Interaction of pancreas extracts with bovine insulin. 1. Purification and chemical properties

Abstract

This paper is concerned with pancreatic substances that can antagonize insulin. Previous work (Marks & Young, 1939; Thorogood & Zinmmermann, 1945; Mirsky, Futterman, Wachman & Perisutti, 1951) has already suggested that insulin antagonists exist in the pancreas. Evidence has been produced for the identity of these antagonists with glucagon, but there is other work suggesting that the pancreas may produce substances that can antagonize insulin in such tissue as muscle (Young, 1962; Antoniades, Renold, Dagenais & Steinke, 1960) when glucagon is without effect (Randle, 1958). Antoniades et al. (1960) suggested that insulin might be combined with a basic protein in pancreas. The studies reported below are an attempt to isolate and characterize at least one of the bovine pancreatic substances that is capable of antagonizing insulin in a biological system such as rat diaphragm. MATERIALS AND METHODS Materials. DEAE-cellulose and CM-cellulose were obtained from Whatman (W. R. Balston Ltd.). Before use DEAE-cellulose was treated with 2N-NH3 and 2M-NaCl; CM-cellulose was treated with 2N-acetic acid and 2M-NaCl; both were washed well with distilled water. The bovine insulin (22-2 i.u./mg.) was obtained from Boots Pure Drug Co. Ltd., Nottingham. The proteolytic enzymes trypsin and oc-chymotrypsin were recrystallized preparations, obtained from L. Light and Co. Ltd., Colnbrook, Bucks. The pancreatic elastase was prepared by the method of Hall & Czerkawski (1959). Lilley & Valiance-Owen (1961) have shown that dialysis tubing contains substances capable of antagonizing insulin, and that these substances can be removed by boiling the tubing in two changes of distilled water. It was deemed wise to take the same precaution with the 1 in. tubing (Visking) used in the present work. Unless otherwise stated all the reagents used were of AnalaR quality. Electrophoresis. The purity of the fractions was frequently checked by use of electrophoresis at high voltage. on paper and at low voltage on cellulose acetate (Oxoid Division of Oxo Ltd., London, E.C. 4) as supporting medium. The electrophoresis was usually performed in a 75 mM-barbiturate (veronal) buffer solution, pH 8-6, which,

Interaction of pancreas extracts with bovine insulin. 2. Reaction in a biological system

“…In the first group of experiments, the crude pancreatic extract (AES1 fraction) and the APP fraction obtained by separation on a CM-cellulose column (Czerkawski & Bingle, 1964) were tested for their activity against bovine insulin by using the rat-diaphragm assay. The pancreatic protein extract or its APP fraction was incubated with bovine insulin for 15 min.…”

“…These results show that the crude pancreatic extract and the fractions of it (c-f) eluted from a CM-cellulose column with 0-1-0-2 M-sodium chloride (APP fraction) were capable of inhibiting the insulin-stimulated uptake of glucose by the rat hemidiaphragm. This active fraction (Czerkawski & Bingle, 1964) has been shown to consist of three protein bands on electrophoresis. Initial experiments suggest that the insulin-antagonistic activity is associated with the major component (APP).…”

“…Pancreatic extracts and their fractions were prepared according to the method of Czerkawski & Bingle (1964) and kept in a solid state at -20°until required. Glassware used in the diaphragm assay was kept separate, and before use cleaned with conc.…”

“…of bovine insulin/ml. in buffer solution was compared with the activity ofa similar concentration of insulin after incubation with the pancreatic extracts and fractions described by Czerkawski & Bingle (1964). In other experiments the actions of the APP fraction on similar concentrations of bovine and porcine insulins were compared.…”

“…A method of extraction and fractionation of bovine pancreas has been detailed by Czerkawski & Bingle (1964). In the prepared extract was present an active pancreatic protein (APP) capable of interacting with and degrading bovine insulin.…”