Biomedical subjects

D F Catanzaro

Publications and source records attributed to D F Catanzaro.

42 records · Page 3Linked to original sources

PubMedDate not supplied

Biosynthesis of preprorenin and intracellular conversion of prorenin to renin.

1. The existence of a biosynthetic precursor of renin (prorenin) has been inferred, but never established experimentally. The aim of the present study was to provide this evidence. 2. In a continuous labelling experiment using submandibular glands of male Quackenbush mice (an extraordinarily rich source of renin) a molecule of Mr 44 500 was immunoprecipitated with antiserum to renin. 3. The Mr 44 500 protein was converted to Mr 40 000 renin in a time-dependent fashion during incubation of tissue at 37 degrees C: 15 min after addition of [35S] methionine only 16% of immunoprecipitable radioactivity was renin, whereas by 4 h this had increased to 86%. The Mr 44 500 protein would therefore appear to be a 'pro' form of renin. 4. Submandibular gland mRNA was translated in a cell-free system derived from rabbit reticulocyte lysate and a molecule of Mr 46 000 was immunoprecipitated with antirenin. This was preprorenin and the Mr of the 'pre' region would thus be 1500, which is consistent with the size generally found for the 'pre' region of secretory proteins. 5. The present study has therefore demonstrated for the first time the biosynthesis of a 'pro' form of renin (prorenin) and its conversion to renin intracellularly.

Animals↗

PubMedDate not supplied

Synthesis of mouse renin as a 2-5-33-5 kilodalton pre-pro-two-chain molecule and use of its cDNA to identify the human gene.

A DNA coding for mouse submandibular gland (MSG) renin was used in studies of the biosynthesis and processing of MSG renin and precursors and as a probe for identification of the human renin gene. A 2 kilodalton signal peptide was demonstrated by cleavage of preprorenin (45 kilodaltons) in the presence of microsomal membranes from dog pancreas. Prior selection of renin mRNA by hybridization with its cDNA obviated the possibility that a protease translated from total MSG mRNA could be activated by the microsomal membranes and then act on the primary translation product. In vitro labelling experiments with female MSG demonstrated that prorenin (43 kilodaltons) is rapidly converted to renin (38 kilodaltons) and that testosterone stimulated synthesis by increasing transcription of renin mRNA. Electrophoresis under non-reducing conditions demonstrated 38 kilodalton renin which when reduced ran as two bands of 33 and 5 kilodaltons. Thus, native MSG renin has two chains linked by disulphide bonding. Hydrolysis of the 38 kilodalton single chain occurred only slowly during in vitro labelling. Prorenin bound only weakly to a pepstatin affinity column and could be activated by adding trypsin to column fractions. Both 38 kilodalton single and two chain renin bound strongly however, suggesting that both are active. Base sequencing of MSG renin cDNA indicated an Arg-Arg residue additional to the published amino acid sequence of the heavy chain. This may account for the two forms seen on isoelectric focusing. Mouse renin cDNA was used as a hybridization probe in screening a human genomic library in order to identify the human renin gene. The DNA in positive colonies had overlapping restriction maps and the coding region was found.

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PubMedDate not supplied

Structure of human renin and expression of the renin gene.

The amino acid sequence of human renin was identified for the first time. This was determined from the nucleotide sequence of exons in the human renin gene identified in a genomic library by recombinant DNA techniques. Examination of amino acid residues involved in the enzymatic hydrolysis by human renin of the unique Leu10-Val11 bond of human angiotensinogen revealed features peculiar to this highly specialized aspartyl protease. The expression of the renin gene was examined with a hybridization probe for renin mRNA in sections and extracts of tissues. In the submandibular gland of mice renin mRNA, like renin, increased during development and in response to testosterone in females; sodium depletion increased renin mRNA in kidney.

Amino Acid Sequence↗

PubMedDate not supplied

Isolation of genomic clones coding for the heavy chains of two human cardiac myosins.

A 14 kilo-base pair DNA clone (lambda HCMHC8) was isolated from a human genomic library by hybridization with a complementary DNA coding for a rabbit cardiac myosin heavy chain. lambda HCMHC8 hybridized to RNA isolated from cardiac but not skeletal muscle and formed heteroduplexes with a genomic clone coding for the fast type of rabbit cardiac myosin heavy chain. lambda HCMHC8 represented at least the 3' half of the gene and contained over 11 exons which together spanned 4 kb of the coding region estimated to be 6 kb. Probes made from lambda HCMHC8 were used to rescreen the library in order to isolate overlapping clones and so extend the sequence (estimated to be approximately 25 kb for the whole gene, including introns). In addition, a clone having a different restriction map was isolated suggesting that man, like rat and rabbit, has two cardiac myosin heavy chain genes. These may code for proteins having different ATPase activity and be expressed in different proportions in different cardiac states, including hypertension.

DNA↗

PubMedDate not supplied

A structural analysis of human renin.

A three-dimensional model of human renin has been used to define amino acid residues involved in interaction with substrate. The structure revealed proteolytic cleavage sites on the surface of the molecule that may help explain numerous experimental observations concerning prorenin and its activation and forms of renin differing in size.

Binding Sites↗

PubMedDate not supplied

Kidney is the only source of human plasma renin in 45-kb human renin transgenic mice.

Prorenin is expressed in certain extrarenal tissues, but normally only the kidneys process prorenin to renin and secrete renin into the circulation. Although transgenic animal lines containing the human renin (hREN) structural gene with either 0.9-kb or 3-kb 5'-flanking DNA express the transgene appropriately in renal juxtaglomerular cells and secrete hREN into the circulation, the source of the circulating renin is not known. In the present study, we observed that 13-kb hREN transgenic mice that contain the structural gene and 0.9-kb 5'-flanking DNA express hREN mRNA in many unusual tissues. We also observed that circulating hREN levels in 13-kb hREN mice increased after bilateral nephrectomy. These results suggested that the hREN gene is expressed at inappropriate locations where prorenin might be processed to renin. To determine if more distal sequences flanking the hREN gene might contribute to cell and tissue specificity, we used a 45-kb hREN genomic fragment that contained the structural gene and about 25-kb 5'- and 8-kb 3'-flanking DNA sequences to generate 3 separate transgenic lines that contained the intact transgene sequences. Ribonuclease protection assays revealed a much narrower tissue distribution of hREN expression than in the 13-kb hREN transgenic mice. In each 45-kb hREN line, hREN mRNA was present only in the kidney, adrenal, lung, eye, ovary, and brain. Moreover, 24 hours after nephrectomy, human plasma renin fell to very low levels, indistinguishable from those of nontransgenic littermates, indicating that their circulating hREN is of renal origin. These studies suggest that sequences flanking the structural gene, missing from previous hREN transgenic lines, suppress renin gene expression at inappropriate extrarenal sites where cellular proteases, to which prorenin is not normally exposed, could convert prorenin to renin, resulting in abnormal secretion of renin into the plasma.

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