New method for analyzing the molecular weights of proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Previously the method for determining protein molecular weights from SDS-PAGE depended on the accidental, only partial linearity of protein movement with the logarithm of its molecular weight. A new, mathematically rigorous method with supporting data is now described demonstrating that such movement is dependent upon the reciprocal of protein size. Experimental data, therefore, follow most closely a hyperbolic curve when plotted directly; it becomes linear and passes through the origin when movement is plotted vs the reciprocal of protein molecular weight. In the earlier method determination of the error of a measurement of molecular weight is very complex and never determined. In the method presented here such error is easily estimated and it is identical in both the hyperbolic and linear forms of data presentation. This method may eventually also allow other less-significant forces controlling movement such as protein charge to be analyzed and understood.

Insulin and IGF-1 manifest differential effects in a clonal capillary endothelial cell line.

125I-insulin (10 fmoles) binding plus internalization (BI) to a clonal capillary endothelial (CE) cell line reached to a steady state after 20 min. Acid-washed fraction accounted for nearly half of the total specifically-bound hormone. Dissociation constants (Kd) for insulin-surface receptor in acid-extractable fraction were 0.04 nM (high affinity) and 4.7 nM (low affinity) with a total number of 210,000 high affinity receptors per cell. When 125I-labeled IGF-1 (15 fmoles) was incubated similarly, BI reached only a quasi-equilibrium by 6 min and continued to increase thereafter. 2-Deoxyglucose transport in these cells was stimulated by insulin whereas IGF-1 inhibited its entry.

The influence of a new compound, PATP, on glucose transport in the isolated fat cell.

A newly synthesized compound, N-(4-pyridylcarbonyl amino) 1, 2, 3, 6 tetrahydropyridine (PATP) was earlier found to elevate the blood glucose level in rats. This suggested that the compound might be accomplishing this by blocking glucose transport into tissue cells. This hypothesis has now been examined in the isolated fat cell system using a modification of the method to improve the accuracy of measurements made. This study indicated that PATP is a competitive inhibitor of glucose transport and metabolism (KI = 0.89 mM) but could not unequivocally prove that the effect was on transporter action alone. That the compounds action was at this level, however was shown by its ability to inhibit the uptake of the transported but non-metabolized sugar, 3-0-methyl glucose (KI = 3 mM). PATP is a nonphenolic inhibitor of glucose transport unrelated in structure to the sugar or to another more potent inhibitor, phloretin.

Degradation of the four monoiodoinsulin isomers by the insulin protease of rat liver.

The cleavage of insulin by the partially purified insulin protease was studied using the four [125I]tyrosine-monoiodoinsulins (tyrosine A-14 and A-19 of the A-chain; tyrosine B-16 and B-26 of the B-chain). The rates of conversion of the four isomers to trichloroacetic acid-soluble form was in the order B-26 greater than A-14 greater than A-19 greater than B-16. The following was observed in experiments which gave 19/14/5/3 percent conversion to trichloroacetic acid-soluble products: the loss of ability to bind to IM-9 lymphocytes was approx. 55% for all four isomers. About 70% of the radioactivity was in the 'insulin' peak, and about 30% was in peptides smaller than insulin as judged by gel filtration on Sephadex G-50. The descending limb of the 'insulin' peak contained significant amounts of radioactive material not binding to IM-9 lymphocytes. This material showed multiple peaks when applied to high performance liquid chromatography. Other experiments were designed to cause an almost complete degradation of the isomers. Under these conditions, the radioactivity eluted on Sephadex G-50 largely as iodotyrosine (and some small peptides) using the A-14, B-16 and B-26 isomers, whereas iodotyrosine was absent using the A-19 isomer. Thus, the insulin protease appears to first degrade insulin to multiple products with molecular sizes slightly smaller than insulin and subsequently to small peptides (e.g containing tyrosine A-19) and amino acids (e.g. tyrosine A-14, B-16 and B-26).

Studies of the properties of the insulin protease of rat liver.

Difficulties encountered in the purification of the cytosolic insulin-degrading protease of rat liver had led to an investigation of its properties related to this problem. A calcium phosphate gel-purified enzyme preparation was dispersed by ion-exchange chromatography into fractions of lowered specific activity and yield (34%). Recombination of active fractions resulted in a 2.5-fold increase in apparent yield with no increase in purification. Only adsorption to the entire length of an anion-exchange column with stepwise salt elution resulted in significant purification. Dispersion of activity was also seen on Sephadex G-150 but not on Sepharose 6B. In the latter system symmetrical elution of activity together with separation from a recently described globin protease activity was achieved. It is concluded that the protease is a complex of rather easily dissociating units, which severely complicates its purification. An earlier study of the enzyme in rat muscle (Duckworth et al. 1972, Proc. Natl. Acad. Sci. U.S.A. 69, 3698-3702) failed to recognize this problem and evidently qualifies the purification reported.

The importance of proteolysis as the initial step of insulin degradation in rat liver homogenates.

It was previously proposed (Varandani, P. T., Proc Natl Acad Sci 69:1681, 1972) that insulin is first degraded by rat liver homogenates in an enzyme-catalyzed reductive process by microsomal glutathione-insulin transhydrogenase before being proteolytically cleaved by the cytosolic enzyme activity designated insulin protease. This study was, however, carried out with concentrations of the hormone 10,000 times the maximal concentration seen in peripheral blood. In the present study, physiological levels of insulin (approximately 0.1 nM) and concentrations of reduced and oxidized glutathione approximating the reductive potentials of normal liver were used. Rates of degradation by separable particulate and soluble components of the homogenate were determined by following enzymatic conversion of [125I]-iodoinsulin to the trichloroacetic acid-solube form. Assessment of the mode of degradation was determined by gel filtration on Sephadex G-50 in the presence of 1 M acetic acid-6M urea. From these studies it was seen that 1) insulin is reduced at a very significant rate nonenzymatically; 2) during short periods of incubation (30 sec) where no significant hormone is reduced nonenzymatically, the rate of cleavage by the insulin protease present in the cytosol is extremely high and the microsomal GIT activity is negligible; and 3) insulin destruction noted in isolated liver cells and perfused liver is most probably due to the insulin protease activity of the cytosol.

Direct measurement of proinsulin in human plasma by the use of an insulin-degrading enzyme.

A method has been described for the direct measurement of proinsulin in human plasma. The method makes use of an insulin-degrading enzyme designated "insulin-specific protease (ISP)", which is obtained from rat skeletal muscle. Under the conditions used, this enzyme rapidly degrades insulin and insulin-like polypeptides to nonimmunoassayable components, whereas proinsulin and proinsulin cleaved at position B(54,55) are not appreciably affected. The incubation of plasma with ISP results in the disappearance of insulin, but not proinsulin, as demonstrated by column chromatography. Immunoassay of the plasma, therefore, before and after incubation, determines the values for the total immunoreactive substance (TIR) and for immunoreactive proinsulin (IRP), respectively. The values obtained for proinsulin levels are reproducible and compare closely with the more complicated column fractionation methods. Proinsulin responses were studied in four normal subjects and one patient with an insulinoma after a glucose load. Fasting proinsulin levels varied widely in the normal subjects, and the levels rose more slowly than TIR levels after glucose. IRP levels in the patient with an insulinoma were very high and fell to normal after removal of the tumor. The ISP method, therefore, appears to be suitable for the direct, accurate, and rapid determination of proinsulin and proinsulin-like materials in human plasma.

Divergent biological effects of adenosine and dibutyryl adenosine 3',5'-monophosphate on the isolated fat cell.

Adenosine 3',5'-monophosphate stimulated production of carbon dioxide and lipid from glucose, whereas its dibutyryl derivative inhibited this conversion. Addition of the dibutyryl derivative to the isolated fat cell further stimulated lipolysis induced by adrenocorticotropic hormone, whereas addition of adenosine 3',5'-monophosphate inhibited this lipolysis. Hence, measured by these two parameters, the biologic properties of adenosine 3',5'-monophosphate and its dibutyryl derivative are distinctly different.