Renin Precursor from Mouse Kidney Identified by Cell-Free Translation of Messenger RNA

1980 ◽  
Vol 59 (4) ◽  
pp. 297-299 ◽  
Author(s):  
K. Poulsen ◽  
J. Vuust ◽  
T. Lund
Keyword(s):  
Molecular Weight ◽  
Messenger Rna ◽  
Submaxillary Gland ◽  
Mouse Kidney ◽  
Single Chain ◽  
Active Renin ◽  
Biosynthetic Precursor ◽  
Mouse Submaxillary Gland ◽  
Free Translation

1. The biosynthetic precursor of renin (pre-prorenin) from mouse kidney is a single chain polypeptide with a molecular weight of 50 000. 2. This is the same value as previously found for mouse submaxillary gland pre-prorenin. 3. Mouse kidney pre-prorenin (mol. wt. 50 000) is larger than the enzymatically active renin (mol. wt. 40 000).

Studies of the Biosynthesis of Renin with a Cell-Free Translation System

1981 ◽  
Vol 61 (s7) ◽  
pp. 241s-243s ◽  
Author(s):  
V. J. Dzau ◽  
A. Ouellette ◽  
R. Pratt
Keyword(s):  
Molecular Weight ◽  
Polyacrylamide Gel Electrophoresis ◽  
Submaxillary Gland ◽  
Translation System ◽  
Mouse Submaxillary Gland ◽  
Free Translation ◽  
Renin Mrna ◽  
A Cell ◽  
Deficient Mice ◽  
Identical Fashion

1. Poly(A)+ mRNA from mouse submaxillary gland encodes a polypeptide of molecular weight 48 000 (48K polypeptide) which is abundant in the male. 2. This polypeptide is selectively absent in the translation products of mRNA from a strain of genetically renin-deficient mice C57 BL/10J. 3. The 48K polypeptide binds and co-elutes in identical fashion with pure authentic renin on pepstatin affinity chromatography. 4. Immunoprecipitation of translation products of male glandular mRNA with renin-specific antibody yielded this 48K band upon analysis by SDS/polyacrylamide gel electrophoresis and fluorography. Pure renin of molecular weight 37 000 blocked the binding of this polypeptide to antirenin antibody. 5. Mouse submaxillary gland synthesizes a renin precursor. The renin mRNA is androgenically regulated.

Immunofluorescent localization of renin in mouse submaxillary gland and kidney.

1978 ◽  
Vol 234 (5) ◽  
pp. E480
Author(s):  
J W Menzie ◽  
L H Hoffman ◽  
A M Michelakis
Keyword(s):  
Direct Evidence ◽  
Submaxillary Gland ◽  
Juxtaglomerular Apparatus ◽  
Mouse Kidney ◽  
Male Mice ◽  
Immunofluorescent Localization ◽  
Submaxillary Glands ◽  
Mouse Submaxillary Gland

An antibody prepared against purified submaxillary renin was used to determine the site of renin concentration in male mice using immunofluorescent localization. The results provide direct evidence that the granular tubules of the submaxillary glands are the source of submaxillary renin. The antibody against submaxillary renin cross-reacts with kidney renin as evidenced by immunofluorescent localization in the juxtaglomerular apparatus of mouse kidney.

Abstract 40: Renin is Produced By a General Lysosomal Degradation Mechanism

Hypertension ◽  
2012 ◽  
Vol 60 (suppl_1) ◽  
Author(s):  
Lucie K Xa ◽  
Marie-Josée Lacombe ◽  
Chantal Mercure ◽  
Timothy Reudelhuber
Keyword(s):  
Messenger Rna ◽  
Degradation Mechanism ◽  
Differential Susceptibility ◽  
Mouse Kidney ◽  
Proteolytic Processing ◽  
Lysosomal Degradation ◽  
Lysosomal Hydrolases ◽  
Expression Array ◽  
Active Renin ◽  
C57bl6 Mouse

Renin is secreted almost exclusively by the juxtaglomerular (JG) cells of the kidney where it is first made as an inactive precursor called prorenin. Conversion of prorenin to active renin requires the proteolytic removal of an N-terminal prosegment by a second protease whose identity is still debated. Active renin is then stored in dense vesicles and secreted in response to stimuli. Using confocal microscopy we found that C57BL6 mouse kidney JG cells are highly enriched in Lamp-1, a biomarker for lysosomes, and that renin and Lamp-1 co-localize in renin storage vesicles. These data suggest that renin is stored in secretory lysosomes. N-terminal sequencing of C57BL6 mouse Ren-1 renin purified from kidney revealed an N-terminus beginning with SPVVLT¼. This is the same amino terminus as that reported for rat renin and mouse As4.1 cells and different from that reported for human renin. This result raises the possibility that rodents and humans use different prorenin processing enzymes (PPE). Treatment of mice with captopril for 7 days increases plasma active renin by 19-fold (control 149 +/- 22 vs. treated 2859 +/- 672 ng AI/ml/hr, P< 0.0001) and kidney renin messenger RNA by 4.81-fold (P< 0.0001). Nevertheless, Illumina expression array analysis of C57BL6 mouse kidney before and after captopril treatment did not reveal candidate PPEs whose expression paralleled that of renin. This result suggests that the PPE is not limited to JG cells. To test the possibility that general lysosomal hydrolases are responsible for renin production, we used a Lamp-1 C-terminal sequence to force the sorting of mouse Ren-1 prorenin into lysosomes of transfected human embryonic kidney (HEK) cells. Transfection resulted in the intracellular retention of renin of the appropriate molecular weight and which lacked the engineered Lamp-1 C-terminal tail, suggesting that the proteolytic processing of prorenin is not carried out by a protease that is restricted to JG cells. Altogether, our results are consistent with mature renin being produced by lysosomal degradation of the prosegment and the selective resistance of mature renin to hydrolysis. The different N-termini of rodent and human renins could be explained by differential susceptibility of their prosegments to lysosomal hydrolysis.

Vast release of submaxillary mouse renin to saliva after stimulation with cholinergic, β-adrenergic but especially α-adrenergic agonists

1982 ◽  
Vol 99 (4) ◽  
pp. 636-640 ◽  
Author(s):  
Erik Bækkelund Pedersen ◽  
Knud Poulsen
Keyword(s):  
Molecular Weight ◽  
Enzymatic Activity ◽  
Adrenergic Receptors ◽  
Submaxillary Gland ◽  
Renin Release ◽  
Adrenergic Agonists ◽  
Cholinergic Stimulation ◽  
Adrenergic Stimulation ◽  
Mouse Submaxillary Gland ◽  
Specific Enzymatic Activity

Abstract. α-adrenergic stimulation with phenylephrine causes release of vast amounts of mouse submaxillary gland renin to saliva. The amount released is about 100-fold higher than that released after β-adrenergic stimulation with isoproterenol and about 1000-fold higher than the amount released after cholinergic stimulation with pilocarpine. α-blockade with phenoxybenzamine before phenylephrine stimulation strongly inhibits the renin release. However, phenoxybenzamine or β-blockade with propranolol before isoproterenol stimulation have no effect on the amount of renin released. Thus, the release of submaxillary mouse renin to saliva is triggered mainly by activation of α-adrenergic receptors. Renin in cholinergic and α- and β-adrenergic stimulated saliva has the same molecular weight and the same specific enzymatic activity as renin in the submaxillary gland. This indicates that renin is secreted to saliva, and probably also to the blood, in its fully enzymically active 40 000 mol. wt. form regardless of the stimulus used. The vast amounts of renin released to saliva (up to 600 Goldblatt Units) demonstrate that submaxillary mouse renin is secreted predominantly to saliva and much less to the blood. The mouse submaxillary gland therefore seems to have an exocrine rather than an endocrine role for release of renin.

Interaction of Plasminogen Activators and Plasminogen with Heparin: Effect of Ionic Strength

1993 ◽  
Vol 70 (05) ◽  
pp. 867-872 ◽  
Author(s):  
Dingeman C Rijken ◽  
Gerard A W de Munk ◽  
Annie F H Jie
Keyword(s):  
Molecular Weight ◽  
Ionic Strength ◽  
Plasminogen Activator ◽  
Plasminogen Activators ◽  
Fibrinolytic System ◽  
Tissue Type ◽  
Single Chain ◽  
Physiological Conditions ◽  
Amino Terminal ◽  
Type Plasminogen Activator

SummaryIn order to define the possible effects of heparin on the fibrinolytic system under physiological conditions, we studied the interactions of this drug with plasminogen and its activators at various ionic strengths. As reported in recent literature, heparin stimulated the activation of Lys-plasminogen by high molecular weight (HMW) and low molecular weight (LMW) two-chain urokinase-type plasminogen activator (u-PA) and two-chain tissue-type plasminogen activator (t-PA) 10- to 17-fold. Our results showed, however, that this stimulation only occurred at low ionic strength and was negligible at a physiological salt concentration. Direct binding studies were performed using heparin-agarose column chromatography. The interaction between heparin and Lys-plasminogen appeared to be salt sensitive, which explains at least in part why heparin did not stimulate plasminogen activation at 0.15 M NaCl. The binding of u-PA and t-PA to heparinagarose was less salt sensitive. Results were consistent with heparin binding sites on both LMW u-PA and the amino-terminal part of HMW u-PA. Single-chain t-PA bound more avidly than two-chain t-PA. The interactions between heparin and plasminogen activators can occur under physiological conditions and may modulate the fibrinolytic system.

Analysis of Intrinsic Fibrinolysis in Human Plasma Induced by Dextran Sulfate

1992 ◽  
Vol 67 (04) ◽  
pp. 440-444 ◽  
Author(s):  
Hiroko Tsuda ◽  
Toshiyuki Miyata ◽  
Sadaaki Iwanaga ◽  
Tetsuro Yamamoto
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Dextran Sulfate ◽  
Tissue Type ◽  
Factor Xii ◽  
Normal Human Plasma ◽  
High Molecular Weight Kininogen ◽  
Single Chain ◽  
Major Pathway ◽  
Normal Human

SummaryThe analysis of normal human plasma by fibrin autography revealed four species of plasminogen activator (PA) activity related to tissue-type PA, factor XII, prekallikrein and urokinase-type PA (u-PA). The u-PA activity increased significantly by incubating plasma with dextran sulfate. This increase was coincident with both the cleavage of factor XII and the complex formation of activated factor XII with its plasma inhibitors, which were determined by immunoblotting procedure. The dextran sulfate-dependent activation of u-PA required both factor XII and prekallikrein, but did not require either plasminogen or factor XI. High molecular weight kininogen was required only at a low concentration of dextran sulfate. Thus the results indicate that the factor XII and prekallikrein-mediated activation of single chain u-PA (scu-PA) operates as a major pathway of scu-PA activation in whole plasma in contact with dextran sulfate.

Properties of Chimeric (Tissue-Type/Urokinase-Type) Plasminogen Activators Obtained by Fusion at the Plasmin Cleavage Site

1993 ◽  
Vol 69 (05) ◽  
pp. 466-472 ◽  
Author(s):  
M Colucci ◽  
L G Cavallo ◽  
G Agnelli ◽  
A Mele ◽  
R Bürgi ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Plasminogen Activator ◽  
Cleavage Site ◽  
Plasminogen Activators ◽  
Tissue Type ◽  
Low Molecular Weight ◽  
Single Chain ◽  
Type Plasminogen Activator ◽  
Kringle 2 ◽  
Fibrin Monomers

SummaryTwo hybrid plasminogen activators (K2tu-PA and FK2tu-PA), linking the kringle 2 domain or the finger plus the kringle 2 domains of tissue-type plasminogen activator (t-PA) to the catalytic domain of single-chain urokinase-type plasminogen activator (scu-PA) were studied. At variance with similar constructs previously reported, they were obtained by fusion of the t-PA and scu-PA derived portions at their plasmin cleavage site (between Arg275 of t-PA and Ile159 of scu-PA), thus eliminating from scu-PA the two peptide bonds (Glu143-Leu144 and Arg156-Phe157) that lead to low molecular weight scu-PA and to thrombin-inactivated tcu-PA. The specific activities of K2tu-PA and FK2tu-PA, as measured by fibrin plate were 2.5 × 106 and 1.0 × 106 t-PA equivalent units/mg, respectively. Activation of plasminogen by hybrid PAs was stimulated by both CNBr-digested fibrinogen (40- and 80-fold) and Des-A-fibrin monomers (6- and 12-fold). The relatively weak stimulation of chimeric PAs by minimally degraded fibrin monomers was consistent with their reduced fibrin binding capacity. Like scu-PA, the chimeric PAs, in the single-chain form, were insensitive to inhibition, as they retained full activity after prolonged incubation in plasma and did not interact with SDS-reactivated recombinant PAI-1. The concentration producing 50% lysis of blood clots in 3 h was 0.5 μg/ml for K2tu-PA and 1 μg/ml for FK2tu-PA, as compared to 0.5 μg/ml and >2 μg/ml for t-PA and scu-PA, respectively. Plasminogen and α2-antiplasmin consumption induced by the hybrid PAs in clot-free plasma was comparable to (K2tu-PA) or lower than (FK2tu-PA) that induced by either t-PA or scu-PA. When exposed to plasmin, the hybrids were completely converted into two-chain molecules with full enzymatic activity. At variance with u-PA, however, the two-chain recombinant activators still required fibrin for full expression of activity. These data indicate that the products of such “artificial” fusion behave like true chimeras without loss of biological activity. The insensitivity to thrombin inactivation and to the proteolytic cleavage leading to low molecular weight scu-PA might confer enhanced stability to the molecules, especially at thrombus level. Moreover, if the thrombolytic activity observed in vitro is maintained in vivo, the prolonged half life of these hybrids should result in higher plasma levels of activator and thus in more extensive and rapid lysis.

Rapid Isolation of High Molecular Weight Urokinase from Native Human Urine

1982 ◽  
Vol 47 (03) ◽  
pp. 197-202 ◽  
Author(s):  
Kurt Huber ◽  
Johannes Kirchheimer ◽  
Bernd R Binder
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Human Urine ◽  
Gel Filtration ◽  
Chain Structure ◽  
The Other ◽  
Single Chain ◽  
Apparent Homogeneity ◽  
Sequential Adsorption

SummaryUrokinase (UK) could be purified to apparent homogeneity starting from crude urine by sequential adsorption and elution of the enzyme to gelatine-Sepharose and agmatine-Sepharose followed by gel filtration on Sephadex G-150. The purified product exhibited characteristics of the high molecular weight urokinase (HMW-UK) but did contain two distinct entities, one of which exhibited a two chain structure as reported for the HMW-UK while the other one exhibited an apparent single chain structure. The purification described is rapid and simple and results in an enzyme with probably no major alterations. Yields are high enough to obtain purified enzymes for characterization of UK from individual donors.

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