Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.23.15 (renin)
 35,795 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Rat renin cDNA was transfected into COS-7 and Chinese hamster ovary (CHO) cells and expressed under the control of the Simian Virus 40 early promoter. Conditioned media of the transfected cells showed renin activity only after trypsin treatment, suggesting prorenin was secreted into the medium. From the trypsinized serum-free culture of the transfected CHO cells active renin was purified to homogeneity by a simple three-step procedure. The active renin had similar specific activity, molecular weight, Km, pH optimum, and isoelectric point compared to native renin. The amino-terminal sequence was the same as that deduced from the renin cDNA. This suggests that the recombinant rat renin is similar to kidney renin in many respects, and is easily obtained by the present procedures.
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PMID:Expression of rat renin in mammalian cells and its purification. 160 Jun 38

Human renin is synthesized as a 406-amino acid preprorenin protein that is processed by a signal peptidase during secretion, to release prorenin as a 386-amino acid zymogen. The 46-amino acid "pro" domain is removed by a renin-processing enzyme, to produce enzymatically active renin, by cleavage at an Arg-Leu bond. The effects of the renin-processing enzyme can be mimicked by trypsin activation, where high concentrations of trypsin are incubated with prorenin for brief periods of time, followed by excess trypsin inhibitor to minimize secondary proteolytic processing by trypsin. In order to study the role of the pro segment in the secretion, folding, and activity of human renin, we engineered a construct where the pro domain from the preprorenin cDNA was deleted. This construct was introduced into mammalian cells and its expression was assayed in transient and stable systems. In COS-1 cells transfected with the prerenin expression vector pREN3, active renin was secreted with a specific activity of 1360 micrograms of angiotensin l/min/mg, compared with trypsin-activated prorenin, which has a specific activity of 818 micrograms of angiotensin l/min/mg. The active renin secreted in this system had a significantly reduced potency for the renin inhibitor SQ 32,970. These results demonstrate that the pro segment is dispensable for the folding and secretion of renin. A permanent cell line expressing the active form of renin was obtained by co-transfection of NRP cells with pREN3 and pHyg. A colony designated B/1 was identified, subcloned, and shown to secrete active renin (110 pg of renin/10(6) cells) optimally when maintained in both G418 and hygromycin.
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PMID:Stable expression, secretion, and characterization of active human renin in mammalian cells. 173 22

When expressed in COS cells, human prorenin was secreted into the medium without being processed to an active renin. Co-expression of furin, a mammalian homologue of the yeast KEX2 gene product, did not affect proteolytic processing of prorenin. A mutant proreninR-4 constructed by site-directed mutagenesis of Pro (-4) to Arg was not cleaved by an endoprotease in the COS cell. However, proreninR-4 was detectably cleaved to yield the active renin upon co-transfection with furin DNA, indicating that Arg at position -4 is important for recognition and processing by furin in addition to the absolute requirement for paired basic amino acids. Another mutant precursor in which Leu (+1) of proreninR-4 was replaced with Ser was found to be much more efficiently processed than proreninR-4, regardless of co-expression of furin. The results suggest that not only a basic amino acid at position -4 but also Leu at position +1 significantly affect the processing of prorenin catalyzed by the COS cell endoprotease or furin.
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PMID:Sequence requirements for proteolytic cleavage of precursors with paired basic amino acids. 193 Jan 63

Human prorenin is an inactive zymogen comprising 43 amino acid residues at the amino terminus of human renin. The aim of this work was to determine why prorenin is inactive at neutral pH. Eighteen different mutant prorenins, in which positively charged residues in the propeptide were substituted with either glutamine (Gln) or lysine (Lys) residues by site-directed mutagenesis, were expressed in COS-7 cells and characterized. By replacing each of the three arginine (Arg) residues (Arg10P, Arg15P, and Arg20P) with Gln residues, partially active prorenins were produced, which exhibited significant but not full renin activity without trypsin activation. The effect of double or triple amino acid substitutions on the appearance of active prorenin was cumulative, the activity reaching about 80% in a mutant in which all the three Arg residues were replaced by Gln residues. In contrast, mutant prorenins with Lys residues substituted for the Arg residues were inactive. These results clearly indicate that the positive charges of the three Arg residues are essential for maintenance of the human prorenin in an inactive form.
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PMID:Site-directed mutagenesis of human prorenin. Substitution of three arginine residues in the propeptide with glutamine residues yields active prorenin. 218 37

To study the role of N-linked oligosaccharides attached to human renin, we generated three kinds of glycosylation-deficient renins in which one or both of two putative N-glycosylation sites was eliminated by amino acid replacement using site-directed mutagenesis. Examination of the three mutant renins (Asn-5 to Ala, Asn-75 to Ala, and both Asn-5 and -75 to Ala) expressed in COS cells demonstrated that both putative sites were certainly glycosylated with heterologous N-linked oligosaccharides. Moreover, the oligosaccharide chain attached at Asn-5 was different from that attached at Asn-75 in its molecular size. In addition, the secreted amount of the three mutant renins were different from one another, although the mutant and wild-type renins had practically the same specific activity. Our results suggest that the N-linked oligosaccharides have no effect on the enzymatic activity, but play an important role in stable secretion of human renin.
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PMID:Characterization of N-linked oligosaccharides attached to human renin expressed in COS cells. 306 25

Renin is an unique aspartyl (acid) protease with optimal activity at neutral pH. It has been suggested that Ala-317 of human renin contributes to neutral optimum pH of the enzyme [(1984) FEBS Lett. 174, 102-111]. The hypothesis was verified by the characterization of mutant renin in which Ala-317 was replaced with Asp by a site-directed mutagenesis. Wild-type and mutant renins, which were expressed in COS cells, exhibited different pH-activity profiles and optimum pH of the mutant enzyme was lower than that of the wild-type enzyme. This result suggests that Ala-317 of human renin plays an important role in the determination of optimum pH of the enzyme.
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PMID:Functional characterization of Asp-317 mutant of human renin expressed in COS cells. 328 Mar 44

One or both of two putative N-glycosylation sites (at asparagine-5 and -75) of human renin was eliminated by amino acid replacement of the asparagine residue with an alanine residue using site-directed mutagenesis. The three glycosylation-deficient renins (Asn-5, Asn-75, Asn-5 and -75 mutants) were expressed in COS cells and secreted into the conditioned media. The secreted amounts of the three mutants were different from one another, although the mutant and wild-type renins had practically the same specific activity. An Asn-5 and -75 mutant which did not contain any glycosylation sites was unstable in the medium, suggesting that the N-linked oligosaccharides play an important role in stabilization of human renin.
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PMID:Role of N-linked oligosaccharides attached to human renin expressed in COS cells. 328 3

Although many in vitro gene transfer methods already exist, such as calcium phosphate precipitation, electroporation, or cationic liposomes, these methods cause significant cell injury and cell death. The study of the biology of endogenous autocrine-paracrine vasoactive systems such as the renin-angiotensin system in vascular cells is limited by the lack of a suitable gene transfer method with high efficiency of transfection and expression that will permit cell biology studies. Recently, the Sendai virus (hemagglutinating virus of Japan, HVJ)-liposome-mediated gene transfer method has been shown to be an efficient and nontoxic method of gene transfer. In this study, we characterized the efficiency and suitability of the HVJ method for vascular biology research. Using SV40 T-antigen complementary DNA (cDNA), we initially compared the efficiency of the HVJ method and lipofection for transfection of cultured vascular smooth muscle cells (VSMCs). We observed that after 35 minutes of incubation, the HVJ method exhibited a 10-fold higher efficiency of transfection than lipofection. We used this method to study vascular angiotensin converting enzyme (ACE) expression in cultured VSMCs and cultured rat carotid arteries in vitro. The HVJ method of transfection of human ACE cDNA into VSMCs and COS cells was significantly more efficient than lipofection. Using this method, we demonstrated that transfection of ACE cDNA resulted in increased DNA synthesis, which was inhibited by the specific angiotensin II receptor antagonist DuP 753 (10(-6) M).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Novel in vitro gene transfer method for study of local modulators in vascular smooth muscle cells. 768 4

The rabbit proximal tubule (PT) has been widely utilized to study the direct effects of angiotensin II (ANG II) on PT function. The purpose of the present study was to characterize the binding properties of PT ANG II receptors, using nonpeptide antagonists, and to clone a rabbit PT ANG II receptor. In rat and rabbit kidney cortical brush-border and basolateral membranes, specific binding of 125I-ANG II was inhibited by the AT1 ANG II-receptor antagonist DuP 753, but not by the AT2 antagonist PD 123319. Using a rabbit kidney cortex cDNA library, we isolated cDNA encoding an ANG II receptor, with an open-reading frame sharing a high degree of sequence homology to previously cloned AT1 ANG II receptors. In transfected COS-1 cells, this rabbit ANG II receptor had properties of the AT1 class. Northern analysis revealed high levels of mRNA expression for this receptor in rabbit kidney cortex and adrenal gland. Within the kidney, message was detected in primary cultures of rabbit PT cells, as well as in freshly isolated rabbit PT segments. Message was also present in cells of the mouse PT line, MCT, and in rat glomerular mesangial cells. Utilizing polymerase chain reaction (PCR) with primers derived from the 1st and 4th transmembrane domains of the rat AT1A ANG II receptor, a 279-bp DNA fragment was amplified from reverse-transcribed RNA from rabbit PT cells. This DNA encoded an amino acid sequence identical to that encoded by the rabbit kidney cDNA clone in the corresponding region and differed by a single base substitution. Southern analysis of rabbit genomic DNA restriction digests with the rabbit ANG II receptor probe revealed hybridization to a single band in each lane. These results indicate that an AT1 ANG II receptor is present in the PT and that a single gene codes for the AT1 receptor in rabbit. The clone isolated in the present study should provide a useful tool with which to study the regulation of the PT renin-angiotensin system.
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PMID:Cloning of a rabbit kidney cortex AT1 angiotensin II receptor that is present in proximal tubule epithelium. 791 79

There are two major isoforms of the angiotensin II receptor, type 1 (AT1) and type 2 (AT2). AT2 is distinguished from AT1 with respect to its ligand selectivity, its insensitivity to non-hydrolyzable GTP analogues, and its as yet unidentified biological functions. In the present study we have expression-cloned AT2 cDNA from a cDNA library of a rat pheochromocytoma cell line (PC12w). Rat AT2 cDNA encodes a 363-amino acid protein that has seven transmembrane domains. AT1 is the closest in homology to AT2 but with only a 32% identity of amino acid sequence. Stably expressed in COS-7 cells, the receptor showed selective binding to AT2-specific ligands PD123319 and CGP42112A but not to the AT1-specific ligand, losartan. Northern blot analysis revealed that the mRNA of rat AT2 was expressed not only in PC12w cells but also in the adrenal glands and in the inferior olive of the brain, both of which are known to contain AT2 type binding sites. The expressed AT2 receptor mediated angiotensin II-induced inhibition of protein tyrosine phosphatase, an action that was dependent on a pertussis toxin-sensitive G-protein-coupled mechanism in COS-7 cells. The AT2-specific ligand CGP42112A was an agonist rather than antagonist in the inhibition of phosphotyrosine phosphatase. AT2 did not cause a decrease in cGMP in PC12w or COS-7 cells expressing AT2 stably. These results indicate that the AT2 receptor is structurally and functionally different from AT1 and suggest novel functional roles of the renin-angiotensin system in cross-talk with phosphotyrosine signaling by modulating protein phosphotyrosine levels.
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PMID:Molecular cloning of a novel angiotensin II receptor isoform involved in phosphotyrosine phosphatase inhibition. 822 11


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