The Nature of Inactive Renin in Human Plasma and Amniotic Fluid

1. The properties of inactive and active renin in human plasma and amniotic fluid were studied chromatographically. Activation was achieved at pH 3.3 with and without added pepsin. 2. Acid activation of renin was time- and temperature-dependent but was inhibited by dilution of the sample. The dilution effect was corrected by adding pepsin. Such characteristics indicate that activation at low pH is catalysed by intrinsic enzymes. 3. Separation and/or dilution of the activating enzyme during ion-exchange chromatography concealed the eluted position of inactive renin and reduced the amount recovered. Only after full activation of the eluted renin was achieved with added pepsin was a distinct peak of inactive renin exposed. 4. At pH 7.5 inactive renin carried a lower negative charge than the active enzyme. This charge difference was lost after activation. 5. No molecular-weight differences between active, inactive renin or the International Renin Standard were detected by gel filtration. No renin of larger molecular weight was present. 6. These findings will be helpful in purification studies of human inactive renin.

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The multiple forms of α-D-mannosidase in human plasma

Biochemical Journal ◽

10.1042/bj1790583 ◽

1979 ◽

Vol 179 (3) ◽

pp. 583-592 ◽

Cited By ~ 23

Author(s):

S Hirani ◽

B Winchester

Keyword(s):

Molecular Weight ◽

Human Plasma ◽

Concanavalin A ◽

Gel Filtration ◽

Deae Cellulose ◽

Exchange Chromatography ◽

The Other ◽

Multiple Forms ◽

Simple Correlation ◽

Multiple Components

The acidic alpha-D-mannosidase in human plasma closely resembles liver acidic alpha-D-mannosidase in its affinity for concanavalin A-Sepharose, molecular weight and resolution into multiple components on DEAE-cellulose. A combination of chromatography on concanavalin A-Sepharose and gel filtration on Sephadex G-200 and Sepharose 6B suggests that four forms of intermediate alpha-D-mannosidase, which differ either in their molecular weight of affinity for concanavalin A, exist in human plasma. A practical classification and nomenclature for the multiple forms of intermediate alpha-D-mannosidase in plasma based on molecular weight and affinity for concanavalin A is proposed. Multiple forms of intermediate alpha-D-mannosidase were also observed by ion-exchange chromatography on DEAE-cellulose, but there was not a simple correlation between these forms and those obtained with the other separation procedures. The form of intermediate alpha-D-mannosidase least abundant in plasma, approx. 7% of the activity, has very similar properties to the neutral alpha-D-mannosidase in human liver. In contrast, the other three forms of intermediate alpha-D-mannosidase, which account for over 90% of the activity, do not appear to be present in liver, except perhaps in trace amounts.

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Reversible Acid Activation of Amniotic Fluid Inactive Renin

Clinical Science ◽

10.1042/cs0640481 ◽

1983 ◽

Vol 64 (5) ◽

pp. 481-486 ◽

Cited By ~ 2

Author(s):

Robert C. Franks ◽

Francis Bodola ◽

Robert D. Renthal ◽

Robert H. Hayashi

Keyword(s):

Amniotic Fluid ◽

Human Plasma ◽

Conformational Change ◽

Deae Cellulose ◽

Acid Activation ◽

Human Amniotic Fluid ◽

Before And After ◽

Inactive Renin ◽

Ph Dependent ◽

Cibacron Blue F3ga

1. Inactive renin was isolated from human amniotic fluid by chromatography with DEAE-cellulose, pepstatin-aminobutyl-agarose, octylagarose and Cibacron Blue F3GA columns. 2. Before and after isolation from amniotic fluid, inactive renin could be activated by incubation with pepsin and by dialysis to pH 3.3, and the acid activation could be reversed by subsequent incubation at pH 7.4 and 37°C. 3. Inactive renin was not activated by procedures expected to dissociate renin from an inhibitor. 4. The results suggest a pH-dependent conformational change as the mechanism of reversible acid activation of inactive renin in amniotic fluid. 5. There are chromatographic and activation similarities of inactive renin from human plasma, kidney and amniotic fluid.

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The Kinin System of Human Plasma

Thrombosis and Haemostasis ◽

10.1055/s-0038-1649349 ◽

1972 ◽

Vol 27 (01) ◽

pp. 141-158 ◽

Cited By ~ 4

Author(s):

Ayşe H. Özge-Anwar ◽

H. Z Movat ◽

J. G Scott

Keyword(s):

Molecular Weight ◽

Human Plasma ◽

Gel Filtration ◽

Anion Exchange Chromatography ◽

Disc Electrophoresis ◽

Exchange Chromatography ◽

Factor Xii ◽

Kinin System ◽

Polyacrylamide Electrophoresis ◽

Contact Exposure

SummaryHuman plasma was adsorbed to celite. The celite eluates were chromatographed on QAE-Sephadex A-50, followed by gel filtration, isoelectric focusing and preparative polyacrylamide electrophoresis.By anion-exchange chromatography, factor XI a, kallikrein and y-globulin were isolated from the excluded protein peak. Factor XIa (MW ∼170,000) corrected the deficiency in plasmas deficient in factors XII or XI, but had no effect on the kininsystem. Its isoelectric point was about 8.0-8.5. Kallikrein had a molecular weight of ∼ 90,000 and a pi of 7.8-8.0 The two substances could be isolated also by polyacrylamide electrophoresis.Highly anionic fractions contained a fragment of factor XII a (prekallikrein activator), which activated prekallikrein and retained some of the clot-promoting property of the intact factor XIIa molecule. Its molecular weight was about 37,000, the pi about 4.4 and it migrated as a prealbumin by disc electrophoresis.It is concluded that, by massive contact exposure, most of factor XII a converts to the highly anionic, low-molecular-weight prekallikrein activator. Since the prekallikrein activator is a fragment of factor XII a, we recommend that it be referred to as “XII f”.

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The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide–adenine dinucleotide phosphate-linked isoenzymes

Biochemical Journal ◽

10.1042/bj1300211 ◽

1972 ◽

Vol 130 (1) ◽

pp. 211-219 ◽

Cited By ~ 14

Author(s):

Colin H. Self ◽

P. David J. Weitzman

Keyword(s):

Molecular Weight ◽

Ph Dependence ◽

Gel Filtration ◽

Deae Cellulose ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Molecular Size ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Low Concentrations ◽

Stimulation Of

Two isoenzymes of NADP-linked isocitrate dehydrogenase have been identified in Acinetobacter lwoffi and have been termed isoenzyme-I and isoenzyme-II. The isoenzymes may be separated by ion-exchange chromatography on DEAE-cellulose, by gel filtration on Sephadex G-200, or by zonal ultracentrifugation in a sucrose gradient. Low concentrations of glyoxylate or pyruvate effect considerable stimulation of the activity of isoenzyme-II. The isoenzymes also differ in pH-dependence of activity, kinetic parameters, stability to heat or urea and molecular size. Whereas isoenzyme-I resembles the NADP-linked isocitrate dehydrogenases from other organisms in having a molecular weight under 100000, isoenzyme-II is a much larger enzyme (molecular weight around 300000) resembling the NAD-linked isocitrate dehydrogenases of higher organisms.

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Purification and characterization of exocellular proteases produced by a clinical isolate and a laboratory strain of Pseudomonas aeruginosa

Canadian Journal of Microbiology ◽

10.1139/m80-012 ◽

1980 ◽

Vol 26 (1) ◽

pp. 77-86 ◽

Cited By ~ 25

Author(s):

S. E. Jensen ◽

L. Phillippe ◽

J. Teng Tseng ◽

G. W. Stemke ◽

J. N. Campbell

Keyword(s):

Molecular Weight ◽

Pseudomonas Aeruginosa ◽

Clinical Isolate ◽

Protease Activity ◽

Gel Filtration ◽

Laboratory Strain ◽

Exchange Chromatography ◽

The Other ◽

Protease Production ◽

Peptide Bonds

Exocellular protease production was examined in two separate strains of Pseudomonas aeruginosa, one a clinical isolate and the other a laboratory strain. Both strains produced two separate proteases (proteases 1 and 2) which were indistinguishable from one strain to the other. The two proteases were purified by a two-step procedure of gel filtration chromatography followed by ion-exchange chromatography. Proteases 1 and 2 were shown to be distinct serologically and unrelated by physicochemical parameters examined. Protease 1 was the major exocellular protein produced and contributed about 95% of the total protease activity of the culture. It was estimated to have a molecular weight of 34 850 and was also shown to contain 10% glucosamine by weight. Protease 2, in contrast, had an estimated molecular weight of 52750 and contained no detectable carbohydrate. Proteases 1 and 2 were both stimulated by Ca2+, and Mg2+ and inhibited by Co2+Zn2+, and 1,10-o-phenanthroline. Protease 1 was also inhibited by EDTA. In addition to protease activity, both proteases 1 and 2 demonstrated elastase activity as well as a limited collagenase activity. Specificity of the two proteases against synthetic peptides was, however, quite different. Protease 1, but not protease 2, showed a preference for peptide bonds in which the amino group was contributed by an amino acid with a hydrophobic R group.

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Separation and partial characterization of guinea-pig caseins

Biochemical Journal ◽

10.1042/bj1730633 ◽

1978 ◽

Vol 173 (2) ◽

pp. 633-641 ◽

Cited By ~ 14

Author(s):

R K Craig ◽

D McIlreavy ◽

R L Hall

Keyword(s):

Molecular Weight ◽

Amino Acid ◽

Guinea Pig ◽

Amino Acid Composition ◽

Acid Composition ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Exchange Chromatography

1. Guinea-pig caseins A, B and C were purified free of each other by a combination of ion-exchange chromatography and gel filtration. 2. Determination of the amino acid composition showed all three caseins to contain a high proportion of proline and glutamic acid, but no cysteine. This apart, the amino acid composition of the three caseins was markedly different, though calculated divergence values suggest that some homology may exist between caseins A and B. Molecular-weight estimates based on amino acid composition were in good agreement with those based on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. 3. N-Terminal analysis showed lysine, methionine and lysine to be the N-terminal residues of caseins A, B and C respectively. 4. Two-dimensional separation of tryptic digests revealed a distinctive pattern for each casein. 5. All caseins were shown to be phosphoproteins. The casein C preparation also contained significant amounts of sialic acid, neutral and amino sugars. 6. The results suggest that each casein represents a separate gene product, and that the low-molecular-weight proteins are not the result of a post-translational cleavage of the largest. All were distinctly different from the whey protein alpha-lactalbumin.

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Possible Mechanisms for the Increase in Alkaline Phosphatase Activity of Lyophilized Control Material

Clinical Chemistry ◽

10.1093/clinchem/18.12.1518 ◽

1972 ◽

Vol 18 (12) ◽

pp. 1518-1523 ◽

Cited By ~ 18

Author(s):

Alistair F Smith ◽

Barbara A Fogg

Keyword(s):

Molecular Weight ◽

Alkaline Phosphatase ◽

Phosphatase Activity ◽

Alkaline Phosphatase Activity ◽

Gel Filtration ◽

Low Molecular Weight ◽

Temperature Dependent ◽

Control Material ◽

Rate Of Increase ◽

Control Procedures

Abstract The increased alkaline phosphatase (EC 3.1.3.1) activity of reconstituted lyophilized control sera has recently been the focus of considerable interest because of its possible implications for quality-control procedures. We confirm that these reconstituted materials show a temperature-dependent increase in alkaline phosphatase activity, but could show no alteration in activity of fresh sera. The rate of increase in activity was unaffected by dialysis of the reconstituted material, and occurred more rapidly in dilute solutions. Studies with acrylamide gel electrophoresis and Sephadex G-200 gel filtration showed that, immediately after reconstitution, a high-molecular-weight alkaline phosphatase component predominated; during subsequent spontaneous activation this component decreased, and there was a concomitant increase in a low-molecular-weight alkaline phosphatase component. The results obtained when the reconstituted material was extracted with butanol suggest that the observed changes in alkaline phosphatase activity may be attributed to the breakdown of a complex between alkaline phosphatase and lipoprotein.

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Partial Primary Structure Of Human α2-Antiplasmin

10.1055/s-0038-1652839 ◽

1981 ◽

Author(s):

H R Lijnen ◽

B Wiman ◽

B Van Hoef ◽

D Collen

Keyword(s):

Amino Acids ◽

Molecular Weight ◽

High Performance Liquid Chromatography ◽

High Performance ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Edman Degradation ◽

Single Chain ◽

Tryptic Digest

α2-Antiplasmin (α2AP), the main physiological inhibitor of plasmin in human plasma, is a single–chain glycoprotein with a molecular weight of 67,000 consisting of about 510 amino acids and containing 13 percent carbohydrate.A tryptic digest on 400 mg of reduced, carboxymethylated and citraconylated purified α2AP was performed. Peptides were separated by combinations of ion exchange chromatography, gel filtration and high performance liquid chromatography, and sequenced using the manual Edman degradation. Some peptides were further digested in order to establish overlaps. At the time of submission of this abstract we have sequenced 7 out of the approximately 21 arginyl peptides completely (each between 3 and 21 residues) and are working on the others. At present we have about 200 residues of sequence. Here we only report the stretches of 10 amino acids or more, which may be useful to compare the structure of α2AP with that of other serine protease inhibitors.

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Physiological zinc-binding proteins of medium molecular weight in the rat gut

British Journal Of Nutrition ◽

10.1079/bjn19860043 ◽

1986 ◽

Vol 55 (2) ◽

pp. 369-377 ◽

Cited By ~ 2

Author(s):

Malcolm J. Jackson ◽

Daphne Holt ◽

Michael Webb ◽

Nicholas D. Carter

Keyword(s):

Molecular Weight ◽

Binding Proteins ◽

Gel Filtration ◽

Deae Cellulose ◽

Exchange Chromatography ◽

Zinc Binding ◽

Intragastric Administration ◽

Ileal Segment ◽

Mucosal Cells ◽

Rat Gut

1. Gel filtration on Sephadex G 75 was used to separate the medium-molecular-weight zinc-binding proteins from the soluble fractions from the duodenal and jejuno-ileal segments of the rat gut at 30 min after the intragastric administration of a tracer dose of 65Zn. These proteins were resolved by ion-exchange chromatography on DEAE cellulose.2. In both the duodenum and jejuno-ileal segment an appreciable fraction of the total soluble Zn was bound in a protein fraction that resembled metallothionein [MT] in its behaviour on gel filtration. These fractions, however, were not homogeneous, but contained several medium-molecular-weight Zn-binding proteins. In the duodenum, but not in the jejuno-ileal segment, two ofthese proteins appeared to be the isometallothioneins, ZnMT-I and ZnMT-11.3. These results suggest a possible role for MT in the binding of newly-absorbed Zn in the duodenal mucosal cells. They also show that gel filtration alone is insufficient for the identification of MT in the intestine.

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A dipeptidylpeptidase secreted by Fasciola hepatica

Parasitology ◽

10.1017/s0031182000077817 ◽

1994 ◽

Vol 109 (1) ◽

pp. 113-118 ◽

Cited By ~ 17

Author(s):

C. Carmona ◽

S. McGonigle ◽

A. J. Dowd ◽

A. M. Smith ◽

S. Coughlan ◽

...

Keyword(s):

Molecular Weight ◽

Ion Exchange ◽

Liver Fluke ◽

Fasciola Hepatica ◽

Gel Filtration ◽

Serine Proteinase ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Substrate Preference ◽

Proteolytic Digestion

SUMMARYA dipeptidylpeptidase (DPP) was isolated from Fasciola hepatica by gel-filtration and ion-exchange chromatography. The exoproteinase is secreted by newly excysted juveniles, immature and mature flukes. The liver fluke DPP is a serine proteinase of molecular weight > 200 kDa and differs from previously characterized mammalian DPPs in its substrate preference and susceptibility to inactivation by inhibitors. The parasite DPP may function in the latter stages of the proteolytic digestion of host macromolecules. In this manner, the enzyme may be important in providing the parasite with dipeptides that could be absorbed through the intestine as nutrient.

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