Degradation products of insulin generated by hepatocytes and by insulin protease

The enzymatic mechanisms for insulin breakdown by hepatocytes have not been established, nor have the degradation products been identified. Several lines of evidence have suggested that the enzyme insulin protease is involved in insulin degradation by hepatocytes. To identify the products of insulin generated by insulin protease and to compare them with those produced by hepatocytes, we have incubated insulin specifically iodinated at either the B-16 or the B-26 tyrosines with insulin protease and with isolated hepatocytes, separated the products on high performance liquid chromatography (HPLC), and identified the B-chain cleavages. Insulin-sized products were obtained by Sephadex G-50 filtration. These insulin-sized products were injected on reverse-phase HPLC, and the peaks of radioactivity were identified. The product patterns generated by the enzyme and by hepatocytes were essentially identical with both isomers. The products were also sulfitolized to prepare the S-sulfonate derivatives of the B-chain and B-chain peptides. Again, the patterns on HPLC generated by the enzyme and by hepatocytes with both isomers were identical. Each of the original product peaks was also sulfitolized and injected separately on HPLC to relate B-chain peptides with product peaks. Again, the peptide compositions of the product peaks for both enzyme and hepatocytes were essentially identical. To identify the cleavage sites in the B-chain of insulin produced by insulin protease, the peptides from the degradation of [125I]iodo(B-26)insulin were purified and submitted to automated Edman degradation to identify the cycle in which radioactivity appeared. Seven peptides with cleavages on the amino side of the B26 residue were identified, and the cleavage sites were determined. Cleavages were found between B-9 and B-10 (Ser-His), B-10 and B-11 (His-Leu), B-14 and B-15 (Ala-Leu), B-13 and B-14 (Glu-Ala), B-16 and B-17 (Tyr-Leu), B-24 and B-25 (Phe-Phe), and B-25 and B-26 (Phe-Tyr). Peptides were also isolated from [125I]iodoinsulin incubated with isolated hepatocytes, and the cleavage sites in several of these were determined. These agreed exactly with the cleavage sites identified generated by the enzyme. The major peptides generated by the degradation of [125I]iodo(B-16)insulin were also isolated and sequenced, again showing identical cleavage sites.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation of insulin degradation products from endosomes derived from intact rat liver

Rats were injected with [125I]iodoinsulin labeled at either the A14 or B26 tyrosine, and the animals were killed and livers subcellularly fractionated to yield light (early or neutral) endosomes and heavy (late or acidic) endosomes. 125I-Labeled material was extracted from endosomes and analyzed by Sephadex G-50 filtration and high performance liquid chromatography (HPLC). Radiolabeled material in both types of endosomes is comprised of high molecular weight, insulin-sized, and low molecular weight components, with B chain-labeled small molecular weight material in two peaks, one corresponding to iodotyrosine and one to small peptides (Mr less than 1500). As compared with A chain label, however, less of the B chain material appears in the degradation components (both high and low molecular weight fractions) suggesting that a fragment of B chain containing the B26 residue is lost from the endosomes. Analysis on HPLC shows that significant amounts of the insulin-sized and high molecular weight material have proteolytic cleavage(s) in the B chain with an intact A chain. The B chain-derived labeled peptides elute from HPLC identically with products generated by insulin protease. These results therefore show substantial insulin degradation occurring in light endosomes prior to endosomal acidification and to receptor dissociation, suggesting receptor-bound insulin is a substrate for insulin protease.     

Conversion of biosynthetic human proinsulin to partially cleaved intermediates by collagenase proteinases adsorbed to isolated rat adipocytes.

Studies of the biological activity of proinsulin have resulted in widely varying conclusions. Relative to insulin, the biological activity of proinsulin has been reported from less than 1% to almost 20%. Many of the assays in vitro for the biological potency of proinsulin have utilized isolated rat adipocytes. To examine further the interaction of proinsulin with rat adipocytes, we prepared specifically-labelled proinsulin isomers that were iodinated on tyrosine residues corresponding to the A14, A19, B16 or B26 residue of insulin. These were incubated with rat adipocytes and their metabolism was examined by trichloroacetic acid precipitation, by Sephadex G-50 chromatography, and by h.p.l.c. chromatography. By trichloroacetic acid-precipitation assay, there was little or no proinsulin degradation. By G-50 chromatography and subsequent h.p.l.c. analysis, however, we found that the labelled proinsulin isomers were converted rapidly and almost completely to materials which eluted differently on h.p.l.c. from intact proinsulin. This conversion was due primarily to proteolytic activity which adsorbed to the fat cells from the crude collagenase used to isolate the cells. Two primary conversion intermediates were found: one with a cleavage at residues 23-24 of proinsulin (the B-chain region of insulin), and one at residues 55-56 in the connecting peptide region. These intermediates had receptor binding properties equivalent to or less than intact proinsulin. These findings show that isolated fat cells can degrade proinsulin to intermediates due to their contamination with proteolytic activity from the collagenase used in their preparation. Thus the previously reported range in biological activities of proinsulin in fat cells may have arisen from such protease contamination. Finally, the present findings demonstrate that a sensitive assay for degradation of hormones is required to examine biological activities in isolated cells.     

Digestion of an exogenous protein by rat yolksac cultured in vitro

Yolk-sacs were removed from 17.5-day pregnant rats injected 2-5h previously with (125)I-labelled bovine serum albumin. The specific activities of acid phosphatase and acid proteinase, and the specific radioactivities (trichloroacetic acid-insoluble and trichloroacetic acid-soluble) were measured in subcellular fractions prepared by homogenization and differential centrifugation. The conversion of acid-insoluble into acid-soluble radioactivity within cultured tissue was followed and the nature of the liberated products was investigated by gel chromatography. The results are consistent with the protein entering lysosomes and being digested there. The radiolabel was released chiefly as free iodotyrosine.     

Contribution of signals and antisignals in the mutational spectrum of the td-intron insertion site]

The insertion sites of the td-intron were studied. It was found that tetranucleotides W-30A-29W-28Y-27, V-19B-18W-17A-16 and A11W12A13W14 predominantly occur at normal sites (signals), and tetranucleotides Y-27W-26M-25G-24, V7B8W9A10 and Y15W16M17G18 occur more frequently at defective sites (antisignals). It was shown that antisignals make the site defective, by blocking its signals. The site becomes defective due to the distortion of signals at a large number of random substitutions, and due to antisignals when the substitutions are few.     

Insulin-like substance and insulin-degrading complex in hemolysates of human erythrocytes

Human erythrocyte lysate was fractionated on various gel filtration media and immunoreactive insulin, insulinase and the influence of individual fractions of the insulin-degrading activity were determined. The hemolysate was shown to contain a complex of substances including an insulin-like substance, insulinase, protease inhibitor and insulinase activator. The insulin-like substance eluted from a Sephadex G-50 column in the same manner as native insulin, and its concentration exceeded the plasma level. Insulinase (Mr 100,000) degraded insulin to the trichloroacetic acid soluble fragments but did not degrade protein or glycoprotein hormones from human pituitaries. Insulinase was inhibited by low temperature, aprotinin and by a newly discovered protease inhibitor from erythrocytes which also inhibits serine proteases--trypsin and chymotrypsin. Another newly discovered substance eluted from a Sephadex G-100 column in the region of low molecular weight substances and showed an insulinase activating activity. The elution patterns of the protease inhibitor and insulinase activator suggest the possibility of the presence of more than one inhibiting and activating factor. The experimental results suggest that the insulin-degrading complex plays a role of a regulator of plasma insulin level. The nonpancreatic origin of the insulin-like substance is also possible.     

Receptor-binding kinetics of A-14 and A-19 125I-labelled insulin

Receptor-binding kinetics and degradation of tyrosine A-14 and A-19 ¹²⁵I-labelled insulin was studied using cultured human lymphocytes. Receptor-binding ability of A-14 insulin was 1.5-times as high as that of A-19 insulin. Dissociation from receptors on lymphocytes showed no difference between these two labelled insulins. In association studies percent bound of A-14 insulin was 1.5-times as high as that of A-19 insulin at any time after incubation. These results suggested that lower binding affinity of A-19 insulin was due to decreased association rate, but not due to increased dissociation rate. Degradation of A-14 insulin by incubation media of lymphocytes was also 1.5-times as high as that of A-19 insulin.     

In Vitro Protein Synthesis by Plastids of Phaseolus vulgaris: V. Incorporation of C-Leucine into a Protein Fraction Containing Ribulose 1,5-Diphosphate Carboxylase

A crude chloroplast preparation of primary leaves of Phaseolus vulgaris was allowed to incorporate ¹⁴C-leucine into protein. A chloroplast extract was prepared and purified for ribulose 1,5-diphosphate carboxylase by ammonium sulfate precipitation, chromatography on Sephadex G-200, and chromatography on Sepharose 4B. The distribution of radioactive protein and enzyme in fractions eluted from Sepharose 4B was nearly the same. The radioactivity in the product was in peptide linkage, since it was digested to a trichloroacetic acid-soluble product by Pronase. Whole cells in the plastid preparation were not involved in the incorporation of amino acid into the fraction containing ribulose 1,5-diphosphate carboxylase, since incorporation still occurred after removal of cells. The incorporation into the fraction containing ribulose 1,5-diphosphate carboxylase occurs on ribosomes of plastids, since this incorporation is inhibited by chloramphenicol. These plastid preparations may be incorporating amino acid into ribulose 1,5-diphosphate carboxylase, but the results are not conclusive on this point.     

Mechanisms of insulin degradation by isolated rat adipocytes

Summary We have examined some of the chemical and biological characteristics of the insulin-derived cell-associated radioactivity following incubation of isolated adipocytes with ¹²⁵I-insulin (10^–10 M) for one hour at 37 °C S ephadex G-50 chromatography of the cell-associated radioactivity demonstrated three peaks: peak I eluted with the void volume and consisted of large molecular weight material; peak II comigrated with 1251-insulin; and peak III consisted of small molecular weight degradation products (probably iodotyrosine). When the insulin peak (peak II) was divided into fourths, it was found that the binding and biologic activity of this material was not homogenous; thus, binding and biologic activity (relative to native insulin) fell markedly from the earliest to the latest eluting fractions of this peak. Furthermore, when the entire peak 11 material was applied to DEAE-Sephacel and eluted with a 0.01–0.2 M NaCl gradient, three distinct peaks were observed. These peaks were all 90% TCA precipitable, whereas the ability of the latter two eluting peaks to precipitate with anti-insulin antiserum was markedly reduced. When similar experiments were performed with chloroquine-treated cells, a large increase in cell-associated radioactivity was observed, and Sephadex G-50 chromatography demonstrated that this increase was entirely confined to peaks I and II. When the insulin peak (peak II) was divided into fourths, it was found that chloroquine markedly inhibited the decreased binding and biologic activity, from the earliest to the latest eluting fraction of this peak. Furthermore, when the peak II material (Sephadex G-50) from chloroquine-treated cells was chromatographed on DEAE-Sephacel, this material eluted in a single peak which was 95% TCA precipitable and 106% precipitable by anti-insulin antiserum. In conclusion, these studies demonstrate that: 1) intermediate insulin-derived products with reduced binding and biologic activity are generated in the process of cellular insulin degradation, and 2) the formation of these intermediate products is mediated by a chloroquine-sensitive pathway.     

Glucose 6-phosphate effects on deoxyglucose, glucose and methylglucose transport in rat adipocytes. Evidence for intracellular regulation of sugar transport by glucose metabolites

Receptor-binding kinetics and degradation of tyrosine A-14 and A-19 125I-labelled insulin was studied using cultured human lymphocytes. Receptor-binding ability of A-14 insulin was 1.5-times as high as that of A-19 insulin. Dissociation from receptors on lymphocytes showed no difference between these two labelled insulins. In association studies percent bound of A-14 insulin was 1.5-times as high as that of A-19 insulin at any time after incubation. These results suggested that lower binding affinity of A-19 insulin was due to decreased association rate, but not due to increased dissociation rate. Degradation of A-14 insulin by incubation media of lymphocytes was also 1.5-times as high as that of A-19 insulin.     

Degradation of surface-labeled hepatoma membrane polypeptides: Effect of inhibitors

When their membrane proteins were labeled with ¹²⁵I by lactoperoxidase, dividing hepatomacells lost radio activity to the medium in a biphasic manner ($\text{T}\text{1}\text{2}\text{= 16–26}\text{h}\text{, > 40}\text{h}$). Lysosomotropic weak bases, chloroquine, and NH₄Cl inhibited the rapid phase by 59%. More than 50% of the radio activity which accumulates in the media from dividing cells during the first 4 h after labeling was trichloroacetic acid-soluble, and was identified as iodotyrosine. Iodotyrosine release from labeled membrane proteins was 60–71% inhibited by lysosomotropic agents chloro quine and NH₄Cl as well as the sodium-proton ionophore, monensin. The inhibitory effect of NH₄Cl and monensin was reversible. Inhibitors of microtubule and microfilament function and transglutamination had no effect on release of iodotyrosine to the medium, but trypsin-like protease inhibitors, p-aminobenzamidine, tosyl-l-lysine/chloromethylketone, and phenylmethylsulfonyl fluoride, as well as the cathepsin B inhibitor, leupeptin, inhibited by21–24%. Iodotyrosine release showed a biphasic Arrhenius plot with an activation energy of 17 kcal/mol above but 27 kcal/mol below 20 °C. These results indicate that cell membrane polypeptides require a temperature-limiting event as well as passage through an ion-sensitive compartment prior to their complete degradation to constituent amino acids. In contrast to other lyososomal-mediated events, however, iodinated membrane proteins of dividing cells are degraded in a manner insensitive to, agents which disrupt the cytoskeleton.     

Metabolism of high-density lipoproteins in cultured rat luteal cells

The uptake of cholesterol from high-density lipoproteins (HDL) labeled with 125I and [3H]cholesterol was examined in cultured rat luteal cells. Luteal cells were incubated with labeled HDL, following which the metabolic fate of the apolipoproteins and cholesterol moieties of the receptor-bound HDL were examined. About 50% of the originally bound HDL apolipoproteins were released into the medium in 24 h by a temperature-dependent process while only 5% of the HDL cholesterol was released unmetabolized. Inclusion of unlabeled HDL in the chase incubation resulted in increased release of apolipoprotein-derived radioactive products without significant change in the release of unmetabolized cholesterol. 60% of the apolipoprotein-derived radioactivity could be precipitated with trichloroacetic acid; the remaining trichloroacetic acid-soluble radioactive fraction was identified as [125I]iodotyrosine. Gel filtration chromatography of the chase-released material showed that the trichloroacetic acid-precipitable products, which contained no detectable amounts of cholesterol, eluted over a range of molecular sizes (9-80 kDa). No intact HDL was retroendocytosed. About 80% of trichloroacetic acid-precipitable products could be immunoadsorbed on anti-apolipoprotein A-I antibody immobilized on CNBr-activated Sepharose, suggesting the presence of fragments containing apolipoprotein A-I. This material was also capable of reassociating with native HDL. Lysosomal inhibitors were partially effective in inhibiting the amount of trichloroacetic acid-soluble products formed. The lysosomal degradation appeared to have no role in the uptake of HDL-derived cholesterol. These studies demonstrate preferential and total uptake of HDL cholesterol by luteal cells, with concomitant degradation of the lipoprotein.     

Characterization of a somatomedin (insulin-like growth factor) synthesized by fetal rat liver organ cultures.

Explants of 19- to 20-day fetal rat liver synthesize polypeptides biochemically and immunologically related to the well characterized somatomedin (insulin-like growth factor) BRL-MSA, multiplication-stimulating activity. Fetal MSA was purified from media conditioned by fetal liver explants by chromatography on Sephadex G-75 under acid conditions. Partially purified fetal MSA: 1) inhibited the binding of BRL-MSA to the MSA receptor of rat liver plasma membranes, to somatomedin-binding proteins from rat serum, and to rabbit anti-BRL-MSA serum; 2) had a molecular weight of 4,500 to 12,500 determined by polyacrylamide gel electrophoresis in sodium dodecyl sulfate; 3) stimulated the incorporation of [3H]thymidine into the DNA of chick embryo fibroblasts and induced cell multiplication; 4) stimulated glucose oxidation in rat adipocytes and weakly inhibited the binding of insulin to the insulin receptors of IM-9 lymphocytes; and 5) stimulated sulfate uptake in costal cartilage from hypophysectomized rats. These activities were associated with the same molecular species in fetal MSA preparations following disc acrylamide electrophoresis and co-migrated with active BRL-MSA peptides.     

Correlation of Carbohydrate Catabolism and Synthesis of Macromolecules During Enzyme Synthesis in Pseudomonas fluorescens.

Kirkland, Jerry J. (Oklahoma State University, Stillwater), and Norman N. Durham. Correlation of carbohydrate catabolism and synthesis of macromolecules during enzyme synthesis in Pseudomonas fluorescens. J. Bacteriol. 90: 23-28. 1965.-Glucose, ribose, and fructose shorten the lag period required for synthesis of protocatechuate oxygenase. Radioactivity from uracil-2-C(14) is incorporated into the hot trichloroacetic acid-soluble fraction after a lag period of approximately 20 min after addition of protocatechuic acid. Addition of glucose or ribose simultaneously with the inducer shortens the lag period to approximately 5 min and increases the rate of uracil incorporation. The inducer must be present to initiate incorporation of radioactivity, and the exogenous carbon source accelerates incorporation but is not sufficient to initiate synthesis by itself. The addition of protocatechuic acid increases the rate and total incorporation of radioactivity from uniformly labeled glucose or ribose-1-C(14) into the hot trichloroacetic acid-soluble fraction. Ribose decreases the incorporation of radioactivity from uniformly labeled glucose into the hot trichloroacetic acid-soluble fraction, and glucose shows a similar effect on incorporation of radioactivity from ribose-1-C(14), indicating the two sugars are serving in the same capacity to enhance enzyme synthesis. Radioactivity from glucose-1-C(14) is not incorporated into the hot trichloroacetic acid-soluble fraction. The results suggest that glucose and ribose shorten the lag period for inducible enzyme formation by serving as a "specific" carbon source for synthesis of macromolecules such as ribonucleic acid.     

Insulin and glucagon degradation by the kidney. II. Characterization of the mechanisms at neutral pH.

Examination of insulin and glucagon degradation by rat kidney subcellular fractions revealed that most degrading activity was localized to the 100 000 X g pellet and 100 000 X g supernatant fractions. Further characterization of the degrading activities of the 100 000 X g pellet and supernatant suggested that three types of enzymatic activity were present at neutral pH. From the cytosol an enzyme with characteristics of the insulin glucagon protease of skeletal muscle was purified. This enzyme appeared to be responsible for insulin degradation by the kidney at physiological insulin concentrations. This enzyme also contributed to glucagon degradation but was not the most active mechanism for this. In the 100 000 X g pellet at least two separate enzymatic activities were present. One of these had properties consistent with those described for glutathione insulin transhydrogenase and appeared to be responsible for insulin degradation at high insulin concentration. The other enzyme was associated with the brush border and had properties consistent with the brush border neutral protease. This enzyme appeared responsible for glucagon degradation at both low and high substrate concentrations. An apparent marked synergism between the 100 000 X g pellet and the 100 000 X g supernatant was noted for insulin degradation at physiological insulin concentrations. Pellet glucagon-degrading activity and soluble insulin-degrading activity were necessary for this. The mechanism was found to be limited insulin degradation by the soluble enzyme resulting in both trichloroacetic acid-precipitable trichloroacetic acid-soluble fragments followed by further degradtion of the fragments by the glucagon-degrading enzyme resulting in an additional increase in trichloroacetic acid-soluble products.     

Insulin degradation. XV. Use of different assay methods for the study of mechanism of action of glutathione-insulin transhydrogenase.

Insulin degradation by glutathione-insulin transhydrogenase has been studied using three different assay procedures: the measurement of the change in insulin immunoreactivity; the formation of 5% trichloroacetic acid-soluble radioactivity from 125 I-labeled insulin and the formation of GSSG via coupling to the oxidation of NADPH with the use of glutathione reductase. The extent of reaction as measured by each assay was different, and the ratios between the assays were not constant with time. Kinetic experiments with the NADPH-coupled assay and the trichloroacetic acid assay yielded similar results: Line-weaver-Burke plots with insulin as variable and GSH as fixed substrate gave a set of straight, intersecting lines, and such plots with GSH as variable and insulin as fixed substrate were parabolic. Apparent Km values for insulin at 1 mM GSH were found to be quite similar by three assay techniques; however, the V values per unit of enzyme protein varied considerably with different procedures. The results are interpreted as indicating that immunoreactivity is lost after reduction of only one of the disulfide bonds of insulin whereas the two interchain disulfide linkages must be broken to produce the trichloroacetic acid-soluble A chain. The results of the NADPH-coupled assay suggest that all three disulfide bonds of insulin are possible substrates for the enzyme. The trichloroacetic acid precipitation assay seems to be the most practicable technique for general use because of the greater ease in performing large number of samples, precision and sensitivity.     

Insulin binding to cultured adult hepatocytes. Effects of bacitracin and chloroquine on the nature of cell-associated radioactivity.

Sephadex (G-50 fine grade)-gel chromatography and trichloroacetic acid (TCA) precipitation were used to investigate the effects of chloroquine and bacitracin on the nature of cell-associated radioactivity in studies on the binding and degradation of 125I-insulin in cultured rat hepatocytes. Sephadex peak I, eluted with the void volume, increased with hepatocyte incubation time and comprised 6% of total cell-bound radioactivity at 120 min. However, all radioactivity in this peak was due to unspecific binding. Peak II, corresponding to intact insulin, represented 95% of specifically cell-associated label at 5 min and decreased to 77% at 120 min. Peak III, containing the final low-Mr degradation products, increased with incubation time (22% of specifically bound label at 120 min). The TCA-precipitable and TCA-soluble fractions of hepatocytes extracted with 0.1% SDS were within 4-7% of the proportions of radioactivity in peaks II and III respectively. Scatchard plots based on insulin-binding data from Sephadex chromatography or TCA precipitation were identical. Dissociation studies revealed that at least 75% of the intact insulin associated with the hepatocytes was bound to receptors at the cell surface. Bacitracin increased the proportion of cell-associated intact hormone and decreased that of ligand degraded when analysed by either Sephadex chromatography or TCA precipitation. The proportion of surface-bound to internalized intact hormone remained unaltered, indicating that bacitracin acted predominantly at the cell surface. In the presence of chloroquine, which dramatically increased the contribution of peak I to specific binding, 'intact' insulin was substantially overestimated when determined as the TCA-precipitable fraction. In addition, all peak I material and 50% of cell-associated label in peak II was trapped intracellularly, thereby pointing to the lysosomal or prelysosomal site of action of this drug.     

Purification and characterization of a trypsin-like proteinase from the midgut of the larva of the hornet, Vespa orientalis

A proteinase from the larval midgut of Vespa orientalis was purified by exchange chromatography on DEAE-Sephadex A-50 and gel filtration on Sephadex G-75. This purified enzyme was proved to be homogeneous by electrophoresis on a cellulose acetate membrane. The molecular weight was calculated to be 27,000 by gel filtration. Optimum pH for the hydrolysis of N-benzoyl-arginine-ethyl ester (BAEE) was 7·5 to 8·5, and optimum temperature with casein as a substrate was 60°C at pH 8·0 for 20 min. According to studies with synthetic inhibitors the hornet protease belongs to the ‘serine proteases’, being inhibited by phenylmethyl sulphonylfluoride (PMSF) and tosyl-lysyl chloromethane (TLCK). The hydrolysis of different amino acid ester bonds and the cleavage specificity on the B chain of oxidized insulin allow us to speak of a trypsin-like protease.     

Collagen synthesis in the muscle of developing chick embryos.

Radioactive protein was prepared from the leg muscle of chick embryos, 11, 14, 16 and 17 days old, each injected with radioactive proline and incubated for 30, 60 or 90 min afterwards. The radioactive protein was incubated with collagenase purified by chromatography on a Sephadex G-100 column. Under this condition, only collagen is digested into products soluble in trichloroacetic acid. The relative rate of collagen synthesis was determined by comparing the amount of radioactivity released into the supernatant fraction and that in the residue, by the method of Diegelmann & Peterkofsky [(1972) Dev. Biol. 28, 443--453]. The results show that the rate of collagen synthesis remains at approx. 10% of the rate of synthesis of other non-collagenous proteins during the development of chick embryonic muscle from 11 to 17 days. This suggests that the synthesis of collagen and that of other proteins are co-ordinately regulated at these stages of development.     

Partial characterization of an endogenous inhibitor of a calcium-dependent form of insulin protease

Insulin protease activity has resisted high-yield purification to homogeneity, due to its low amount in tissues, its instability, and its erratic recovery from several types of chromatography. This report outlines the preliminary characterization of a naturally-occurring insulin protease inhibitor that accounts for some of these problems in rat skeletal muscle. In these experiments, inhibitory activity was assayed by its effect upon hydrolysis of 125I-(A14)-insulin by the partially purified insulin protease activity of rat skeletal muscle cytosol. During Sephadex G-200 chromatography of cytosol at pH 7.5, inhibitory activity copurifies with insulin protease activity, and the incomplete resolution of the two activities contributes to the impression that insulin protease exists in distinct 180,000-dalton and 80,000-dalton forms. By contrast, during DEAE-Sephacel chromatography of cytosol at pH 7.5, inhibitory activity and insulin protease activity are resolved by eluting the resin with 50 mM NaCl and 200 mM NaCl, respectively. Post-DEAE-Sephacel inhibitor has an Mr(app) of 67,000 daltons or 80,000-120,000 daltons, as determined by high-performance liquid chromatography or Sephadex G-150 chromatography, respectively. Post-DEAE-Sephacel insulin protease activity exhibits a Km for insulin of 15 nM and resides in a 200,000-dalton neutral thiol protease which requires 50 micromolar calcium for its maximum insulin-degrading activity. The inhibitor reduces the enzyme's activity reversibly, nonprogressively, and non-competitively with respect to insulin, but it does not alter the enzyme's sensitivity to calcium ion. These observations suggest that calcium and an endogenous protease inhibitor may influence cellular degradation of insulin via previously unrecognized effects upon cytosolic insulin protease activity.