Purification and characterization of a human recombinant T-cell protein-tyrosine-phosphatase from a baculovirus expression system.

A 48-kDa human T-cell protein-tyrosine-phosphatase (TC.PTPase) and a truncated form missing an 11-kDa C-terminal segment (TC delta C11.PTPase) were expressed by using the baculovirus system and characterized after extensive purification. The full-length PTPase was restricted to the particulate fraction of the cells from which it could be released by a combination of salt and detergent. The enzyme was entirely specific for phosphotyrosine residues. It displayed a low level of activity toward phosphorylated, reduced, carboxamidomethylated, and maleylated lysozyme (RCML), but was 12 times more active toward phosphorylated myelin basic protein (MBP). By contrast, the 37-kDa form localized in the soluble fraction, and its activity toward RCML was 5 times higher than that observed with MBP. The autophosphorylated cytoplasmic domain of the EGF receptor served as substrate for both enzymes. Limited proteolysis of either protein gave rise to a 33-kDa fragment displaying the substrate specificity of the truncated form. These data lend further support to the view that the C-terminal segment of the T-cell PTPase serves a regulatory function, playing an important role in the localization and substrate specificity of the enzyme.     

Influence of sulphur dioxide on the fermentation of sulphite spent liquor

In fodder yeast production from sulphite spent liquor, sulphur dioxide is well-known as a yeast poison. The present paper deals with the detailed influence of sulphur dioxide. The experimental work was done in a laboratory fermenter under technical conditions. A decrease in biomass yield, and an increase in oxygen consumption, caused by increasing carbon dioxide production, were found. During the fermentation, there is no destruction of the carbonyl-bisulphite adduct to be observed. Sulphur dioxide has no influence on the crude protein content of yeast cells, and there is also no influence on the morphology of cells.     

Ferredoxin-dependent methane formation from acetate in cell extracts of Methanosarcina barkeri (strain MS).

Cell extracts of Methanosarcina barkeri grown on acetate catalyzed the conversion of acetyl-CoA to CO2 and CH4 at a specific rate of 50 nmol min-1 mg-1. When ferredoxin was removed from the extracts by DEAE-Sephacel anion exchange chromatography, the extracts were inactive but full activity was restored upon addition of purified ferredoxin from M. barkeri or from Clostridium pasteurianum. The apparent Km for ferredoxin from M. barkeri was determined to be 2.5 M. A ferredoxin dependence was also found for the formation of CO2, H2 and methylcoenzyme M from acetyl-CoA, when methane formation was inhibited by bromoethanesulfonate. Reduction of methyl-coenzyme M with H2 did not require ferredoxin. These and other data indicate that ferredoxin is involved as electron carrier in methanogenesis from acetate. Methanogenesis from acetyl-CoA in cell extracts was not dependent on the membrane fraction, which contains the cytochromes.     

Purification and characterization of catalytic fragments of phosphorylase kinase gamma subunit missing a calmodulin-binding domain

A catalytic fragment preparation of rabbit muscle phosphorylase kinase produced by limited chymotryptic digestion was isolated and identified as the NH2-terminal region of the gamma subunit by Edman degradation. Mass spectral analysis, gas phase sequence analysis, and amino acid analysis of the active fragment carboxyl-terminal peptides revealed multiple COOH termini generated at residues Tyr290, Arg296, and Phe298 in the gamma subunit sequence. These active fragment species are about 24% smaller than the gamma subunit (Mr 44,673) and range in size from Mr 33,279 to Mr 34,275. The active fragment preparation exhibits a specific activity about 6-fold higher than that of the gamma subunit-calmodulin complex. Calmodulin confers calcium sensitivity to the gamma subunit but has no effect on the enzymatic properties of active fragment. Affinity measurements demonstrated a dissociation constant of 0.7 microM for active fragment binding to dansylcalmodulin, a value about 28-fold weaker than reported for the gamma subunit. These data support the presence of a calmodulin binding domain in the COOH-terminal region of the gamma subunit.     

Esmolol: effects on isoprenaline- and exercise-induced cardiovascular stimulation in conscious dogs

Esmolol, a recently developed ultra-short acting beta-adrenoceptor blocking agent, was evaluated in 12 conscious chronically instrumented dogs with intact autonomic reflexes. The significance of its beta 1-adrenoceptor selectivity was examined at various cardiovascular activation levels established by either incremental isoprenaline infusion or graded treadmill exercise. The observed parameters were heart rate, systolic and diastolic arterial blood pressure, left ventricular dp/dtmax, and left ventricular end-diastolic pressure. Intravenous infusion of esmolol (25 and 250 micrograms.kg-1.min-1) led to a dose-dependent reduction of the isoprenaline-induced increase in positive dp/dtmax. The concomitant increase in heart rate was suppressed to a lesser extent. Characteristically of a beta 1-selective agent, esmolol had only a slight effect on the isoprenaline-induced reduction in diastolic blood pressure. The impact of esmolol on exercise-induced hemodynamic activation was much smaller. Exercise-induced increase in positive dp/dtmax was more sensitive to beta-adrenoceptor blockade than the concomitant increase in heart rate. Diastolic blood pressure was not influenced significantly. beta-Adrenoceptor blockade was virtually reversed within 20 min of discontinuation of esmolol infusion.     

A sequence-specific protease cleaves a maternal, cortical protein during early embryogenesis in Sciara coprophila (Diptera)

One of the first events of egg activation in Sciara coprophila (Diptera) is the disappearance of an abundant maternal 38-kDa protein (p38) and the simultaneous emergence of an abundant 35-kDa protein (p35). Western blotting experiments using monoclonal antibodies directed against p38 reveal that p38 and p35 are serologically related and indicate that maternal p38 is transformed into p35 during early development. This transition is possibly accompanied by a conformational change in the part of the protein that is common to both protein species. The processing of p38 to p35 can be mimicked by trypsin treatment in vitro, suggesting that a trypsin-like protease is responsible for this conversion in vivo. Immunostaining indicates that the p38 class of antigens is evently distributed in the periplasm of early cleavage embryos. After the arrival of nuclei in the periplasm, the antigens become associated with the infolding cellular membranes. A similar membrane association of actin can be observed with anti-actin antibodies. Nevertheless, p38 and actin are clearly distinct from each other. We presume that p38 is a product of a maternal effect gene necessary for early dipteran development.     

Cerebroside sulfatase activator deficiency induced metachromatic leukodystrophy

Two siblings of consanguineous parents had presented with a variety of findings indicative of juvenile metachromatic leukodystrophy (MLD). However, instead of the expected profound deficiency of arylsulfatase A (ARS A), their enzyme levels were about half-normal, and enzyme from fibroblasts had properties identical with the properties of enzyme from normal fibroblasts. Nevertheless, the hydrolysis of cerebroside sulfate by growing fibroblasts was markedly attenuated. Supplementation of the fibroblasts with cerebroside sulfatase activator normalized the response in the loading test. These results imply that the fibroblasts, and by extension the patients, are deficient in activator. Although the defective catabolism of cerebroside sulfate and the clinical manifestations in these patients mimic MLD, the molecular basis is distinct from the classical forms of the disorder.     

Preparation and characterization of a cell-free system from Chinese hamster ovary cells that translates natural messenger ribonucleic acid and analysis of intermediary reactions

A cell-free system from cultured Chinese hamster ovary cells has been developed, which translates endogenous mRNAs, exogenous natural mRNAs, and synthetic polynucleotide templates. The analysis of most of the reactions involved in initiation, elongation, and termination of protein synthesis can be carried out in this system. The postmitochondrial fraction, containing ribosomal 40 and 60 S subunits, 80 S ribosomes, polysomes, and cytosol proteins, incorporates amino acids into protein. The preparation is capable of recycling endogenous mRNA by initiating protein synthesis on polysomal mRNA, and of initiating protein synthesis on exogenous templates. When endogenous mRNA is degraded with micrococcal nuclease, polysomes are no longer evident and protein synthesis is markedly depended on added mRNA, ATP, GTP, and a nucleoside triphosphate-generating system. Amino acid incorporation is linear for over 2 h, polysomes containing nascent polypeptide chains are reformed and, with time, most of the protein synthesized is released into the media. Gel electrophoretic analysis of the product formed in response to globin mRNA indicates that most of the radioactivity migrates as a single peak, in the region corresponding to globin. Comparison of the electrophoretic pattern obtained from labeled Chinese hamster ovary cells with that from incubations of cell extract and Chinese hamster ovary mRNA indicates that essentially all of the polypeptides formed by the intact cell are synthesized by the cell-free system. Sucrose gradient centrifugation of incubations containing mRNA-depleted extract and [³⁵S]methionine, in the absence of added mRNA, is used to detect initiation intermediates in the formation of the [40 S Met-tRNA_f] complex and, with added natural mRNA plus cycloheximide, to detect intermediates in the formation of the 80 S initiation complex. Chain elongation reactions are measured by the incorporation of [³H]phenylalanine into polyphenylalanine in extracts supplemented with poly(U), or by the formation of nascent polypeptide chains on polysomes with natural mRNA. Chain termination is measured by analyzing the amount of radioactive protein released into the cytosol.     

Effects of retinoic acid on protein synthesis in cultured melanoma cells

Retinoic acid reduces the growth rate of mouse S91 melanoma cells in culture and increases the proportion of cells in the G₁ phase of the cell cycle. Because of the integral role protein synthesis has been shown to play in growth control we studied the effect of retinoic acid on the protein synthesis machinery with a cell-free system developed from the melanoma cells. This system was capable of translating endogenous mRNA, exogenous globin mRNA, and the synthetic template poly(U). Of the above activities of the protein synthesis system only the translation of endogenous mRNA was reduced significantly in the cell-free system prepared from retinoic acid-treated cells. Analyses of the amount and function of RNA revealed that treatment with retinoic acid leads to reductions in total RNA content, in the proportion of ribosomes in polysomes, in the amount of poly(A)RNA, and in the amount of polysome-associated mRNA. All these effects of retinoic acid contribute to the decrease in protein synthesis activity of treated cells. Two-dimensional electrophoresis anlaysis of L-[³⁵S]methionine-labeled proteins produced by untreated and treated cells revealed only a few quantitative differences. We suggest that retinoic acid-induced suppression of protein synthesis activity may be the cause for growth inhibition.