Linkage and expression of the Eg locus controlling inclusion of β-glucuronidase into microsomes

In the mouse, one structural gene codes for the amino acid sequence of the -glucuronidase found in both lysosomes and microsomes. The function of a second, independently segregating locus, Eg, is required for the inclusion of -glucuronidase into microsomes. In microsomes, the enzyme, which contains four subunits, is found in a macromolecular complex with up to four additional protein chains; the attachment of these chains is defective in the Eg ⁰ mutant lacking microsomal glucuronidase. The Eg gene has now been linked with Es-1 (1.1±0.3% recombination) on chromosome 8. The -glucuronidase structural gene Gus is on chromosome 5. Thus the gene responsible for processing the polypeptide chain is not genetically linked to the gene directing the synthesisof the enzyme itself.This work was supported in part by the Training Program in Cancer Research, CAO 5016 16, and by U.S. Public Health Service Grant GM 19521.     

Studies on the interaction of bleomycin A2 with rat lung microsomes. III. Effect of exogenous iron on bleomycin-mediated DNA chain breakage

The interaction of bleomycin A₂ with rat lung microsomes results in bleomycin-mediated DNA chain breakage due to the mixed-function oxidase catalyzed activation of bleomycin. This study demonstrates that the addition of exogenous Fe³⁺ significantly enhances the bleomycin-mediated cleavage of DNA deoxyribose, that the enhancing effect of Fe³⁺ is maximum when a 1:1 ratio of bleomycin to Fe³⁺ is achieved and that either NADPH or NADH can serve as pyridine cofactors for this reaction. Since the activation of bleomycin can be facilitated by iron in the Fe²⁺ form, these observations support the hypothesis that the mixed-function oxidase system may serve to maintain either adventitious or exogenous iron in the Fe²⁺ form. In the absence of DNA, the interaction of bleomycin with rat lung microsomes results in the self-inactivation of bleomycin, a reaction which is also enhanced by the addition of exogenous Fe³⁺. Thus, the microsomal mixed-function oxidase system represents an efficient biological system for the ‘activation-inactivation’ of bleomycin.     

Phorbol ester and the actions of phosphatidylinositol 4,5-bisphosphate specific phospholipase C and protein kinase C in microsomes prepared from cultured cardiomyocytes

Microsomes were prepared from cultured neonatal rat cardiomyocytes. Incubation of microsomes in buffer containing 5µM CaCl₂, 5 mM cholate and 100 nM [3H-]Phosphatidylinosito14,5-bisphosphate (PtdIns(4,5) P₂) resulted in the formation of [³H-]InsP ₃. GTP-gamma-S (125 µM) stimulated the production of [³H-]InsP ₃. Microsomes prepared from phorbol ester-treated (100 nM phorbol 12-myristate 13-acetate, PMA) cardiomyocytes showed decreased activities of basal as well as GTP-gamma-S-stimulated [³H-]Ptdlns(4,5)P ₂ hydrolysis. In the microsomes a 15 kD protein was demonstrated to be the major substrate phosphorylated by intrinsic protein kinase C, which was activated by 0.5 mM Ca²⁺. Addition of phorbol ester (100 nM PMA) enhanced the ³²P-incorporation into the 15 kD protein. Protein kinase C, purified from rat brain, in the presence of Ca²⁺, diglyceride, and phosphatidylserine did not change the phosphorylation pattern any further. In conclusion, it was shown that phorbol ester pretreatment of neonatal rat cardiomyocytes reduces microsomel GTP-gamma-S-stimulated Ptdlns(4,5)P ₂-specific phospholipase C activity, as estimated with exogenous substrate, and that in cardiomyocyte microsomes phorbol ester activates protein kinase C-induced 15 kD protein phosphorylation. The results indicate that phorbol ester may down-regulate -adrenoceptor mediated Ptdlns(4,5)P ₂ hydrolysis by activation of protein kinase C-induced 15 kD protein phosphorylation.List of abbreviations ATP Adenosine 5-Trphosphate - CSU Catalytic Subunit of cyclic AMP-dependent protein kinase - DG Diacylglycerol - DMSO Dimethylsulfoxide - DTT DL-dithiothreitol - EDTA Ethylenedinitrilotetraacetic Acid - EGTA Ethyleneglycol-0,0-bis(aminoethyl)-N,N,N,N,-tetraacetic acid - GTP-gamma-S Guanosine 5-O-(3-thiotriphosphate) - HPTLC High Performance Thin Layer Chromatography - InsP ₃ Inositol monophosphate - InsP ₂ Inositol bisphosphate - InsP ₃ Inositol trisphosphate - MES 2-Morpholinoethanesulfonic acid - MOPS 3-[N-Morpholino]Propanesulfonic acid - PAGE Polyacrylamide-gel Electrophoresis - PKC Protein Kinase C - PLase C Phospholipase C - PMA Phorbol 12-Myristate 13-Acetate - PMSF Phenylmethylsulfonyl Fluoride - PtdSer Phosphatidylserine - PtdIns Phosphatidyl inositol - PT Pertussis Toxin - Ptdlns(4)P Phosphatidylinositol 4-monophosphate - Ptdlns (4,5)PZ-Phosphatidylinositol4,5-bisphosphate - SDS-Sodium Dodecyl Sulfate Tris-Tris(hydroxymethyl) aminomethane     

Calcium and plant organelles

Abstract. The role of intracellular organelles in the regulation of cytosolic Ca²⁺ levels and whether changes in these levels affect organelle metabolism is considered. We have assessed the biochemical properties of the Ca²⁺ transporting systems in mitochondrial, chloroplast and microsomal fractions. It is proposed that although all of these organelles can transport Ca²⁺ to varying extents it would appear that in some tissues at least mitochondria do not play a significant role in the maintenance of cytosolic Ca²⁺. The most important Ca²⁺ transporting systems are probably the ATP dependent Ca²⁺ extrusion across the plasma membrane and Ca²⁺ uptake by endoplasmic reticulum, as well as light driven Ca²⁺ uptake by chloroplasts. Changes in cytoplasmic [Ca²⁺] do appear to regulate the activity of NAD kinase in chloroplasts, the mitochondrial external NADH dehydrogenase and intra-mitochondrial glutamate dehydrogenase, all of which play a key role in plant cell metabolism. Since some of these enzymes are affected by primary stimuli such as light or hormones, it is concluded that Ca²⁺ may act as a second messenger mediating some of the primary responses.     

Signal recognition particle triggers the translocation of storage globulin polypeptides from field beans (Vicia faba L.) across mammalian endoplasmic reticulum membrane

Hybridization-selected mRNAs coding for individual storage globulin polypeptides of field beans (Vicia faba L.) were translated in a cell-free system. Added mammalian signal recognition particle (SRP) recognizes cleavable signal peptides of the major vicilin and both legumin polypeptide precursors and induces translational arrest. The latter can be released by potassium-washed membranes (K-RM) leading to shortened polypeptides protected against proteases. Thus, SRP and K-RM function in a similar way with plant polypeptides as described for mammalian secretory proteins [(1981) J. Cell Biol. 91, 557-561]. Obviously, the initial steps in the biosynthesis and processing of plant storage globulin polypeptides are principally identical to those of animal secretory proteins.     

In vitro labeling of peroxisomal cholesterol with radioactive precursors

Peroxisomes isolated from rat liver were incubated with [³H]squalene and [³H]mevalonate and the subsequent incorporation of radioactivity into cholesterol studied. The isolated lipids became labeled after incubation with both precursors. In contrast to findings with microsomes, trypsin and detergent treatment of peroxisomes did not influence the rate of cholesterol synthesis. In addition, the luminal content of peroxisomes could alone mediate this synthetic process. Upon treatment of rats with various inducers of peroxisomes and of the endoplasmic reticulum, as well as upon feeding with cholesterol and cholestyramine, large differences in the pattern ofin vitro incorporation of [³H]mevalonate into the cholesterol of peroxisomes and microsomes were observed. Injection of this precursor also resulted in high initial labeling of peroxisomal cholesterolin vivo. These experiments indicate that cholesterol synthesis may also occur in peroxisomes.     

亚硒酸钠诱发大鼠白内障晶状体前列腺素生物合成的研究

 用亚硒酸钠诱发大鼠产生白内障后,将晶状体微粒体与外源性花生四烯酸共同孵育,用放射免疫方法测定白内障晶状体前列腺素E_2(PGE_2)及前列腺素F_2α(PG-F_2α)的生物合成情况,并与正常晶状体进行了比较,结果表明大鼠晶状体具有酶促合成PGs的能力。正常晶状体及白内障晶状体合成PGE_2的能力分别为687.75±113.97及1095.00±79.39pg/100mg晶状体湿重/15分钟,PGE_2α则分别为51.45±36.72及158.83±115.94pg/100mg晶状体湿重/15分钟(平均数±S.D.)。这说明大鼠白内障晶状体合成PGs的能力明显增高,与正常晶状体相比有显著性差异(PGE_2P<0.001,PGF_2αP<0.02)。在前2次注射亚硒酸钠后,大鼠白内障晶状体PGs的合成能力逐渐高于正常晶状体,并随注射亚硒酸钠的次数增加和白内障晶状体混浊程度加重,PGs在晶状体内的含量增加。     

The effect of dietary polyenylphosphatidylcholine on microsomal delta-6-desaturase activity, fatty acid composition, and microviscosity in rat liver under oxidative stress

Polyenylphosphatidylcholine is a choline-glycerophospholipid containing up to 80% of total fatty acids as linoleic acid and may be an important factor in ensuring normal functioning of cell membranes. We tested the effect of a polyenylphosphatidylcholine-supplemented diet and compared it with both a trilinolein-supplemented and a laboratory chow diet on the fatty acid composition, microviscosity, and delta-6-desaturase activity of liver microsomal membranes of 12-month-old rats, in the absence or presence of oxidative stress induced by adriamycin. Polyenylphosphatidylcholine- and trilinolein-supplemented diets showed a similar increase in linoleic acid content and delta-6-desaturase activity in liver microsomes, indicating that low amounts of linoleic acid are able to partially restore the enzyme activity in old rats, independent of the source of linoleic acid. After adriamycin treatment, delta-6-desaturase activity increased in polyenylphosphatidylcholine and trilinolein groups, indicating a protective mechanism against the damage induced by polyunsaturated fatty acid peroxidation. The measurement of malondialdehyde production showed a protective effect on adriamycin-induced lipid peroxidation by polyenylphosphatidylcholine supplementation only. Microsomal membrane microviscosity did not change independent of diet and adriamycin treatment, suggesting that the response of microsomes to lipid peroxidation might be the maintenance of a given membrane order. Administration of polyenylphosphatidylcholine can prevent or minimize the liver damage induced by adriamycin treatment.