Intact chromaffin granules isolated from bovine adrenal medulla were incubated at 37TC for up to 22 hr. Processing of enkephalin-containing (EC) peptides in the granules was followed by the change in their size distribution as shown by chromatography on Sephadex G-75 columns. A gradual shift toward lower molecular weight EC peptides was observed during the incubation, indicating processing of the higher molecular weight to lower molecular weight EC peptides. The total amount of [Met]-enkephalin, free and in peptide linkage, remained constant indicating that little -or no nonspecific degradation occurred during the experiment. HPLC resolution of the fraction containing the low molecular weight EC peptides showed that free enkephalins as well as [Met]enkephalin-Arg6-Phe7 and [Met]enkephalin-Arg6-Gly7-Leu8 accumulated while [Met]enkephalin-Arg6 and [Metlenkephalin-Lys6 disappeared. All the above data indicate the press ence of an atypical trypsin activity and the presence of a carboxypeptidase B-like activity within the granules. From the rates of accumulation of the low molecular weight EC peptides and the disappearance of the higher molecular weight EC peptides, a processing rate of 65-70 pmol/g tissue per hr was estimated, which calculates to a lifetime of 6-8 days for EC peptides in the granules. Under steady-state conditions this rate of processing appears to be too low to produce significant amounts of free enkephalins from larger EC peptides. This is well in accord with previous observations that relatively small amounts of free enkephalins are found in bovine adrenal medulla.Enkephalin-containing (EC) peptides represent one of the more recently discovered groups of peptide hormones (1,2). In mammals there are at least three families of EC peptides, each derived through processing of distinct prohormones, coded for by three distinct genes (3-6). As in many other prohormones, internal sequences of the active peptides are bracketed by paired basic amino acid residues, lysine or arginine, that serve as signals for processing. Although the sites of cleavage are known, the responsible enzymes have not yet been. characterized. Cleavage at paired basic residues by a "trypsin-like" enzyme, followed by removal of the generated carboxyl-terminal lysine or arginine by a "carboxypeptidase B-like" enzyme, has been suggested and there have been reports of partial purification of such enzymatic activities from extracts of bovine adrenal chromaffin granules (7-11). However, the reported activities were low and contamination by lysosomal proteases could not be ruled out entirely. In addition, a purely trypsin-like activity would not possess the specificity required for this processing, because single internal basic residues [for example Arg6 of the EC octapeptide (12) and heptapeptide (13) Processing of EC Peptides in Intact Chromaffin Granules. The 1.8 M sucrose layer of the gradient was diluted with 0.3 M sucrose to a final concentration of 0.6 M, placed on top of a 2.2 M sucrose layer (0.5 ml), and recentrifuged...

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Incubation of purified rat brain tubulin with cholera toxin and radiolabeled [32P] or [8‐3H]NAD results in the labeling of both alpha and beta subunits as revealed on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE). Treatment of these protein bands with snake venom phosphodiesterase resulted in quantitative release of labeled 5′‐AMP, respectively labeled with the corresponding isotope. Two‐dimensional separation by isoelectric focusing and SDS‐PAGE of labeled and native tubulin revealed that labeling occurs at least in four different isotubulins. The isoelectric point of the labeled isotubulins was slightly lower than that of native purified tubulin. This shift in mobility is probably due to additional negative charges involved with the incorporation of ADP‐ribosyl residues into the tubulin subunits. SDS‐PAGE of peptides derived from [32P]ADP‐ribosylated alpha and beta tubulin subunits by Staphylococcus aureus protease cleavage showed a peptide pattern identical with that of native tubulin. Microtubule‐associated proteins (MAP1 and MAP2) of high molecular weight were also shown to undergo ADP‐ribosylation. Incubation of permeated rat neuroblastoma cells in the presence of [32P]NAD and cholera toxin results in the labeling of only a few cell proteins of which tubulin is one of the major substrates.

A transient rise in cyclic guanosine 3' : 5' monophosphate (c-GMP) in the liver was observed in rats in vivo 10--20 min after partial hepatectomy. A similar increase in c-GMP in the liver was also found in rats in vivo 15 min after infusion of TGH solution (a mixture of triiodothyronine, glucagon, and heparin). In both cases, inductions of ornithine decarboxylase [EC 4.1.1.17] and tyrosine aminotransferase [EC 2.6.1.5] were found 4 hr after the beginning of the experiments. Later, 22 hr after the surgical intervention or hormone infusion, thymidine kinase [EC 2.7.1.21] was activated and liver slices were able to incorporate [3H]thymidine into DNA. These biochemical phenomena were observed commonly in regenerating liver as well as in the liver of rats infused with TGH solution. c-GMP, but not c-AMP, could induce ornithine decarboxylase and tyrosine aminotransferase in isolated, perfused liver.

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