RESUMEN
A Ca2+-independent phospholipase A2 (PLA2) maximally active at pH 4 and specifically inhibited by the transition-state analogue 1-hexadecyl-3-trifluoroethylglycero-sn-2-phosphomethanol (MJ33) was isolated from rat lungs. The sequence for three internal peptides (35 amino acids) was used to identify a 1653-base pair cDNA clone (HA0683) from a human myeloblast cell line. The deduced protein sequence of 224 amino acids contained a putative motif (GXSXG) for the catalytic site of a serine hydrolase, but showed no significant homology to known phospholipases. Translation of mRNA produced from this clone in both a wheat germ system and Xenopus oocytes showed expression of PLA2 activity with properties similar to the rat lung enzyme. Apparent kinetic constants for PLA2 with dipalmitoylphosphatidylcholine as substrate were Km = 0.25 mM and Vmax = 1.89 nmol/h. Activity with alkyl ether phosphatidylcholine as substrate was decreased significantly compared with diacylphosphatidylcholine. Significant lysophospholipase, phospholipase A1, or 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine acetylhydrolase activity was not observed. Enzyme activity was insensitive to p-bromophenacyl bromide, bromoenol lactone, trifluoromethylarachidonoyl ketone, mercaptoethanol, and ATP, but was inhibited by MJ33 and diethyl p-nitrophenyl phosphate, a serine protease inhibitor. SDS-polyacrylamide gel electrophoresis with autoradiography of the translated [35S]methionine-labeled protein confirmed a molecular mass of 25.8 kDa, in good agreement with the enzyme isolated from rat lung. By Northern blot analysis, mRNA corresponding to this clone was present in both rat lung and isolated rat granular pneumocytes. These results represent the first molecular cloning of a cDNA for the lysosomal type Ca2+-independent phospholipase A2 group of enzymes.
Asunto(s)
ADN Complementario/química , Lisosomas/enzimología , Fosfolipasas A/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , Electroforesis en Gel de Poliacrilamida , Humanos , Concentración de Iones de Hidrógeno , Pulmón/enzimología , Datos de Secuencia Molecular , Oocitos/enzimología , Mapeo Peptídico , Fosfolipasas A1 , Fosfolipasas A2 , Biosíntesis de Proteínas , Ratas , Xenopus laevisRESUMEN
In Clostridium thermoaceticum, the synthesis of acetyl-CoA from methyl tetrahydrofolate occurs via a series of enzymatic reactions involving methyl transferase, corrinoid/Fe-S protein (corrinoid), carbon monoxide dehydrogenase (CODH) and ferredoxin. We have investigated the possibility of one or more of these proteins existing as multi-enzyme complexes in vivo with higher catalytic activity. A protein complex consisting of CODH and corrinoid was isolated from the cell-free extracts of Clostridium thermoaceticum. The acetyl-CoA synthesis was found to be approximately 1.8-fold higher with the complex than that observed with the isolated protein components. HPLC gel filtration analyses of the native and DTE reduced complex suggested that the CODH:corrinoid complex is held together primarily by an inter disulfide bond. By differential labeling of thiols with [14C]N-ethylmaleimide it was found that Cys-506 of the alpha subunit of CODH was involved in the disulfide linkage with the corrinoid of the complex.
Asunto(s)
Aldehído Oxidorreductasas/metabolismo , Clostridium/enzimología , Cisteína , Proteínas Hierro-Azufre/metabolismo , Vitamina B 12/metabolismo , Acetilcoenzima A/biosíntesis , Aldehído Oxidorreductasas/química , Secuencia de Aminoácidos , Sitios de Unión , Catálisis , Cromatografía Líquida de Alta Presión , Corrinoides , Disulfuros/metabolismo , Ditioeritritol , Datos de Secuencia Molecular , Complejos Multienzimáticos/metabolismoRESUMEN
Significant rise in urea and D-aspartate aminotransferase and inhibition of alkaline phosphatase in serum were observed in rats fed garlic extract (2 ml/100 g body wt, intragastrically) for 10 days. The liver showed histological changes. Garlic oil feeding (10 mg/100 g body wt, intragastrically) after 24 hr fasting was found lethal. The cause of death appears to be acute pulmonary oedema. On histological examination, all the organs of the dead rats revealed severe congestion. However, similar feeding of garlic oil was well tolerated by rats in the fed state. Also, 24 hr fasted rats could tolerate this dose of garlic oil, provided they were previously adapted to garlic oil feeding.