Purification of porcine proinsulin by high-performance liquid chromatography.

A procedure has been developed for purification of porcine proinsulin by high-performance liquid chromatography from a preparation obtained as a side product during the Sephadex G-50 gel filtration of an impure porcine insulin preparation. Reversed-phase chromatography was carried out on octadecylsilica as the stationary phase with graded mixtures of acetonitrile or methanol-acetonitrile and phosphate buffer pH 2.4 as the mobile phase. The crude preparation separated into five different groups of proteins, the proinsulin-containing peak being identified by the co-eluting internal proinsulin marker. After purification by conventional procedures (separation, pooling, freeze drying, desalting, reprecipitation and drying) this peak fraction was rechromatographed by high-performance liquid chromatography (for final purification) to give a single peak protein which had identical electrophoretic mobility to that of commercial porcine proinsulin, and which converted to a protein with electrophoretic mobility similar to that of porcine insulin.

Comparison of carboxymethyl-cellulose cation-exchange chromatography and high-performance liquid chromatography in the purification of guinea-pig insulin from pancreatic extracts.

Guinea-pig insulin was purified from pancreatic extracts either by carboxymethyl-cellulose cation-exchange chromatography with a sodium chloride gradient or by high-performance liquid chromatography (HPLC) on octadecyl silica with mixtures of acetonitrile and phosphate buffer. HPLC proved to be superior to ion-exchange chromatography in the purification of insulin with respect both to time saving and to the purity of the product.

Cation-activated phosphatase activities in islet cell plasma membrane preparations.

Pancreatic islets from rats or ob/ob mice were homogenized and fractionated either by a two-step or one-step sucrose gradient centrifugation. A plasma membrane enriched fraction was obtained at a sucrose density of about 1.10. The distribution of the plasma membrane probe 125I-wheat germ agglutinin was parallel to that of other plasma membrane markers. Hydrolysis of Mg-ATP-gamma-3Pp in rat islet membranes was of high specific activity, but was little affected by K+ and/or Na+. K+-activated, ouabain-sensitive phosphatase activities were, on the other hand, readily demonstrated in both rat and ob/ob mouse islet membranes. The K+-activated hydrolysis of organophosphate indicators were markedly inhibited by ATP. The binding of 45Ca2+ to mouse islet plasma membranes was increased by ATP. Cation transport and the interaction of Ca2+ with the islet cell plasma membrane may be dependent on phosphoryl-transfer reactions with ATP as the physiological substrate.

Effects of hypophysectomy and short-term growth hormone replacement on insulin release from and glucose metabolism in isolated rat islets of Langerhans.

The effects of hypophysectomy and short-term GH replacement on insulin release and on some aspects of glucose metabolism in isolated rat islets of Langerhans were investigated. The effects on body, pancreas and adrenal gland weights, and on the levels of blood plasma constituents were also measured. Three to four weeks after hypophysectomy the early and late phases of insulin release from islets incubated with high concentrations of glucose, but not with low concentrations of glucose or with xylitol, leucine, arginine, tolbutamide, citrate or butyrate, were significantly lowered. Short-term GH replacement partially reversed the depression in glucose-stimulated insulin release. This reversal effect was not dependent on the increase in body weight of rats after GH replacement when the fall in adrenal gland but not in pancreas weight was also reversed. Nine out of the 12 plasma constituents measured, including glucose, were maintained in the control range of levels, but albumin, inorganic phosphate and urea nitrogen levels were altered after hypophysectomy or GH replacement. Three to four weeks after hypophysectomy, total glucose oxidation and glucose utilization by the islets were slightly depressed. Hypophysectomy appeared to slow down glucose 6-phosphate utilization in the islets. However, the functional capacity of the glucose phosphorylating, glucose-6-phosphate and 6-phosphogluconate dehydrogenase activities were not changed. Short-term GH replacement caused improvements in these islet functions.

Changes in insulin content of, and insulin and RNA synthesis in, isolated rat islets of Langerhans after hypophysectomy and short-term growth hormone replacement.

Three to four weeks after hypophysectomy, the insulin content and glucose-stimulated insulin or total RNA synthesis in the pancreatic islets were estimated. The ratio of the glucose-stimulated insulin release to the islet insulin content was lowered. After short-term GH replacement, insulin release and insulin content were slightly increased such that the ratio of release to content remained virtually unaltered. Glucose-stimulated insulin synthesis was also depressed but improved slightly after GH replacement. However, there was no significant change in the incorporation of [5-3H]uridine into islet total RNA after hypophysectomy or subsequent GH replacement.

Quantitation of isolated rat islets of Langerhans on the basis of deoxyribonucleic acid content under metabolic conditions of altered protein synthesis.

A micromethod was employed to estimate quantitatively and reproducibly the deoxyribonucleic acid (DNA) content of isolated rat islets of Langerhans. The DNA content per islet varied linearly with the mean diameter or the dry weight of the islets isolated. The DNA in the freeze-dried islets of male Sprague-Dawley or Wistar rats was about 21.0 mug/mug islet dry weight. Three to four weeks after hypophysectomy, with or without short term bovine growth hormone replacement, the DNA content per unit dry weight of islets was not significantly altered. Islet DNA content and islet dry weight are proposed as an interconvertible and reliable basis of reference for measurements of different islet functions.