Active form and total activity of pyruvate dehydrogenase were measured in rat liver homogenates. The activity obtained immediately after homogenization was considered to represent the active form, i. e. dephospho pyruvate dehydrogenase, originally present in the liver. Enzyme activity found after incubation of the homogenate with Mg2+ and partially purified pyruvate dehydrogenase phosphatase from pig heart, whereby inactive dehydrogenase is dephosphorylated to give the active form, was regarded as total pyruvate dehydrogenase activity. In the livers from normal fed rats, the active form accounts for only one sixth of total pyruvate dehydrogenase activity. This differs from other tissues such as heart muscle, kidney, brain and adipose tissue where about two thirds of total pyruvate dehydrogenase are present as the active form To elucidate the possible role of pyruvate dehydrogenase interconversion in the regulation of pyruvate metabolism in liver, the activity of active and total pyruvate dehydrogenase were determined in livers of rats subjected to various metabolic conditions. After fasting and refeeding with glucose as well as after treatment with nicotinic acid or insulin there were significant changes of active dehydrogenase activity without gross alterations of total activity. In general, metabolic states associated with decreased plasma fatty acids resulted in an increase of active pyruvate dehydrogenase whereas a rise in plasma fatty acids led to a lowering. It is concluded that in liver, similar to heart muscle and kidney (Wieland et al., 1971), fatty acids play an important role in the control of pyruvate dehydrogenase interconversion. The significance of this mechanism for the regulation of pyruvate metabolism in liver relative to the feedback control of pyruvate dehydrogenase by acetyl-CoA is discussed.The finding that mammalian pyruvate dehydrogenase is an interconvertible enzyme [l --61 has raised appreciable interest regarding the physiological significance of this process for the regulation of pyruvate metabolism. It has been demonstrated that pyruvate dehydrogenase in various rat tissues occurs in the active and the inactive form and that the ratio of the two changes very markedly after special treatment of the animals. Thus, for instance, two thirds of total pyruvate dehydrogenase is present as the active form in heart muscle and kidney from normal fed rats. It decreases to one sixth of total dehydrogenaseactivity after fasting the animals for 24 h and returns to its original level 2 h after refeeding glucose [7-91. From these and other studies with diabetic rats it became apparent that situations of increased supEnzymes. Lactate dehydrogenase (EC 1.1.1.27); Arylamine acetyltransferase (EC 2.3.1.5).ply of free fatty acids due to increased lipolysis are characterized by low levels of the active dehydrogenase and vice versa. Furthermore perfusion experiments of the isolated rat heart [lo] strengthened the view that fatty acid oxidation, by some still unknown mechanism, directs the pyruvate dehydrog...

Immunoprecipitation and tryptic peptide analysis of newly synthesized proteins from rat islets have identified an 18,000 molecular weight (MW) protein as proglucagon. Conversion of this precursor was kinetically similar to the conversion of proinsulin and resulted in the formation of both pancreatic glucagon and a 10,000-MW protein lacking this hormonal sequence.

Newly synthesized rat islet proteins have been analyzed by polyacrylamide slab gel electrophoresis and fluorography. A minor component having an apparent molecular weight of 11,100 was identified as preproinsulin by the sensitivity of its synthesis to glucose, the pattern of NH2-terminal leucine residues, and the rapidity of its appearance and disappearance during incubation of islets or islet cell tumors. A small amount of labeled peptide material which may represent the excised NH2-terminal extension of preproinsulin or its fragment was also detected. The kinetics of formation and processing of the preproinsulin fraction were complex, consisting of a rapidly turning over component having a half-life of about 1 min and a slower minor fraction that may have bypassed the normal cleavage process. The electrophoretic resolution of the preproinsulin and proinsulin fractions into two bands each is consistent with the presence of two closely related gene products in rat islets rather than intermediate stages in the processing of these peptides.Many recent studies have shown that the in vitro translation products of the mRNAs for various secretory proteins carry a 20-to 30-residue NH2-terminal extension that contains a high proportion of hydrophobic amino acids (1-4). This region is believed to assist in the formation of the ribosome-membrane junction leading to the vectorial discharge and segregation of the nascent polypeptides (1, 2). Several studies with reconstituted systems have provided additional evidence that this signal sequence is rapidly cleaved from the nascent secretory product before polypeptide chain completion by a protease(s) associated with the rough microsomes (5, 6), thus accounting for the apparent difficulty in detecting these presecretory forms in intact tissues (7,8). In the present studies, intact rat islets of Langerhans or insulinoma cells were incubated for brief periods with labeled amino acids and then analyzed by sodium dodecyl sulfate (NaDodSO4)/acrylamide slab gel electrophoresis and fluorography in an attempt to detect preproinsulin (3, 9, 10) and to assess its kinetic behavior as a precursor of proinsulin.METHODS AND MATERIALS Tissue Sources. Islets were isolated from Sprague-Dawley rats as described (11). Lobules of exocrine pancreatic tissue were collected during this isolation procedure. Insulin-producing tumors were either maintained from an x-ray-induced insulinoma in NEDH inbred rats (12) or induced by injection of streptozotocin and nicotinamide in Holtzman rats (13). Samples of 25-100 islets or equivalent amounts of exocrine tissue, or of tumor cells, were incubated in 50 Ml of Hanks' buffer supplemented with 2.5 or 25 mM glucose, amino acids according to Eagle's minimal essential medium (omitting leucine), and 0.5 ,MCi of [3H]leucine per islet, at 370 ([3,4,5-The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate...

Colchicine inhibited amylase secretion by isolated rat parotid glands only 6 h after administration of the drug in vivo. This delayed effect was not the result of the inability of the drug to reach its reaction site. When parotid glands were emptied of their secretory granules by isoproterenol treatment, the subsequent replenishment of cells with granules was inhibited by colchicine. Colchicine concomitantly produced alterations of the Golgi complexes, the cisternae of which were reduced in size and surrounded by clusters of microvesicles.

A B S T R A C T Livers of normal mice were prefused in situ and the secretion of newly synthesized (i.e. labeled) proteins into the perfusate were measured. In control livers, the secretion of newly synthesized proteins was found to be linear with time. In marked contrast, when livers were perfused with vinblastine, vincristine, or colchicine, drugs known to interfere with the hepatic microtubular system, the release of newly synthesized proteins was either strongly inhibited or completely suppressed although total hepatic protein synthesis (estimated by the incorporation of labeled amino acids into hepatic plus perfusate proteins) remained unaltered. Chromatographic separation of the various secreted proteins showed that the release of albumin, globulins, and small polypeptides was decreased to a similar extent by vincristine or colchicine. In the particular case of albumin, it was further observed that total (i.e. liver plus perfusate) labeled amino acid incorporation into albumin was not altered by either vincristine or colchicine, whereas the incorporation of these amino acids into liver albumin was markedly increased but incorporation into perfusate albumin was decreased, suggesting that the translocation of this particular protein from the liver to the perfusate had been affected by the presence of these drugs. It is proposed that the functional integrity of microtubules is necessary for the intracellular movement and eventual release of albumin and other proteins by the liver, and suggested that microtubules might possibly be a site of regulation of hepatic protein secretion.