Properties of the activation by pepsin of inactive renin in human amniotic fluid.

1. The renin present in human amniotic fluid was found to have an apparent Mr of 58 000 by gel filtration and is thus bigger than renin in untreated kidney extracts and plasma (Mr approximately 40 000). 2. Treatment with pepsin (40 microgram/ml pH 4.8, 2 h, 22 degrees C) caused a 6-fold increase in activity of this renin species, although Mr was not very different (57 000). 3. Unlike renal renin, renin in human amniotic fluid was not a glycoprotein and behaved similarly on concanavalin A-Sepharose before and after activation by pepsin. 4. Ion-exchange chromatography demonstrated a small change in the ionization properties of human amniotic fluid renin after activation by pepsin. 5. Pepsin-mediated activation resulted in a five-fold increase in V, but only a small decrease in the Km of renin to 39% of normal, so that the increase in activity observed was not due to an increase in the affinity of the enzyme for its substrate. The kinetic data were consistent with the theory of noncompetitive inhibition. 6. The activation of human amniotic fluid renin by pepsin may be caused by a change in the tertiary structure of the molecule subsequent to a proteolytic action that does not remove detectable polypeptide components.     

Pure human inactive renin. Evidence that native inactive renin is prorenin

To clarify contradicting observations on the identity of inactive renin and prorenin, inactive renin was completely purified from native human chorion laeve and the culture medium of human chorion cells. A 720,000-fold purification with 14% recovery was achieved from chorion laeve in 6 steps, including immunoaffinity chromatography on a monoclonal antibody to human renin coupled to Protein A-Sepharose CL-4B. A 3,100-fold purification with 40% recovery was achieved from chorion culture medium in 4 steps, including immunoaffinity chromatography. Inactive renin purified from the two different sources migrated as a single protein band with the same molecular weight of 47,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and consisted of multiple components that could be resolved by isoelectric focusing. Both had the same pI values which shifted downward upon activation by trypsin; however, relative peak heights were different between the two preparations. The purified inactive renin from chorion laeve was completely inactive and did not bind to pepstatin-aminohexyl-Sepharose; however, that from chorion culture medium was partially active and completely bound to the pepstatin gel, indicating that each molecule is partially activated. Trypsin-activated inactive renins from both sources were identical with human renal renin in terms of pH optimum and Km. Specific activities of trypsin-activated inactive renin from chorion laeve and chorion culture medium were 529 Goldblatt units/mg of protein and 449 Goldblatt units/mg of protein, respectively. Amino acid sequence analysis of both of the purified inactive renin preparations demonstrated a leucine residue at the amino terminus. The sequence of 11 additional amino acids was identical in both and agreed with that predicted from the base sequence of the renin gene. These findings indicate that preprorenin is converted to prorenin following removal of a 23-amino acid signal peptide and that the native inactive renin, whose amino acid sequence commences with Leu-Pro-Thr..., is prorenin.     

Physicochemical properties of non-activated and activated renin from human amniotic fluid.

The main physicochemical and enzymic properties of non-activated and activated human amniotic renin (EC 3.4.99.19) were studied in order to clarify the relationships between the two enzymes. Human amniotic renin was activated by dialysis against acidic buffer (pH 3.3), direct acidification or trypsin treatment. All procedures produced similar activation. The physicochemical characteristics of non-activated and activated renin were compared to those of human renal renin. Non-activated renin had a molecular weight of 45,500. A similar molecular weight was obtained by gel eluate activation and by acid treatment of renin prior to gel filtration. Similar isoelectric points were also found for non-activated and activated renin. One major renin peak focused at pH 6.6, whereas no similar renin peak was detected in extracts from normal human kidney. In addition, non-activated and activated renin forms were found to have the same optimal pH, the same Km and the same inhibiting pepstatin concentrations.     

Comparative enzymatic studies of human renin acting on pure natural or synthetic substrates

Some of the essential structural requirements for the enzymatic reaction of pure human renin acting on pure human and rat angiotensinogen and on their synthetic tetradecapeptide substrates were investigated. The five carboxy terminal amino acids of synthetic tetradecapeptides played a significant role in substrate recognition and/or hydrolysis by human renin. Kinetic constants Km, Kcat and kcat/Km of the various human renin assays were different according to the substrate used. The presence of either an asparagine or a threonine residue in the S'4 renin subsite did not affect significantly the kinetic constant values. A tyrosine residue, rather than a histidine residue, in the S'3 renin subsite gave the best synthetic substrate studied. When tyrosine residue was present in the S'2 renin subsite an important decrease in kcat was observed. Human angiotensinogen was hydrolysed by human renin with lower Km and kcat values than those measured with human and porcine synthetic substrates, suggesting that the 3-dimensional structure of human angiotensinogen plays a key role in the hydrolysis. This finding was supported by assays performed with rat angiotensinogen, which was cleared by human renin with the same kcat value as rat tetradecapeptide, but with a 49-fold lower Km. Between human and rat angiotensinogen a kcat/Km value of only 2-fold higher has been found in the renin assay using human substrate.     

Active and inactive renin in human forearm of hypertensive patients.

Although many in vitro and animal studies indicate the existence of a local renin--angiotensin system, data regarding its physiological role are quite controversial, and moreover, evidence suggesting inactive and active renin release from vascular tissue in vivo is lacking both in animal and humans. The aim of our study was to evaluate whether beta-adrenoceptor stimulation, a well-known stimulus to renin production, through isoproterenol might cause local renin production from vessels of the forearm of hypertensive patients. Drugs were infused into the brachial artery at systemically ineffective rates, while forearm blood flow (FBF, venous plethysmography), mean intra-arterial pressure, and heart rate were monitored throughout. Active and inactive vessel renin production was measured by calculating venous-arterial (V-A) differences by simultaneous sampling from brachial artery and an ipsilateral deep vein. Active renin (PRA) and total renin (Sepharose bound trypsin activation) were measured by radioimmunoassay while inactive renin was calculated as the difference between total and active renin. V-A differences were corrected for FBF to calculate renin extraction or production. In a group of 10 patients, isoproterenol, which was infused at increasing cumulative rates (0.03, 0.1, 0.3 micrograms.100 mL-1 forearm tissue.min-1 for 5 min each), caused a dose-dependent increment in FBF that was blunted by intra-arterial propranolol (n = 5) pretreatment (10 micrograms.100 mL-1 forearm tissue.min-1 for 10 min). beta-Adrenoceptor stimulation caused a dose-dependent outflow of both active and inactive renin, an effect antagonized by propranolol. In conclusion, our data represent the first evidence in humans of tissue active and inactive renin production in the forearm vascular bed.     

Identification and quantitation of human amniotic fluid components using capillary zone electrophoresis

A capillary electrophoresis method was used to measure albumin, immunoglobulin G (IgG), transferrin, and uric acid in 230 amniotic fluid (AF) samples collected at 15.15 ± 0.06 weeks gestation. Species were quantified by external calibration using thiamine as internal standard. All major components were detected within 10 min. Migration time reproducibility was 3.0% relative standard deviation (RSD) and normalized peak areas were 12% RSD or better at 190 nm from 81 measurements of a pooled AF sample. The separation profile was not affected by 10 h of storage at room temperature or by 10 freeze-thaw cycles, suggesting that frozen AF samples are suitable for protein biomarker studies.     

In vitro biosynthesis of human renin and identification of plasma inactive renin as an activation intermediate

The biosynthesis and post-translational modifications, including proteolytic processing and core glycosylation, of the human renin precursor have been studied in vitro in a cell-free system. For this purpose, highly enriched renin mRNA was isolated from a renin-producing juxtaglomerular cell tumor and translated in rabbit reticulocyte lysate containing [35S]methionine in the presence or absence of dog pancreas microsomal membranes. Fluorographic analysis of the radioactive translation products, immunoprecipitated and then resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, revealed that the primary translation product, preprorenin (Mr = 45,000), is initially processed to glycosylated prorenin (Mr = 47,000) during or shortly after its sequestration into the lumen of the microsomal membranes. The vectorial translocation across the membrane was confirmed by the observation that the proform was resistant to digestion with trypsin while preprorenin was sensitive. Radiosequencing and the use of prorenin-specific antibodies established the cleavage points of the pre- and profragment and showed that the in vitro precursor of human renin contains a 23-residue signal peptide and a 43-residue prosegment. The post-translational modification which, despite the removal of signal peptide, resulted in an increase in apparent Mr, reflects the glycosylation as examined using Xenopus oocytes microinjected with renin mRNA in the presence of tunicamycin, an inhibitor of protein glycosylation. Four anti-peptide antibodies which specifically recognize the NH2 terminus (Pro 1), two middle parts (Pro 2A and Pro 2B), and COOH terminus (Pro 3) of the prosegment, respectively, have been raised and used to characterize plasma prorenin. Renin precursors (pre- and prorenin) synthesized in vitro or in the kidney reacted with these antibodies (anti-Pro 1, anti-Pro 2A, anti-Pro 2B, and anti-Pro 3). However, quite unexpectedly, human plasma prorenin was recognized only by anti-Pro 3, indicating that plasma prorenin is a truncated version of intact prorenin, which lacks a large portion of the NH2 terminus of the prosegment and may represent an activation intermediate. This somewhat surprising result may lead to a better understanding of the exact roles and activation mechanisms of plasma prorenin existing in a relatively large amount.     

Investigation of human and rat inactive renin in plasma and kidney.

Under an initial interval of immobilization stress in rats, reciprocal changes of plasma active and inactive renin were observed, suggesting activation of circulating inactive renin. Molecular weight (MW) studies revealed that this activation might proceed via a MW shift from inactive renin with MW of 50,000 to active renin of MW 43,000. In a later interval of stress, under stimulated renin secretion, a lower MW form (38,000) of active renin was released into the circulation. This MW is close to that of active renin (39,000) found in rat kidney renin granules. In renin granules, equilibrated in fractions of 1.6 and 1.7 mol/L sucrose in discontinuous density gradient, trypsin-activatable renin activity formed 36 and 16% of total activity, respectively. In humans, under acute bicycle exercise, a lower MW form (39,000) of active renin was released into the circulation, while the content of inactive renin with MW in the range of 51,000-58,000 and at 47,000 did not substantially change. There was a slight decrease in circulating inactive renin passing through the kidney. The data suggest that, at least in rats, in vivo pathways for activation of inactive renin might exist, other than that proceeding before secretion from renin granules. Under the conditions of increased renin secretion, a lower MW form of active renin is mainly released into the circulation in both rats and humans.     

Active and inactive renin after exercise

The effects of graded exercise on plasma concentrations of active and inactive renin were studied in seven healthy men. Exercise was performed on a cycle ergometer at four different exercise intensities (corresponding to 30%, 50%, 80% and 87% of VO2max) for 10 min each. Concentrations of active renin and total renin after activation by trypsin were measured by direct immunoradiometric assay. Non-trypsin-activated renin concentration (inactive) was obtained by subtraction. Active renin concentrations at 30%, 50%, 80% and 87% of VO2max were 1.2, 1.9, 3.1 and 4.6 times higher than the control concentration, respectively. Similar increases in plasma renin concentration, determined by conventional enzymatic assay, were observed at every stage. In contrast, changes in inactive renin concentration were not significant at any stage. Significant increases in noradrenaline concentration were found at every exercise stage, but adrenaline, aldosterone and lactate concentrations were significantly elevated only after exercise at 50%, 80% and 87% of VO2max. The similarity between the changes in concentration of active renin and noradrenaline would suggest that sympathetic nerve activity may have been responsible either for the release of active renin or for the conversion of inactive renin to its active form in the kidney.     

Glycosaminoglycan composition of human amniotic fluid

The glycosaminoglycan composition of human amniotic fluid between 12–21 weeks gestation has been studied by Dowex column chromatography coupled with enzymatic analyses of the specific glycosaminoglycan in each column fraction. The total uronic acid recovered from the columns consisted of “glycopeptides” (7%), hyaluronic acid (34%), nonsulfated chondroitin (14%), chondroitin-4-sulfate (13%), chondroitin-6-sulfate (20%), dermatan sulfate (5%), and heparan sulfate (6%). Based on these studies a simple screening procedure was devised to detect increased quantities of heparan sulfate and dermatan sulfate in 5–10-ml samples of amniotic fluid and tested in the antenatal diagnosis of Hurler and Hunter's syndrome. A false negative result was recorded in a Hunter fluid obtained early gestation and a false positive result recorded in a normal fluid obtained at weeks. These data suggest that the time in gestation when amniotic fluid is sampled for chemical analysis is an important variable affecting glycosaminoglycan composition in both normal and pathological pregnancies.     

Proteomics analysis of human amniotic fluid

Amniotic fluid is a dynamic and complex mixture that reflects the physiological status of the developing fetus. In this study, the human amniotic fluid (AF) proteome of a 16-18-week normal pregnancy was profiled and analyzed to investigate the composition and functions of this fluid. Due to the complexity of AF, we utilized three different fractionation strategies to provide greater coverage. Two types of two-dimensional LC/MS/MS as well as an LC-SDS-PAGE-LC-MS/MS platform were used. A total of 16 AF samples between gestational ages of 16 and 18 weeks from women carrying chromosomally normal fetuses were analyzed by one of the three fractionation methods followed by a common reverse phase LC-MS/MS step. Mascot and The Global Proteome Machine engines were used to search the International Protein Index human database for peptide sequence identification. The list of proteins was generated by combining the results of both engines through the PeptideProphet of Scaffold software. All identified proteins were combined to generate the AF proteome comprising 1,026 unique gene matches or 842 non-redundant proteins. This list includes most of the currently used biomarkers for pregnancy-associated pathologic conditions such as preterm delivery, intra-amniotic infection, and chromosomal anomalies of the fetus. The subcellular localization, tissue expression, functions, and networks of the AF proteome were analyzed by various bioinformatic tools. These data will contribute to the better understanding of amniotic fluid function and to the discovery of novel biomarkers for prenatal diagnosis of fetal abnormalities.     

Regulation of N-acetylglucosaminyltransferase V activity. Kinetic comparisons of parental, Rous sarcoma virus-transformed BHK, and L-phytohemagglutinin-resistant BHK cells using synthetic substrates and an inhibitory substrate analog

Baby hamster kidney (BHK) cells transformed with Rous sarcoma virus, RS-BHK cells, demonstrate a 2.5-fold increase in the activity of N-acetylglucosaminyltransferase V (GlcNAc-T V, EC 2.4.1.155), and this increase in activity appears to be specific for this enzyme. By contrast, a lectin-resistant BHK cell line selected for its ability to grow in high levels of L-phytohemagglutinin, LP3.3, is characterized by a specific decrease in its GlcNAc-T V activity. To test if these alterations in the apparent Vmax of GlcNAc-T V are due to changes in the efficiency of populations of enzymes in RS-BHK and LP3.3 cells compared to the parental BHK cells, we have compared the kinetic properties of the enzymes from these three sources. The Km constants observed for both the sugar nucleotide donor (UDP-GlcNAc) and two synthetic trisaccharide acceptors were indistinguishable. The Vmax values toward three synthetic acceptors were also determined first for the BHK GlcNAc-T V, and they varied by over 5-fold. When these values were measured for the variant and transformed cell enzymes, however, similar 5-fold differences were still observed, although the absolute values for these acceptors were all higher or lower for the RS-BHK and LP3.3 enzymes, respectively. In addition, we have synthesized a deoxygenated analog of the specific GlcNAc-T V acceptor, beta GlcNAc(1,2) alpha Man(1,6) beta ManOR, where the reactive 6'-OH group has been removed, and the resulting trisaccharide was found to be a competitive inhibitor of the enzyme. The Ki for this inhibitor was near 70 microM for the GlcNAc-T V from all three sources. These kinetic comparisons demonstrate that the enzymes from the three cell types have kinetically indistinguishable active sites. These results suggest that the differences in the apparent Vmax values among the cell types are most likely due to alterations in the number of active molecules rather than in the modulation of either their catalytic activities or specificities.     

Analysis of inactive renin by renin profragment monoclonal antibodies

Two peptides were synthesized, corresponding to the sequences (-19 to -7) and (-26 to -17) of the prorenin prosegment. Monoclonal antibodies were raised to these sequences and used to characterize human plasma inactive renin. Only anti (-19 to -7) reacted with inactive renin, as measured by direct assay or affinity chromatography. The data were used to evaluate two possible inactive renin stuctures: plasma inactive renin is a truncated prorenin lacking the prosegment N-terminal portion; its spatial conformation masks the N-terminal extremity, preventing interaction of this region with specific antibodies.     

Stem cells in human amniotic fluid

Amniotic fluid (AF) contains a heterogeneous population of cells of fetal origin in which stem cells are present. These cells are characterized by the expression of mesenchymal (CD73, CD90, CD105) and neural (Nestin, β3-tubulin, NEFH) markers, and also some markers of pluripotency (Oct4, Nanog), and they are capable of differentiating into diverse derivatives in vitro. We have shown that epithelial markers (Keratin 19, Keratin 18, and p63) are expressed in AF stem cells simultaneously with mesenchymal ones. During cloning, colonies of cells with fibroblastoid and epithelioid cells are formed. The status and differentiation potential of stem cells from AF have been discussed.     

Kinetic investigation of the staphylococcal protease-catalyzed hydrolysis of synthetic substrates.

In investigating the staphylococcal protease-catalyzed hydrolysis of N-tert-butoxycarbonyl-L-glutamate alpha-phenyl ester, N-benzyloxycarbonyl-L-glutamate alpha-phenyl ester and N-benzyloxycarbonyl-L-glutamate alpha-p-nitroanilide, we obtained kinetic evidence consistent with the formation of an acyl-enzyme intermediate. We found that addition of a nucleophile, such as methanol, led to the partition of the common acyl-enzyme intermediate between water and the alcohol. With N-benzyl-oxycarbonyl-L-glutamate alpha-phenyl ester, a specific ester substrate, deacylation was shown to be the rate-limiting step. By studying the kcat/Km ratio of these hydrolyses as a function of pH, we have shown that two ionizable groups on the enzyme are essential to the catalytic process. One of these groups has a pK of 6.58 and the other, a pK of 8.25. The assignment of these pK values is discussed in connection with the known features of the serine proteinase reaction mechanism. In addition, monovalent anions were shown to inhibit staphylococcal protease hydrolyses. They seem to compete with the negative charge of the substrate, thus inhibiting its binding on the enzyme molecule. Finally we compared the kinetic parameters obtained with five proteases isolated from different strains of Staphylococcus aureus.     

Purification of human plasma inactive renin by immunoaffinity chromatography on profragment-specific IgG

Inactive renin was purified to apparent homogeneity from human plasma by ion exchange, gel filtration, Affi-Gel blue, immunoaffinity chromatography on profragment-specific IgG coupled to Sepharose, and preparative HPLC. By this method, a 460000-fold purification was obtained. The purified renin was totally inactive and was activated by trypsin.