Ethanol-elicited alterations in the oligomycin sensitivity and structural stability of the mitochondrial F0 . F1 ATPase

Liver mitochondria from rats fed ethanol chronically demonstrated a 35% decrease in mitochondrial ATPase activity. Moreover, the ATPase activity was inhibited only 61% by addition of oligomycin. Treatment of mitochondria from ethanol-fed rats with the detergent, Lubrol-WX, caused the release of 36% of the F1 from the resulting inner membrane particles. In comparison, only 5% of the F1 was dissociated when control mitochondria were subjected to the Lubrol treatment. However, when the units of ATPase activity from the supernatant and particles obtained after Lubrol treatment were added together, their sums were equivalent in preparations from control and ethanol-fed animals. Moreover, polyacrylamide gel electrophoresis analyses indicated equal amounts of the alpha + beta subunits of F1 in mitochondria from control and ethanol-fed rats. Reconstitution experiments with urea particles and F1 prepared from both control and ethanol mitochondria revealed a decrease in oligomycin sensitivity which could be attributed to an alteration in the functioning of either the oligomycin sensitivity conferring protein or a membrane sector subunit that interacts with oligomycin. Analysis by reconstitution also demonstrated that there were no ethanol-elicited alterations in the properties of the F1 portion of the ATP synthase complex. These observations indicate that the activity of the ATP synthase complex is altered significantly by ethanol-elicited changes in the functioning of those polypeptides involved in modulating both oligomycin sensitivity and the association of F1 with membrane sector subunits.     

Evidence for ligand- and pH-dependent conformational changes in liposome-associated mannose 6-phosphate receptor

Digestion of mannose 6-phosphate receptor preparations with trypsin and chymotrypsin was found to produce characteristic polypeptide "fingerprints" of the receptor. Lengthy digestions with both proteases produced fragments of the receptor which appeared to be resistant to further proteolysis. This suggests the occurrence of distinct structural domains within the receptor protein. Liposome-associated mannose 6-phosphate receptor preparations were made using phosphatidylcholine and purified receptor. Receptor molecules were oriented in the liposomes with greater than 90% of ligand-binding sites on the outside surfaces of the liposomes. Liposome-associated mannose 6-phosphate receptor was labeled with 125I at pH 7.5 and 5.4 in the presence or absence of sugar phosphate ligands. Limited trypsin digestion was used to analyze 125I-labeled receptor preparations. Peptide fragments having molecular weights of approximately 60,000 and 23,000 were found to be most prominently labeled. At pH 7.5 the labeling of the 60-kDa fragment was enhanced strongly by the presence of mannose 6-phosphate. This ligand-induced enhancement of 125I-labeling was saturable, had a K1/2 value of 0.4 mM, required the presence of phosphatidylcholine, and did not occur at pH 5.4. Incorporation of 125I into both polypeptide fragments was significantly reduced at pH 5.4. These results suggest the occurrence of ligand- and pH-dependent conformational changes in domains of the mannose 6-phosphate receptor which may be necessary for proper function of this membrane receptor in receptor-mediated endocytosis.     

Synthesis and processing of alpha-galactosidase A in human fibroblasts. Evidence for different mutations in Fabry disease

The synthesis and processing of the human lysosomal enzyme alpha-galactosidase A was examined in normal and Fabry fibroblasts. In normal cells, alpha-galactosidase A was synthesized as an Mr = 50,500 precursor, which contained phosphate groups in oligosaccharide chains cleavable by endoglucosaminidase H. The precursor was processed via ill-defined intermediates to a mature Mr 46,000 form. Processing was complete within 3-7 days after synthesis. In the presence of NH4Cl and in I-cell fibroblasts, the majority of newly synthesized alpha-galactosidase A was secreted as an Mr = 52,000 form. For comparison, the processing and stability of alpha-galactosidase A were examined in fibroblasts from five unrelated patients with Fabry disease, which is caused by deficient alpha-galactosidase A activity. In one cell line, synthesis of immunologically cross-reacting polypeptides was not detectable. In another, the synthesis, processing, and stability of alpha-galactosidase A was indistinguishable from that in normal fibroblasts. In a third Fabry cell line, the mutation retarded the maturation of alpha-galactosidase A. Finally, in two cell lines, alpha-galactosidase A polypeptides were synthesized that were rapidly degraded following delivery to lysosomes. These results clearly indicate that Fabry disease comprises a heterogeneous group of mutations affecting synthesis, processing, and stability of alpha-galactosidase A.     

Lipid asymmetry induced by transmembrane pH gradients in large unilamellar vesicles

We have investigated the influence of transmembrane pH gradients across large unilamellar vesicle membranes on the transbilayer distributions of simple lipids with weak base and weak acid characteristics. Trinitrobenzenesulfonic acid labeling results consistent with a rapid and complete migration of stearylamine and sphingosine to the inner monolayer of the large unilamellar vesicles are observed when the large unilamellar vesicles' interior is acidic. Alternatively, when the vesicle interior is basic, oleic and stearic acid cannot be removed by external bovine serum albumin, indicating a localization in the inner monolayer. Moreover, effects corresponding to the decrease in external surface charge predicted upon the migration of stearylamine or stearic acid to the inner monolayer are readily detected employing ion exchange chromatography. These results are consistent with transbilayer distributions of these agents dictated by a Henderson-Hasselbach equilibrium. The possible implications for metabolic regulation by pH gradients, as well as factors giving rise to phospholipid transbilayer asymmetry, are discussed.     

Two independent growth factor-generated signals regulate c-fos and c-myc mRNA levels in Swiss 3T3 cells

Polypeptide growth factors that stimulate cell proliferation bind to cell surface receptors and activate intracellular signal transduction pathways. One major signalling pathway, initiated by phosphatidylinositol (PI) turnover, involves activation of protein kinase C. Some polypeptide growth factors, including mitogens that activate protein kinase C, induce a rapid increase in expression of the proto-oncogenes, c-myc and c-fos. In order to characterize the signal transduction pathways responsible for proto-oncogene activation, we treated Swiss 3T3 cells with the tumor promoter phorbol dibutyrate to generate cells deficient in protein kinase C. These cells were then stimulated with platelet extract, bombesin, or epidermal growth factor (EGF) and the levels of c-myc and c-fos mRNA were determined. Platelet extract or bombesin, which stimulate PI turnover, were substantially weaker inducers of c-myc and c-fos mRNA levels in the protein kinase C-depleted cells, although some variability with platelet extract was noted. EGF, which does not stimulate PI turnover in several cell systems, was by contrast a potent inducer of both proto-oncogenes whether or not the cells were deficient in protein kinase C. Pretreatment of cells with phorbol dibutyrate caused little or no change in the basal levels of c-myc or c-fos mRNA, but led to a small but significant increase in basal levels of ornithine decarboxylase mRNA. These results demonstrate that EGF and growth factors that activate PI turnover induce expression of the c-myc and c-fos proto-oncogenes through different pathways.     

Stimulated neutrophils produce an ethanolamine plasmalogen analog of platelet-activating factor

Human neutrophils stimulated by ionophore A23187 incorporate [3H]acetate into platelet-activating factor and an additional product which is chromatographically similar to phosphatidylethanolamine and accounts for approximately 25% of the [3H]acetate-containing lipids. Three general approaches indicated the sn-1 moiety of the unknown phospholipid is primarily alk-1'-enyl-linked: 1) approximately 80% of the intact phospholipid as well as its derivatives was highly sensitive to hydrolysis by HCl, 2) 80% of the product which resulted from treating the unknown with phospholipase C and acetylating the free hydroxyl group at the sn-3 position, chromatographed with authentic 1-O-alk-1'-enyl-2,3-diacetylglycerol, and 3) catalytic hydrogenation of the diacetylglycerol product described in 2) resulted in a product which chromatographed with alkyldiacetylglycerol and was not sensitive to strong acid. Treatment of the intact phospholipid with phospholipase A2 resulted in the release of 88% of the radiolabel into the acidified aqueous phase of the extraction mixture, indicating the moiety in the sn-2 position remained as acetate and had not been elongated to fatty acid. The head group was determined to be phosphoethanolamine based upon its complete conversion to the dinitro- and trinitrophenyl derivatives by the amine-derivatizing reagents fluorodinitrobenzene and trinitrobenzenesulfonic acid, respectively. From these data is was concluded that the unknown product is 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine (80%), and 1-O-alkyl-2-acetyl-sn-glycero-3-phosphoethanolamine (10%). Sonicates prepared from neutrophils stimulated with ionophore A23187 contained an acetyltransferase activity capable of utilizing 1-O-alk-1'-enyl-2-lyso-sn-glycero-3-phosphoethanolamine and [14C]acetyl-CoA to produce the product identified as 1-O-alk-1'-enyl-2-acetyl-sn-glycero-3-phosphoethanolamine.     

Precursor supply for insect juvenile hormone III biosynthesis in a cockroach

The biosynthesis of the sesquiterpenoid juvenile hormone III (JH III) was studied using corpora allata of the cockroach Diploptera punctata incubated in vitro and a radiochemical assay for the hormone produced. The influence of several exogenous precursors such as glucose, trehalose, acetate, amino acids, and mevalonate on JH synthetic rates was studied. Glucose or trehalose were needed for an optimal rate of JH synthesis. Highest rates were achieved at trehalose concentrations below the normal hemolymph levels (35-40 mM). About one-third of the glucose utilized for the biosynthesis of JH III was metabolized through a pentose pathway, but acetyl-CoA derived from glucose was significantly diluted by acetyl-CoA from other sources. Amino acids provided both a source of carbon for JH III synthesis and a source of energy that allowed JH III synthesis from acetate and stimulated JH III synthesis from glucose. Acetate was a poor substrate, because it could not support JH III synthesis in long term incubations. The incorporation of exogenous mevalonate into JH III was dependent on the physiological state of the glands, but there was a significant dilution with endogenous mevalonate. This dilution reflected in part the poor penetration of mevalonate into the corpora allata cells, because JH synthesis in mevinolin-treated cells was not fully rescued by mevalonate.