Characterization of the Ss sialoglycoprotein and its antigens in Rhnull erythrocytes

The Ss sialoglycoprotein (glycophorin B) and its antigens in Rhnull erythrocytes, which lack the Rhesus blood group antigens, due to apparently silent (amorphic type) or independent suppressor (regulator type) genes, were investigated. The quantity of the molecule in amorphic and in regulator type red cell membranes was found to be decreased by about 60%-70%, as judged from sodium-dodecylsulfate polyacrylamide gel electrophoresis. The Ss glycoprotein content in the erythrocytes from heterozygotes (regulator type) was diminished to an extent of about 30%. Confirming and extending previous studies, the S, s, Ux, Uz and 'N' antigens were slightly weakened in Rhnull erythrocytes. The U and Duclos receptors were only slightly or not depressed in amorphic Rhnull cells, but almost absent from or not detectable in those of the regulator type. This demonstrates that an additional alteration, apart from the decreased Ss glycoprotein content of the membranes, accounts for the weakness of these receptors in regulator type cells. We propose the hypothesis that (a) protein(s) encoded by the Rhesus locus form(s) a complex with the Ss glycoprotein. Thus, it (they) might facilitate the incorporation of the Ss glycoprotein into the membrane and also contribute to the complete expression of the U and Duclos antigens in normal cells.     

The effects of experimental hypo- and hyperthyroidism on blood viscosity and other blood parameters in the rat

Three groups of male Sprague Dawley rats received methimazole without or with Na-thyroxine in drinking water (3 and 0.33 mg T4/l, respectively) to induce characteristic alterations of their thyroid status (hypothyroid, hyperthyroid, euthyroid). A fourth group served as an untreated control without any additive to the drinking water. With respect to the different thyroid status, the following changes in the blood parameters were found: increasing plasma-T3-levels caused a reduction in plasma viscosity, in total plasma protein and in alpha 1-globulin, but an increase in hematocrit, whole blood viscosity, the number of erythrocytes and leukocytes, alpha 2-globulin and beta-globulin. It was concluded that the increase in the plasma viscosity in the hypothyroid status is mainly due to an alteration of the plasma protein pattern, and that the increase in whole blood viscosity in the hyperthyroid rat is a consequence of increased hematocrit.     

Generation of a transmembrane gradient of Na+ in Methanosarcina barkeri

A transmembrane Na+ gradient was generated by Methanosarcina barkeri during methanogenesis. The intracellular Na+ concentration amounted to approximately one fifth of the extracellular one. A secondary Na+/H+ antiport system was shown to be responsible for Na+ extrusion. This system could be inhibited by amiloride. In the presence of amiloride the delta pH across the cytoplasmic membrane increased and a transmembrane Na+ gradient could neither be generated nor maintained. The possible role of Na+ in the oxidation of methanol to the level of formaldehyde is discussed.     

Three different proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity from Alcaligenes eutrophus

The existence of three different proteins exhibiting NAD-dependent acetaldehyde dehydrogenase activity was confirmed in Alicaligenes eutrophus. The fermentative alcohol dehydrogenase, which also exhibits acetaldehyde dehydrogenase activity, is one of these proteins. The other two proteins were purified from A. eutrophus N9A mutant AS4 grown on ethanol applying chromatography on DEAE-Sephacel and triazine-dye affinity media. Acetaldehyde dehydrogenase II, which amounts to about 14% of the total soluble protein in cells grown on ethanol, was purified to homogeneity. The relative molecular masses of the native enzyme and of the subunits were 195,000 or 56,000, respectively. This enzyme exhibits a high affinity for acetaldehyde (Km = 4 microM). Acetaldehyde dehydrogenase I amounts only to less than 1% of the total soluble protein. The relative molecular masses of the native enzyme and of the subunits were 185,000 and 52,000, respectively. This enzyme exhibits a low affinity for acetaldehyde (Km = 2.6 mM). Antibodies raised against acetaldehyde dehydrogenase II did not react with acetaldehyde dehydrogenase I. Two different strains, A. eutrophus N9A mutant AS1, which represents a different mutant type and can utilize both ethanol or 2,3-butanediol, and the type strain of A. eutrophus (TF93), which can utilize ethanol, form two acetaldehyde dehydrogenases during growth on ethanol, too. As in AS4, one of these enzymes from each strain amounted to a substantial portion of the total soluble protein in the cells. These major acetaldehyde dehydrogenases were purified from both strains; they resemble acetaldehyde dehydrogenase II isolated from AS4 in all relevant properties. Antibodies against the enzyme isolated from AS4 gave identical cross-reactions with the enzymes isolated from AS1 and TF93.     

Fluorescent and photoactive probes for the study of protein kinase C

A fluorescent diacylglycerol, 2-(12-N-dansylaminododecanoyl)-1-myristoyl-sn-glycerol (dansyl-DAG) and a photoactive diacylglycerol, 2-(12-[N-(4-azido-2-nitrophenyl)] aminododecanoyl)-1-myristoyl-sn-glycerol (azido-DAG) have been synthesized. Both have been shown to bind to protein kinase C by inhibition of phorbol dibutyrate binding. Dansyl-DAG was able to activate protein kinase C at low calcium concentrations. Stimulation of neutrophils with dansyl-DAG resulted in a large release of superoxide radicals from the cells. The physicochemical properties of dansyl-DAG and azido-DAG may allow one to label and follow specifically changes in the location of protein kinase C and to understand some aspects of its function and regulation.     

Ethanol induces release of arachidonic acid but not synthesis of eicosanoids in mouse peritoneal macrophages

Exposure of mouse peritoneal macrophages to ethanol induces a rapid release of arachidonic acid to the extracellular medium. All major classes of phospholipids, phosphatidylcholine, phosphatidylethanolamine and phosphatidylinositol contribute to this release. Ethanol-induced mobilization of arachidonic acid occurs by deacylation, but it is not accompanied by eicosanoid synthesis. These data suggest that at least two signals are necessary for the release and metabolism of arachidonic acid. Ethanol also activates a phospholipase C which hydrolyzes only phosphatidylinositol, and not its phosphorylated derivatives.     

Influence of Ca2+ and Mg2+ on the turnover of the phosphomonoester group of phosphatidylinositol 4-phosphate in human erythrocyte membranes.

In isolated erythrocyte membranes, increasing the free Mg2+ concentration from 0.5 to 10 mM progressively activates the membrane-bound phosphatidylinositol (PtdIns) kinase and leads to the establishment of a new equilibrium with higher phosphatidylinositol 4-phosphate (PtdIns4P) and lower PtdIns concentrations. The steady-state turnover of the phosphomonoester group of PtdIns4P also increases at high Mg2+ concentrations, indicating a simultaneous activation of PtdIns4P phosphomonoesterase by Mg2+. Half-maximum inhibition of PtdIns kinase occurs at 10 microM free Ca2+ in the presence of physiological free Mg2+ concentrations. Increasing free Mg2+ concentrations overcome Ca2+ inhibition of PtdIns kinase. In the presence of Ca2+, calmodulin activates Ca2+-transporting ATPase 5-fold, but does not alter pool size and radiolabelling of PtdIns4P. In intact erythrocytes, adding EGTA or EGTA plus Mg2+ and the ionophore A23187 to the external medium does not exert significant effects on concentration and radiolabelling of polyphosphoinositides when compared with controls in the presence of 1.4 mM free Ca2+.