The membrane of the rough endoplasmic reticulum contains cytoplasmically exposed high affinity GTP-binding sites

Binding experiments using [14C]GTP and rat liver rough microsomes gave Scatchard plots with Kd congruent to 0.1 microM and a binding capacity congruent to 40 pmol/mg microsomal protein. Removal of the ribosomes did not modify the binding parameters. GTP was competed out by GTP analogues but not by ATP. Limited proteolysis of rough microsomes decreased the GTP-binding capacity and prevented GTP from suppressing the latency of mannose-6-phosphatase and of the synthesis of dolichol-linked chitobiose. The GTP-binding protein is probably involved in these effects of GTP. Its function could be to act in the association-dissociation of membrane components at the ribosome-membrane junction.     

Developmental regulation of myelin basic protein expression in mouse brain

Developmental regulation of myelin basic protein expression in mouse brain has been examined by comparing the myelin basic protein coding potential of mRNA in vitro with the accumulation of myelin basic protein-related polypeptides in vivo. In vitro translation of mRNA isolated from mouse brain generated eight myelin basic protein-related polypeptides with apparent molecular weights of 34K, 30K, 29K, 26K, 21.5K, 18.5K, 17K, and 14K. A similar set of eight myelin basic protein-related polypeptides with corresponding molecular weights was identified in vivo when total brain proteins were analyzed by immunoblotting. Each of the myelin basic protein-related polypeptides shows a characteristic developmental profile in terms of mRNA level and rate of accumulation implying a complex developmental program of myelin basic protein gene expression with regulation and modulation at several different biosynthetic levels.     

Hydrolysis of dansyl-peptide substrates by leucine aminopeptidase: origin of dansyl fluorescence changes during hydrolysis

The origin of the fluorescence changes observed in stopped-flow experiments of the hydrolysis of three 5-(dimethylamino)naphthalene-1-sulfonyl-(dansyl) peptide substrates by porcine kidney cytosol leucine aminopeptidase has been investigated. The substrates used all have the potential to accept energy from aromatic residues of the enzyme via resonance energy transfer when they are bound as enzyme-substrate complexes, indicating that fluorescence changes due to the buildup and decay of such intermediates are possible. However, the fluorescence of these substrates differs from that of the products, and direct excitation of their dansyl groups during hydrolysis can also be responsible for the observed fluorescence changes due to changes in the concentrations of free substrate and product. The dansyl fluorescence changes observed with excitation wavelengths near 280 nm are not accompanied by quenching of the enzyme fluorescence, as would be expected if there were enzyme-to-substrate energy transfer. The magnitude of the maximal fluorescence change at a fixed concentration of substrate is also independent of the enzyme concentration. Furthermore, the excitation profile for the fluorescence changes shows that they arise from direct excitation of the dansyl group. Thus, there is no energy transfer in these reactions, and the fluorescence changes observed arise from direct excitation of the dansyl group and reflect the instantaneous concentration of substrate. This behavior contrasts sharply with that for the reaction of carboxypeptidase A with dansyl-Gly-Tyr, which has been studied as a positive control for an energy-transfer system.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification of almond emulsin alpha-L-fucosidase I by affinity chromatography.

An affinity chromatographic method to purify α-l-fucosidase I from almond emulsin was developed. A derivative of lacto-N-fucopentaose II, ?-aminocaproyl-lacto-N-fucopentaosylamine, was coupled to sepharose 4B and packed in a column. By adopting this column for affinity chromatography, the enzyme was purified a hundredfold. The enzyme preparation was free from any other exoglycosidases which act on natural substrates.     

Sensitivity to X-irradiation in relation to cell size of CHO cells synchronized in early G1

Abstract. A population of line CHO Chinese hamster cells was synchronized by mitotic selection and allowed to enter early G₁, after which the largest and smallest cells in the population were sorted, irradiated, and their viability determined. Despite sizeable differences in volume, metabolic capability and cell cycle progression rates, an equivalent level of survival was obtained for the two populations, indicating that the factors responsible for the volume, metabolic and progression heterogeneity do not contribute greatly to radiation sensitivity.     

Synergism between diacylglycerols and calcium ionophore in the induction of human B cell proliferation mimics the inositol lipid polyphosphate breakdown signals induced by crosslinking surface immunoglobulin

Resting human tonsillar B cells were stimulated to divide by heat killed Staphylococcus aureus Cowan strain 1 which was shown to induce hydrolysis of phosphatidylinositol 4, 5-bisphosphate known to give rise to diacylglycerol and an increase in cytosolic free calcium. Addition of the diacylglycerols, 1-oleoyl-2 acetyl glycerol or sn-1, 2-dioctanoylglycerol, together with the calcium ionophore ionomycin to B cell cultures induced marked cell proliferation whereas these agents were ineffective when used alone. Both diacylglycerols were shown to compete with [3H] phorbol 12,13 dibutyrate in binding to protein kinase C. These data support the hypothesis that synergism between cytosolic calcium and endogenous diacylglycerol, which activates protein kinase C, is involved in signal transduction in the proliferation of human B cells.     

Human hypoxanthine-guanine phosphoribosyltransferase

A mutant form of human hypoxanthine-guanine phosphoribosyltransferase (HPRTToronto) was isolated from erythrocytes of a male patient with gout due to a partial deficiency of enzyme activity. The tryptic peptides of HPRTToronto were mapped by reverse-phase high pressure liquid chromatography in an attempt to define the precise abnormality in its primary structure. Sequence analysis of the single aberrant peptide in HPRTToronto revealed an arginine to glycine amino acid substitution at position 50. A single nucleotide change in the codon for arginine 50 (CGA leads to GGA) could explain this substitution.     

Chronic exposure to inhaled anesthetics increases cholesterol content in Acholeplasma laidlawii

Acholeplasma laidlawii cells were grown in cholesterol-enriched medium and exposed continuously to either air (control), 4.0 vol.% halothane in air at 1 atm pressure (4% atm halothane), or 80% cyclopropane in oxygen for 24 h at 37°C. Cells grown in the presence of 4% atm halothane or 80% cyclopropane had approximately twice as much membrane cholesterol content/mg protein as the control cells. Cells grown in an anesthetic environment also tended to have a higher membrane cholesterol/phospholipid molar ratio compared to control cells. Membranes isolated from halothane-exposed cells grown in a cholesterol-enriched medium were more ordered at 37°C (measurements were made with no anesthetic present) than membranes from control cells grown in an identically enriched medium. This difference in membrane physical state between control and anesthetic-exposed cells decreased as the temperature decreased, and disappeared at approx. 23°C. Continuous exposure of A. laidlawii to 4% atm halothane or 80% cyclopropane for 24 h did not markedly affect membrane fatty acid composition, either in cells grown on an unsupplemented medium or in cells grown in a medium enriched in myristic, palmitic or stearic acids. These results further support the hypothesis that an increased membrane cholesterol content may play a role in the tolerance or dependence that develops after chronic exposure to anesthetic agents.