Hormonal and drug effects on the degradation of human myelin basic protein peptide 43-88 by alkaline proteolytic activity in the rat kidney.

The microsomal brush-border fraction of rat renal tissue contains enzymatic activity, optimally active at pH 9, that is capable of degrading human myelin basic protein (BP) peptide 43-88. In the present study, this degradation and the effect on it of selected drugs and hormones were examined further. Of the substances tested, 10(-2) M chloroquine and 10(-5) M ACTH 1-24 were found to be the most effective inhibitors followed by 10(-5) M ACTH 1-39; parathormone, glucagon and insulin were found to be inhibitors an order of magnitude weaker than ACTH 1-24. Hydrocortisone, dexamethasone, maleic acid and ACTH 4-10 were found to have minimal or no inhibitory effect on the peptide degrading activity. Gel filtration of the degradation products indicated that the rate of degradation of BP peptide 43-88 at pH 9 had been retarded by ACTH 1-24. These studies indicate that the clearance and catabolism of this peptide may be altered by available therapeutic agents.

The degradation of human myelin basic protein peptide 43-88 by human renal neutral proteinase.

Normal human kidney contains a neutral endopeptidase that can degrade human myelin basic protein peptide 43-88. In the present study, renal homogenates prepared from postmortem tissue obtained from four persons with multiple sclerosis, two with amyotrophic lateral sclerosis, one each with olivopontocerebellar atrophy, subacute sclerosing panencephalitis, and Guillain-Barré syndrome, and four controls were analyzed for the enzymes present that degrade human myelin BP peptide 43-88. There was no evidence that the activity in renal tissue for degrading human BP peptide 43-88 is qualitatively different in persons with MS, other neurologic diseases, or controls. Gel filtration of digested peptide demonstrated the action of an endopeptidase capable of hydrolyzing BP peptide 43-88 into large fragments.

Species comparison of renal proteolytic activity for human myelin basic protein peptide 43-88.

1. A species comparison was conducted on the proteolytic activity in human, dog, rabbit, guinea-pig and rat kidney which can degrade human myelin basic protein peptide 43-88. 2. In rat kidney the degrading activity occurred over a pH range of 4-11.5 with the greatest activities at pH 5 and 9. The peptide degrading activity in human, dog, rabbit and guinea-pig kidney was considerably less than in the rat and occurred predominantly at pH 7 with lesser activity at pH 9. 3. The effects of inhibitors of proteolytic enzymes indicated that the peptide degrading activities at the same two pH's of dog, rabbit and guinea-pig were similar to one another but differed from that of human. 4. These results indicate that the activity for degrading a potential autoantigenic material is widespread in renal tissue among different species and that different enzymes are involved. More generally, these findings suggest that renal proteinases differ among commonly used laboratory animals and also differ from the human enzymes.

The renal degradation of myelin basic protein peptide 43-88 by two enzymes in different subcellular fractions.

Previous studies have demonstrated that the kidney is the major site for clearance and catabolism of a peptide (residues 43-88) of encephalitogenic or basic protein (BP) derived from central-nervous-system myelin. In the present investigation rat renal tissue was shown to be capable of degrading human BP peptide 43-88 over the pH range 4-11.5 with peaks of activity at pH5 and pH9. The enzymic activity at pH5 was localized mainly to the 5900 g pellet (crude mitochondrial fraction) and, on the basis of its sulphydryl features, was inferred to be cathepsin B. The enzyme activity at pH9 was greatly enriched in the 100 000 g pellet (microsomal brush-border fraction), and its sensitivity to inhibitors suggested that it was a metalloproteinase. The activity at alkaline pH in the 100 000 g pellet was stimulated 3-fold by non-ionic detergents and 20-fold by ATP and polyphosphates. Through a series of experiments the ATP stimulation of the alkaline proteinase activity was concluded to be the result of a reversal of inhibition imposed by the presence of another cationic protein, methylated bovine serum albumin. Inhibition by certain bivalent cations, the irregular effects of chelators and the effects of poly-L-lysine supported this conclusion. These studies indicate the availability of renal enzymes of different types and in different cellular compartments that are capable of degrading BP peptide 43-88. In particular, the relative amounts of bivalent cations, anions and charged proteins and peptides are likely to be major influences on the activity of the alkaline proteinase in vivo. The control of this degradation as well as the features of the smaller fragments of the peptide formed may determine biological and immune events subsequent to the release of this potentially autoantigenic material.

Purification of insulin-specific protease by affinity chromatography.

A single enzyme that proteolytically degrades insulin was isolated from rat skeletal muscle. This enzyme was purified 1000-fold by a series of steps, including affinity chromatography on insulin bound to agarose at the NH(2)-terminal phenylalanine of the B chain. Insulin linked to agarose at the B-29 lysine residue did not bind the enzyme and, therefore, was not suitable for purification procedures. Insulin linked at the phenylalanine residue was a substrate for the enzyme and was degraded by it; insulin attached to agarose at the lysine residue was not degraded by the enzyme. The purified enzyme preparation yielded one major band on polyacrylamide gel electrophoresis, and elution of this area of the gel yielded insulin-degrading activity. The purified enzyme degraded insulin but not proinsulin, with a K(m) for insulin of 22 nM and a K(i) for proinsulin of 40 nM. The enzyme is sulfhydryl-dependent, with a physiological pH optimum.