Multiple forms of immunoreactive renin in human pituitary tissue

Immunoreactive renin was demonstrated in pituitary tissues of postmortem human subjects with different diseases. The specific immunoreactive renin activity comprised the majority of the tissue renin-like activity (mean, 83%), indicating the absence of nonspecific actions of proteases such as cathepsin D. We used three pituitary specimens with high levels of the specific renin activity for further biochemical characterization of the enzyme. Small differences were found in the molecular mass (45 K, 42 K and 37 K), binding to concanavalin A-Sepharose, and isoelectric points (pI) (4.72, 4.78, 4.86, 5.06, 5.28 and 5.44). These results seem to be interpreted as evidence for the presence of specific renin in the human pituitary with microheterogeneity.     

Renin and Angiotensin-Converting Enzyme in Human Neuroblastoma Tissue

High activity of renin was demonstrated in human neuroblastoma tissue. This activity was inhibited by specific antibody raised against human renal renin, indicating that it was not due to the nonspecific action of proteases. The specific activity of renin was 122.8 ng of angiotensin I generated mg of protein-1 h-1. It shared some biochemical features with well-known kidney renin, such as molecular weight, optimum pH, the presence of trypsin-activatable inactive renin, and glycoprotein nature. Furthermore, angiotensin-converting enzyme (ACE) activity (2.64 nmol mg of protein-1 min-1) was found in the tissue. This activity was inhibited by captopril, a specific ACE inhibitor, or by omission of chloride ion. These results suggest that true renin in addition to ACE exists in human neuroblastoma tissue.     

A study on renin binding protein (RnBP) in the human kidney

We purified, from human kidney, a protein that reacts with rabbit anti-porcine kidney renin binding protein (RnBP) antiserum by trapping with porcine kidney renin. The purified preparation showed a single protein peak on gel filtration by high performance liquid chromatography (HPLC) and two protein bands on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The latter two kinds of protein were identified as the porcine renin and human kidney protein from their electrophoretic mobilities and reactivity toward rabbit anti-porcine kidney renin and RnBP antisera. The molecular weights of the purified preparation and the human kidney protein were estimated to be 56,000 by HPLC and 43,000 by SDS-PAGE, respectively. The specific activity of porcine renin in the purified preparation was 8.6 mg angiotensin I per mg of protein per h at 37 degrees C and pH 6.5. This specific activity was about one-fifth that of free porcine renin. Therefore, it is suggested from the reactivity toward the anti-porcine RnBP antiserum and inhibitory action toward porcine renin that the human kidney protein is RnBP and that the human RnBP is purified as a complex with porcine renin.     

Chromatofocusing of mammalian estrone sulfate sulfohydrolase activity

Estrone sulfate sulfohydrolase (estrogen sulfatase) activity was solubilized by treatment with Triton X-100 from 105,000 g pellets of guinea pig uterus, testis and brain, as well as from rat liver and human placenta. The solubilized forms were subjected to chromatofocusing in the fast protein liquid chromatography (FPLC) system and on conventional columns packed in our laboratory. The guinea pig tissue pattern was complex. Uterus showed peaks of activity with apparent pI's of 9.11 and 7.6; testis contained 3 peaks with pI's of 9.18, 8.7 and 7.5; brain possessed peaks with pI's of 9.28 and 8.6. In each case the major activity peak was that with pI greater than 9. Rat liver activity chromatofocused as a single peak of apparent pI = 6.87 and the human placental enzyme also showed a single, though broad, peak, of pI = 6.57. This suggests not only that the guinea pig enzyme(s) differs markedly from those of rat liver and human placenta, but that there may be qualitative differences between the forms in the three guinea pig tissues. Chromatofocusing behaviour was not independent of the specific exchange resins and ampholytes utilized. The recovered enzyme activity was fairly stable and it seems that chromatofocusing could be a useful step in purification of the guinea pig enzyme(s), particularly the main form possessing a pI greater than 9.     

Evidence for the glycoprotein nature of kidney renin.

The glycoprotein nature of renin isolated from either rabbit or human kidney has been demonstrated by affinity chromatography on concanavalin A-Sepharose. The bulk of rabbit renin activity bound to concanavalin A is released by 20 to 50 mM alpha-methyl-D-mannoside. Adsorption of renin is prevented by periodate oxidation prior to chromatography. Mild acid treatment (pH 2.5) prior to chromatography does not alter the concanavalin A binding profile although the pI values of native rabbit renin (5.1-5.6) are shifted into a broader distribution (4.7-6.4). The molecular weight values of rabbit renin obtained by gel filtration and those from zone centrifugation are identical (37000 +/- 1000), consistent with a low percent of carbohydrate in the glycoprotein. A hydrophobic contribution to the binding of renin by concanavalin A is evident since, in the presence of mM Ca2+ and Mn2+, higher concentrations of alpha-methyl-D-mannoside are required to affect the same release of renin at 23 degrees C compared to that at 4 degrees C. Furthermore, 25% ethylene glycol releases renin in the absence of alpha-methyl-D-mannoside. It is concluded that renin contains a small number of carbohydrate residues in relatively close proximity to a hydrophobic surface which enhances the interaction with concanavalin A.     

Blockade of AT1 receptors by losartan did not affect renin gene expression in kidney medulla

This study was designed to determine particular changes in the renin gene expression and activity in renal cortex and medulla after AT(1) receptor blockade. It was found that two-week-treatment with AT(1) blocker losartan induced an increase in tissue renin activity in both parts of kidney causing subsequent elevation of plasma renin activity. Renin mRNA in losartan-treated rats was increased only in cortex, suggesting cortex origin of elevated renin activity in medulla. Medullary renin mRNA indicated local synthesis of renin within the whole kidney and supported the idea of the presence of tissue renin-angiotensin system. Our results show that gene expression of renin in kidney medulla is insensitive to AT(1) receptor blockade and this points out that the regulation of kidney renin-angiotensin system probably differs from that in cortex.     

Purification and characterization of human kidney renin

By means of a new rapid and small scale purification method, human kidney renin has been purified from a single kidney in a homogeneous state, as judged on SDS-PAGE. The kidney which showed unusually high renin activity was from a patient with cardiomyopathy. 8,000-fold purification was attained by means of only pepstatin-aminohexyl-Sepharose chromatography and FPLC on a Mono Q column, and the yield was 34%. The specific activity was 5.63 mg angiotensin I per mg protein per h at 37 degrees C and pH 6.5 with porcine angiotensinogen as the substrate. The molecular weight was estimated to be 37,000 by SDS-PAGE and 38,000 by HPLC on a TSK G-3000 SW column. The preparation showed three bands on isoelectric focusing. The molecular weight and the profile on isoelectric focusing of the purified renin agreed with those found for the extracts of both the patient's kidney and a kidney with the usual low renin activity.     

A fluorogenic near-infrared imaging agent for quantifying plasma and local tissue renin activity in vivo and ex vivo

The renin-angiotensin system (RAS) is well studied for its regulation of blood pressure and fluid homeostasis, as well as for increased activity associated with a variety of diseases and conditions, including cardiovascular disease, diabetes, and kidney disease. The enzyme renin cleaves angiotensinogen to form angiotensin I (ANG I), which is further cleaved by angiotensin-converting enzyme to produce ANG II. Although ANG II is the main effector molecule of the RAS, renin is the rate-limiting enzyme, thus playing a pivotal role in regulating RAS activity in hypertension and organ injury processes. Our objective was to develop a near-infrared fluorescent (NIRF) renin-imaging agent for noninvasive in vivo detection of renin activity as a measure of tissue RAS and in vitro plasma renin activity. We synthesized a renin-activatable agent, ReninSense 680 FAST (ReninSense), using a NIRF-quenched substrate derived from angiotensinogen that is cleaved specifically by purified mouse and rat renin enzymes to generate a fluorescent signal. This agent was assessed in vitro, in vivo, and ex vivo to detect and quantify increases in plasma and kidney renin activity in sodium-sensitive inbred C57BL/6 mice maintained on a low dietary sodium and diuretic regimen. Noninvasive in vivo fluorescence molecular tomographic imaging of the ReninSense signal in the kidney detected increased renin activity in the kidneys of hyperreninemic C57BL/6 mice. The agent also effectively detected renin activity in ex vivo kidneys, kidney tissue sections, and plasma samples. This approach could provide a new tool for assessing disorders linked to altered tissue and plasma renin activity and to monitor the efficacy of therapeutic treatments.     

Multiple forms of human renin. Purification and characterization.

Human renin was purified from a juxtaglomerular cell tumor with a high renin content, 24.2 Goldblatt units/mg of protein. The purification procedure comprised three steps: gel filtration, DEAE-cellulose chromatography, and preparative isoelectric focusing. Five forms of renin amounting to 5.3 mg of enzyme were obtained with isoelectric points of 4.95, 5.10, 5.35, 5.55, and 5.70. They were all glycoproteins. The three major fractions had very similar specific activities, 868, 860, and 809 Goldblatt units/mg of protein. These fractions produced a single band on analytical isoelectric focusing and a single arc on immunoelectrophoresis. On polyacrylamide gel electrophoresis at pH 7.8, each fraction consisted of two renin bands with the same molecular weight, but different net charges. The molecular weight determined by gel filtration and Fergusson plot analysis on polyacrylamide gel was 38,000 to 42,000. The optimum pH determined on N-acetyltetradecapeptide substrate was 6.5, and the Km was 6.8 x 10(-6) M. These parameters were identical with those for standard human kidney renin. Antibodies raised against tumor renin completely inhibited the activity of both tumor and standard renin. Under dissociating conditions (sodium dodecyl sulfate-polyacrylamide gel electrophoresis and gel electrophoresis in the presence of 6 M urea), part of the purified enzyme dissociated into two smaller fragments (Mr = 20,000 and 25,000) containing renin activity.     

Comparative studies on extracellular penicillinases of the same structural gene, penP, expressed in Bacillus licheniformis and Bacillus subtilis

Extracellular penicillinases produced by Bacillus licheniformis ATCC 9945A and Bacillus subtilis from the same structural gene, penP, were compared. The two strains secreted the same exo-large penicillinase (mol. wt, 305000; isoelectric point, pI = 5.00-5.04; NH2-terminal amino acid, Ser). In contrast, the exo-small enzyme from Bacillus subtilis (mol. wt, 29500; pI = 5.00-5.04; NH2-terminal amino acid, Glu or Asn) was slightly different from that of Bacillus licheniformis (mol. wt, 29500; pI = 5.13; NH2-terminal amino acid, Lys). The difference in the NH2-terminal residue is most probably due to differences in degradation by host-specific proteolytic enzymes.     

Superoxide dismutase, glutathione peroxidase, and glutathione reductase in sheep organs

The enzyme activities of the superoxide dismutase (SOD), glutathione peroxidase (GSHPx), glutathione reductase (GR) and thiobarbituric acid reactive substances (TBARS) content were measured in tissue extracts of the liver, kidney and lung of sheep in a nonpolluted control area (C), a polluted area pasture (PP) and those from polluted areas but fed in the laboratory with an experimental emission supplement diet (EEF). Compared with the control SOD, activity was significantly increased (1.75 times) only in the liver of the PP group. In the EEF group there was a tendency toward lower activities in all organs. The Cu,Zn-SOD isoenzymes pattern analyzed by isoelectrofocusing was different in the organs of the animals exposed to pollutants when compared with those of the controls. In the liver, two new isoenzymes with pI 5.30 and 5.70 were found in the PP group and an additional isoenzyme with pI 5.10 in the EEF group. The kidney isoenzymes with pl 5.30 and 5.40 were inhibited in the EEF group. In the lung, two new isoenzymes appeared with pl 5.30 and 5.40 in the PP group and two new isoenzymes with pI 6.10 and 6.50 in the EEF group. GSHPx activity was inhibited in the liver and kidney of the sheep exposed to pollutants. GR activity was significantly changed only in the liver. The activity in the PP group was 2.30 and 2.10 times higher than in the C and EEF groups, respectively. TBARS content was increased in the liver and kidney of the EEF group compared with the control.     

Molecular cloning and nucleotide sequence of a human renin cDNA fragment.

We have studied human renin messenger RNA by hybridization with the mouse submaxillary gland (SMG) renin cDNA probe. The human kidney messenger RNA is about 1.6 kilobase (kb) long, similarly to the mouse SMG renin mRNA. A kidney renin cDNA clone of 1.1 kb length was obtained. A comparison of nucleotide sequences of mouse and human cDNA clones reveals conservation of residues involved in catalytic mechanisms and a potential glycosylation site. The human renin molecular probe allowed us to study renin expression in human chorionic tissue. The chorionic and kidney renin messenger RNAs are similar in length. The Southern blot analysis reveals the presence of a single renin gene in human DNA.     

Renin inhibits N-acetyl-D-glucosamine 2-epimerase (renin-binding protein)

Renin-binding protein (RnBP) is a highly specific renin inhibitor first isolated from porcine kidney. Our recent studies demonstrated that the human RnBP is the enzyme N-acetyl-D-glucosamine (GlcNAc) 2-epimerase [Takahashi, S. et al. (1999) J. Biochem. 125, 348-353]. We have developed a new assay method for GlcNAc 2-epimerase activity using a system of N-acyl-D-hexosamine oxidase coupled with peroxidase and employed this method to study the effects of renin on GlcNAc 2-epimerase activity. The recombinant human (rh) RnBP existed as a dimer and its GlcNAc 2-epimerase activity was strongly inhibited by the purified renin concomitant with the formation of RnBP-renin heterodimer, so-called high molecular weight (HMW) renin. The renin activity was also inhibited by rhRnBP in a dose-dependent manner. These results indicate that renin is an inhibitor of GlcNAc 2-epimerase, and the renin-RnBP heterodimer HMW renin is an inactive form of both renin and GlcNAc 2-epimerase activities.     

Acidic and basic forms of glutathione S-transferases from human placenta and comparison with human kidney glutathione S-transferase

Glutathione S-transferase (GSH-transferase) was purified from human placenta and kidney by affinity chromatography on S-glutathione-carbamidomethyl-epsilon-aminolysyl-Sepharose CL 4B and gel filtration chromatography on Sephades G-75. Electrophoretically pure enzyme with the specific activities of 50.7 and 55.9 U/mg, respectively, were obtained. In addition to the known acidic isoenzyme from human placenta (isoelectric point, pI, 4.5), we describe here for the first time the presence of 6 basic forms with pI values between 8.0 and 9.0. The kidney GSH-transferase contained 2 acidic forms with isoelectric points at 4.6 and 4.65, and 6 basic forms with pI values between 8.7 and 9.4. The basic and acidic isoenzymes from placenta were separated by ion exchange chromatography on Sephadex DEAE A-25. The acidic form accounted for 36% of the total GSH-transferase activity from placenta. Antibodies against the kidney enzyme were raised in rabbit. Total cross-reactivity of placental GSH-transferase with antikidney-GSH-transferase antibodies was obtained, suggesting that the kidney and placental enzymes are immunologically closely related.     

Purification of human renin by affinity chromatography using a new peptide inhibitor of renin, H.77 (D-His-Pro-Phe-His-LeuR-Leu-Val-Tyr).

A new affinity column for renin was prepared by coupling the isosteric peptide inhibitor of renin, H.77 (D-His-Pro-Phe-His-LeuR-Leu-Val-Tyr, where R is a reduced isosteric bond, -CH2-NH-), to activated 6-aminohexanoic acid-Sepharose 4B. Chromatography of a crude extract of human kidney cortex on this material resulted in a 5500-fold purification of renin in 76% yield. The purified enzyme (specific activity 871 units/mg) was free of non-specific acid-proteinase activity and was stable at pH 6.8 and -20 degrees C over a period of several weeks.     

Purification and characterization of human truncated prorenin.

Posttranslational processing of enzymatically inactive prorenin to an active form participates in the control of the activity of a key system involved in blood pressure regulation, growth, and other important functions. The issue is complicated because renin can be produced by a number of tissues throughout the body, in addition to the kidney, but the mechanism by which they process prorenin to renin is unknown and difficult to determine because of the small amounts of renin present. In the juxtaglomerular cell of the kidney, a 43 amino acid prosegment is cleaved from the amino terminus of prorenin to generate renin of molecular weight 44,000 [Do, Y. S., Shinagawa, T., Tam, H., Inagami, T., & Hsueh, W. A. (1987) J. Biol. Chem. 262, 1037-1043]. Using human uterine lining or a recombinant human prorenin system, we employed the same approach as that used in kidney, ammonium sulfate precipitation at pH 3.1 followed by pepstatin and H-77 affinity chromatography or gel filtration, to purify to homogeneity a 45,500-MW totally active renin. The specific activity of the active truncated prorenin was 850 Goldblatt units (GU)/mg of protein for chorion-decidua renin and 946 GU/mg of protein for recombinant renin, both similar to that reported for pure human renal renin. Both forms of renin cross-reacted with an antibody generated against 44,00-MW pure human renal renin and with an antibody generated against a peptide identical to the carboxy-terminal one-third of the prosegment.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and properties of multiple forms of dihydrodiol dehydrogenase from human liver

Two acidic and three basic forms of monomeric dihydrodiol dehydrogenase with molecular weights in the range of 36,000-39,000 were purified from human liver. One acidic enzyme (pI 5.2), which was specific for NADP- and dihydrodiols of benzene and naphthalene, was immunologically identified as aldehyde reductase. The other four enzymes oxidized alicyclic alcohols as well as the dihydrodiols using both NADP+ and NAD+ as cofactors, but showed differences in specificity for hydroxysteroids and inhibitor sensitivity. Two of the basic enzymes (pI 9.7 and 9.1) exhibited a 20 alpha-hydroxysteroid dehydrogenase activity and sensitivity to 1,10-phenanthroline, whereas the third basic enzyme (pI 7.6) oxidized some 3 alpha-hydroxysteroids at low rates and was inhibited by cyclopentane-1,1-diacetic acid. Another acidic enzyme, which accounted for the largest amount of enzyme activity in the tissue and appeared in two heterogenous forms with pI values of 5.9 and 5.4, showed a high 3 alpha-hydroxysteroid dehydrogenase activity and was the most sensitive to inhibition by medroxyprogesterone acetate. The Km values of the enzymes, except the pI 5.2 enzyme, for hydroxysteroids (10(-6) to 10(-7) M) were lower than those for xenobiotic alcohols.     

Purification and properties of a megakaryocyte stimulatory factor present both in the serum-free conditioned medium of human embryonic kidney cells and in thrombocytopenic plasma

Megakaryocyte stimulatory factor (MSF) has been purified to homogeneity (7.5 X 10(5)-fold) from serum-free conditioned medium obtained from cultured human embryonic kidney cells and to near homogeneity (1.44 X 10(7)-fold) from thrombocytopenic rabbit plasma. MSF activity from either source was assayed by its ability to enhance the rate of synthesis of platelet factor 4-like proteins in a rat promegakaryoblast cell line. The 125I-labeled factor prepared from human embryonic kidney cell conditioned medium is homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and isoelectric focusing in the presence of 9.2 M urea. MSF obtained from the above source is an acidic protein (pI = 5.1) with an Mr = 15,000 which stimulates platelet factor 4-like protein synthesis in rat promegakaryoblast cells by as much as 7-fold, and exhibits half-maximal activity at a concentration of 0.8 pM. MSF was also purified from thrombocytopenic rabbit plasma by a nearly identical isolation procedure, and 125I-labeled factor prepared from this source also possessed an Mr = 15,000. MSF exhibited no biologic activity corresponding to other known hemopoietic growth factors, and appears to be specific for the megakaryocyte lineage.     

Characterization of pure human renal renin. Evidence for a subunit structure

Renin was completely purified from human kidney cortex employing a rapid three-step procedure which included homogenization and ammonium sulfate precipitation, aminohexyl-pepstatin affinity chromatography, and affinity chromatography using a synthetic octapeptide renin inhibitor (H-77) with a reduced peptide bond (-CH2-NH- instead of -CO-NH-) between Leu5-Leu6, Three kg of cortex dissected from 10 kg of human cadaver kidney yielded 1.7 +/- 0.5 mg of protein (mean +/- S.E. for five procedures) with a specific activity of 1094 +/- 166 Goldblatt units/mg of protein and an overall recovery of 52 +/- 2%. Both gel filtration high performance liquid chromatography and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a molecular weight of 44,000, although Mr = 22,000 and 18,000 bands were also identified by SDS-PAGE. The pH optima with sheep angiotensinogen were 5.5 and 7.8 and the Km was 0.31 microM. With pure human substrate the pH optimum was 6.0 and the Km was 1.15 microM. Enzyme activity was inhibited by two different anti-human renal renin antibodies. Amino-terminal sequencing demonstrated a leucine residue at the 1-position. Sequencing of 15 additional amino acids agreed with that predicted from the gene sequence and indicated that prorenin is converted to renin following cleavage at the carboxyl end of two basic residues, Lys-2 Arg-1. As with SDS-PAGE analysis, high performance liquid chromatography in the presence of 6 M urea demonstrated Mr = 44,000, 22,000, and 18,000 bands. Immunoblot studies revealed that all of these bands cross-reacted with antihuman renin antibody. Amino-terminal sequencing indicated the Mm = 22,000 band is the amino terminus and the Mr = 18,000 band the carboxyl terminus of Mr = 44,000 renin. In the aqueous phase, these subunits bound to H-77 suggesting that they represent components of the active enzyme complex. Unlike mouse renin, there was no evidence of disulfide bonds. These results raise the question of whether human renin circulates as a subunit aggregation as well as a single chain protein. This may serve as a possible mechanism to regulate renin activity in plasma and tissues.