyrate uptake by cells grown in serum to that exhibited by serum-starved cells. Aminoisobutyrate uptake by cells grown in serum was reduced by glycine, proline, alanine, serine, glutamine, methylaminoisobutyrate and 2-aminonorbornane-2-carboxylate, the effects of methylaminoisobutyrate and 2-aminonorbornane-2-carboxylate being additive. However, similar inhibition phenomena were not seen for cells deprived of serum where aminoisobutyrate uptake tended to a relatively constant level insensitive to inhibitory influences, yet substantially greater than that arising by simple diffusion. The comparative insensitivity of our hepatoma line when starved of serum to competition and repression phenomena is in contrast to findings of others. Our results also suggest a lack of clear delineation of specificities for the A and L transport systems as usually defined.

Amino Acids↗
PubMed1981

The influence of nerve section on the metabolism of polyamines in rat diaphragm muscle.

Concentrations of spermidine, spermine and putrescine have been measured in rat diaphragm muscle after unilateral nerve section. The concentration of putrescine increased approx. 10-fold 2 days after nerve section, that of spermidine about 3-fold by day 3, whereas an increase in the concentration of spermine was only observed after 7-10 days. It was not possible to show enhanced uptake of either exogenous putrescine or spermidine by the isolated tissue during the hypertrophy. Consistent with the accumulation of putrescine, activity of ornithine decarboxylase increased within 1 day of nerve section, was maximally elevated by the second day and then declined. Synthesis of spermidine from [14C]putrescine and either methionine or S-adenosylmethionine bt diaphragm cytosol rose within 1 day of nerve section, but by day 3 had returned to normal or below normal values. Activity of adenosylmethionine decarboxylase similarly increased within 1 day of nerve section, but by day 3 had declined to below normal values. Activity of methionine adenosyltransferase was elevated throughout the period studied. The concentration of S-adenosylmethionine was likewise enhanced during hypertrophy. Administration of methylglyoxal bis(guanylhydrazone) produced a marked increase in adenosylmethionine decarboxylase activity and a large increase in putrescine concentration, but did not prevent the rise in spermidine concentration produced by denervation. Possible regulatory mechanisms of polyamine metabolism consistent with the observations are discussed.

Adenosylmethionine Decarboxylase↗
PubMed1981

Attempts to demonstrate short-term metabolic effects of vinyl chloride in normal rat liver.

The present study examines whether VCM or one of its metabolites, chloracetaldehyde, has a short-term effect on cell metabolism. ATP, ADP, and AMP levels were measured in regenerating rat liver after 4 and 16 hours. No change in the levels of these nucleotides was evident after i.v. injection of VCM immediately after partial hepatectomy. A second technique, that of measuring transmembrane potentials with microelectrodes, was applied both to cells in rat liver slices and pulmonary alveolar macrophages. The addition of VCM and chloracetaldehyde with the electrodes in position in rat liver slices had no significant effect on transmembrane potentials. The same was true for a series of measurements on macrophages before and after addition of the test chemicals. It therefore appears that VCM does not alter general cell metabolism but could act on a specific intracellular target.

Adenine Nucleotides↗
PubMed1980

Determination of magnesium and potassium binding constants to phosphoenolpyruvate, 2- and 3-phosphoglycerate and a number of other anions.

The stability constants for the binding of magnesium ions to phosphoenolpyruvate, 2- and 3-phosphoglycerates, 2,3-diphosphoglycerate, Pi, glucose 6-phosphate, isocitrate, glutamate and NAD have been determined spectrophotometrically in the presence of 8-hydroxyquinoline at 25 and 37 degrees C. Estimates of potassium binding constants have also been calculated for phosphoenolpyruvate and 2- and 3-phosphoglycerate and compared with previously published values.

Chemical Phenomena↗
PubMed1980

Resolution with a protein synthesising system of conflicting estimates for the stability constant for KATP3- formation.

Published stability constants for the formation of KATP3- at I = 0.1 range from 14 to 218 M-1. By studying the incorporation of [14C]leucine into protein by rat liver ribosomes at various K+ and ATP concentrations it is concluded that 14 M-1 is more likely to be the correct stability constant for KATP3- formation. Evidence for the formation of Mg2ATP and Mg(NO3)2 is also presented.

Adenosine Triphosphate↗