The mechanism by which secretory proteins are segregated within the cisternal space of microsomal vesicles was studied using dog pancreas mRNA which directs the synthesis of 14 well-characterized nonglycosylated pancreatic exocrine proteins . In the absence of microsomal membranes, each of the proteins was synthesized as larger polypeptide chains (presecretory proteins), 1,000-2,000 daltons larger than their authentic counterparts as judged by polyacrylamide gel electrophoresis in SDS. Conditions-optimal for the study of reconstituted rough microsomes in the reticulocyte lysate system were examined in detail using mRNA and microsomal membranes isolated from dog pancreas . Functional reconstitution of rough microsomes was considerably more efficient in the presence of micrococcal nuclease-treated membranes than in the presence of EDTA-treated membranes . Analysis for segregation of nascent secretory proteins by microsomal vesicles, using post-translational incubation in the presence of trypsin and chymotrypsin, 50 g,g/ml each, was shown to be inadequate, because of the disruption of vesicles by protease activity . Addition of 1-3 mM tetracaine or 1 mM dibucaine stabilized microsomal membranes incubated in the presence of trypsin and chymotrypsin at either 0°or 22°C. Each of the pancreatic presecretory proteins studied was correctly processed to authentic secretory proteins by nuclease-treated microsomal membranes, as judged by both one-dimensional and two-dimensional gel electrophoresis . Post-translational addition of membranes did not result in either segregation or processing of nascent polypeptide chains . Posttranslational proteolysis, carried out in the presence of 3 mM tetracaine, indicated that each of the 14 characterized dog pancreas secretory proteins was quantitatively segregated by nucleasetreated microsomal vesicles . Segregation of nascent secretory proteins was irreversible, since radioactive amylase, as well as the other labeled secretory proteins, remained quantitatively sequestered in microsomal vesicles during a 90-min incubation at 22°C after the cessation of protein synthesis. Studies employing synchronized protein synthesis and delayed addition of membranes indicated that all pancreatic presecretory proteins contain amino terminal peptide extensions . These peptide extensions are shown to mediate the cotranslational binding of presecretory proteins to microsomal membranes and the transport of nascent secretory proteins to the vesicular space. The maximum chain lengths which, during synthesis, allow segregation of nascent polypeptide chains varied between 61 (pretrypsinogen 2 + 3) and 88 (preprocarboxypeptidase A1) amino acid residues among dog pancreas presecretory proteins . Reconstitution studies using homologous and heterologous mixtures of mRNA (dog, guinea pig, and rat pancreas ; rat liver) and micrococcal nuclease-treated microsomal membranes (dog, guinea pig, and rat liver; dog pancreas), in the presence of placental ribonuclease inhibitor, suggest that the translocation mec...

The detection of bacterial lipoic acid by a modified g.c.-m.s. procedure is reported. Cells were hydrolysed in HCl to release protein-bound lipoic acid, which, after extraction into benzene, was reduced with NaBH4. The dihydrolipic acid so generated was then isolated by covalent chromatography on dithiolspecific p-aminophenylarsenoxide-agarose and, after elution by 2,3-dimercaptopropane-1-sulphonic acid and extraction into benzene, was allowed to O2-oxidize to the disulphide form. The isolated lipoic acid was allowed to react with diazomethane, and the methyl ester so produced was detected by g.c.-m.s. Analysis of the mass spectrum showed the characteristic molecular ion and seven fragmentation ions, which, along with the identification of those ions retaining the two sulphur atoms, allows the definitive detection of lipoic acid. The methodology has been successfully tested with authentic lipoic acid, the 2-oxoglutarate dehydrogenase multienzyme complex and with whole cells of Escherichia coli. In addition, it has been used to search for and identify lipoic acid in the archaebacterium Halobacterium halobium. The significance of this discovery and the possible roles of the cofactor in H. halobium are discussed.

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Phosphoglyceromutase (PGM) from chicken breast muscle was titrated with p-mercuribenzoate (PMB), 5,5'-dithiobisnitrobenzoate (Nbs2), N-ethylmaleimide (NEM), iodoacetate and iodoacetamide. The effect of all of the sulfhydryl reagents, with the exception of NEM was to cause a loss in enzymatic activity. Addition of KCN following reaction with Nbs2 resulted in the recovery of a small amount of enzymatic activity. In the absence of substrate (3-phosphoglyceric acid) or cofactor (2,3-diphosphoglyceric acid) and in the presence or absence of 6 M guanidine hydrochloride, six sulfhydryl groups per mole of enzyme were titrated with PMB.

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