Isolation and activation of inactive renin from human kidney and plasma. Plasma and renal inactive renins have different molecular weights.

Inactive renin and active renin from human kidney and human plasma were prepared in highly purified forms by three steps of chromatography on Octyl-Sepharose, immunoaffinity chromatography, and pepstatin-amino hexyl Sepharose CL-4B. The inactive renin and active renin from human kidney had molecular weights of 51,000 and 44,000 as measured by a calibrated gel filtration column run with internal molecular weight standards. Molecular weights of plasma inactive renin and active renin were 56,000 and 51,000 respectively. Both inactive and active renins were found to be heterogeneous, consisting of several components with different isoelectric points. Renal inactive renin has higher pI values of 6.40, 6.10, 5.90, 5.61, and 5.40. Renal active renin has pI values of 5.73, 5.40, 5.25, and 5.13. The pI values of plasma inactive renin were 6.37, 6.08, 5.77, 5.36, and 5.25; of plasma active renin, 5.68, 5.40, 5.33, and 5.25. Trypsin activation and plasmin activation of plasma inactive renin produced an active enzyme with similar molecular weight but lower pI values. Acid activation of inactive renin did not change the molecular weight and pI values.

Expression and excretion of human pancreatic secretory trypsin inhibitor in lipoprotein-deletion mutant of Escherichia coli.

We constructed a gene coding for the 56-amino acid human pancreatic secretory trypsin inhibitor (PSTI), and ligated it on a plasmid downstream from the trp promoter and the signal peptide sequence of alkaline phosphatase. The resulting plasmid was transfected into a lipoprotein deletion mutant (Escherichia coli JE5505) and the plasmid-carrying cells were induced with 3-indoleacrylic acid. A considerable amount (50 micrograms/ml culture) of the mature PSTI protein was detected in the culture supernatant. The excreted PSTI was identical to the natural PSTI protein with respect to the trypsin-inhibiting activity, the N-terminal and the C-terminal amino acid sequences and the amino acid composition.

A novel purification method of human renin.

Human renal renin was isolated from a partially purified preparation, Haas' preparation step 5 material, with a remarkably high yield of 28% by a newly developed method. This method consisted of only four steps including affinity chromatography on pepstatin-aminohexyl-agarose, chromatofocusing, gel filtration and DEAE-chromatography after the initial extraction and concentration. This method enabled us to obtain pure human renin without another affinity column used by other investigators. Pure human renin had a molecular weight of 40,000 daltons as estimated by gel filtration and sodiumdodecylsulfate-polyacrylamide gel electrophoresis. The pI of purified renin was 5.6.

Evolutionary origin of a calcium-dependent protease by fusion of genes for a thiol protease and a calcium-binding protein?

Calcium-dependent protease (calcium protease) is apparently involved in a variety of cellular processes. Here we have attempted to clarify the role and regulatory mechanism of calcium protease by analysing its structure. The complete primary structure of calcium protease (relative molecular mass (Mr) 80,000 (80K), 705 amino acids) was deduced from the nucleotide sequence of cloned complementary DNA. The protein contains four distinct domains, and we have observed a marked similarity between the second and fourth domains and the papain-like thiol proteases and calmodulin-like calcium-binding proteins, respectively. This finding suggests that calcium protease arose from the fusion of genes for proteins of completely different function and evolutionary origin. Further, it provides functional insight into cellular regulatory mechanisms mediated by Ca2+ through calcium-binding proteins.

Molecular characterization of inactive renin: complete purification of prorenin in hog kidney and isolation of inactive renin from neuroblastoma cells: evidence for 2 different types of inactive renin.

To determine the molecular properties of inactive renin and its relationship to active renin, inactive renin in hog kidney was purified by devising affinity chromatography. Electrophoretically homogeneous inactive renin was prepared by 3 million-fold purification. It consists of a single polypeptide chain and undergoes reduction in molecular weight from 50,000 to 38,000 upon activation by proteases but not by dissociative treatment. This type of inactive renin is considered as a zymogen. However, a stable complex of renin and its inhibitor with a molecular weight of 110,000 was found in cultured neuroblastoma cells indicating the presence of a second type of inactive renin.

Renin: structural features of active enzyme and inactive precursor.

To determine the structural basis for the unique catalytic mechanism of renin and the mechanism of activation of inactive renin, renin and inactive renin were isolated in pure form. The active site of renin consists of two aspartyl residues, two tyrosyl residues, and one arginyl residue, analogous to pepsin and other acid proteases. The complete amino acid sequence of mouse submaxillary gland renin was determined. Of the amino acids, 43% were identical to those in porcine pepsin. Combination of various chromatographic techniques permitted the separation of inactive renin from active renin in human plasma and kidney. Inactive renin of hog kidney was completely purified. Inactive renin consists of a single polypeptide chain and is activated by proteolysis but not by dissociative reagents such as 4 M NaCl or detergent. Thus it was concluded that the inactive renin in these tissues is renin zymogen rather than a renin-inhibitor complex.