Renin Precursor and Its Activation Mechanism in Hog Kidney

1980 ◽  
Vol 59 (s6) ◽  
pp. 21s-24s ◽  
Author(s):  
Kazuo Murakami ◽  
Saori Takahashi ◽  
Shigehisa Hirose ◽  
Yukio Takii ◽  
Tadashi Inagami
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Activation Mechanism ◽  
Net Charge ◽  
Isoelectric Points ◽  
Binding Substance ◽  
Inactive Renin ◽  
Hog Kidney

1. A completely inactive renin was isolated from hog kidney extract by affinity chromatography on pepstatin-aminohexyl-Sepharose and on an Affi-Gel Blue column. 2. This inactive renin had a molecular weight of 43 000 ± 1500 as determined by gel filtration on Ultrogel AcA 44. Upon activation with trypsin, its molecular weight fell to 41 000 ± 1400. 3. The inactive renin lacked the ability to bind renin-binding substance whereas trypsin-activated renin was able to bind the renin-binding protein and to form high-molecular-weight renin. 4. Chymotrypsin as well as trypsin could activate the inactive renin although less effectively. 5. The active renins generated from the inactive renin by the action of different proteolytic enzymes differed in their net charge, reflecting the specificities of the proteases used; the isoelectric points of the native, the trypsin-activated and the chymotrypsin-activated forms of renin occurred at pH 5.3, 5.1 and 4.8 respectively.

The Different Forms of Antithrombin III in Serum

1977 ◽  
Vol 38 (02) ◽  
pp. 0494-0503 ◽  
Author(s):  
D. S Pepper ◽  
D Banhegyi ◽  
J. D Cash
Keyword(s):  
Molecular Weight ◽  
Affinity Chromatography ◽  
Human Serum ◽  
Degradation Product ◽  
Antithrombin Iii ◽  
Gel Filtration ◽  
Free Form ◽  
Polymeric Form ◽  
At Iii ◽  
Human Thrombin

SummaryAntithrombin III (AT III) complexes were isolated from human serum by affinity chromatography and gel filtration. In the first step of the preparation, using heparin-agarose chromatography, we observed that the complexed form of AT III bound less strongly to the gel than the free form and that about half of the AT III was free. With further purification a 2.5 × 105 molecular weight complex was isolated. Using 125I labelled human thrombin, this complex was radioactive indicating the presence of thrombin. Only in a synthetic thrombin-AT III system was a 9 × 104 molecular weight complex detected, but not in serum. These facts suggest that in serum AT III complexes may exist in a polymeric form. Also, an AT III antigen derived from the original AT III molecule, but not complexed, was isolated which may be a degradation product.Abbreviations used: AT-III, antithrombin III. Hepes, N-2-Hydroxyethylpiperazine-N-2-Ethanesulphonic acid.

scholarly journals Complex Formation of Covalently Crosslinked Fibrin Oligomers with Agarose Coupled Fibrinogen and Fibrin

1977 ◽  
Author(s):  
R. von Hugo ◽  
R. Hafter ◽  
A. Stemberger ◽  
H. Graeff
Keyword(s):  
Molecular Weight ◽  
Complex Formation ◽  
Affinity Chromatography ◽  
High Molecular Weight ◽  
Calcium Chloride ◽  
Gel Filtration ◽  
Void Volume ◽  
Soluble Fibrin ◽  
Derivatives Of

Crosslinked high molecular weight derivatives of fibrin (fibrinoligomers) were observed during intravascular coagulation. It was the purpose of this study to investigate the complex formation of fibrin oligomers with fibrinogen and fibrinmonomer. Fibrinogen coupled to agarose (Fg-ag) as well as fi-brinmonomer coupled to agarose (Fm-ag) was used as substrate. Soluble oligomers of fibrin were produced by incubating fibrinogen with thrombin, calcium-chloride, cystein and F XIII. They were separated from fibrinogen by gel filtration. Γ-dimers were demonstrated in fractions from the void volume and the shoulder prior to the fibrinogen peak. These fractions were subjected to affinity chromatography. Crosslinked oligomers of fibrin were not adsorbed on Fg-ag, yet adsorption occured on Fm-ag. This indicates that fibrin oligomers have no affinity to fibrinogen, yet readily form complexes with fibrin. This could mean that in vivo they compete with fibrinogen for the fibrinmonomer part of soluble fibrin monomer complexes, and hence have a tendency to increase in size.

scholarly journals Partial purification and characterization of chick-embryo prolyl 3-hydroxylase

1979 ◽  
Vol 183 (2) ◽  
pp. 303-307 ◽  
Author(s):  
K Tryggvason ◽  
K Majamaa ◽  
J Risteli ◽  
K I Kivirikko
Keyword(s):  
Molecular Weight ◽  
Chick Embryo ◽  
Ethylene Glycol ◽  
Affinity Chromatography ◽  
Concanavalin A ◽  
Gel Filtration ◽  
Partial Purification ◽  
Purification And Characterization ◽  
Chick Embryo Extract

Prolyl 3-hydroxylase was purified up to about 5000-fold from an (NH4)2SO4 fraction of chick-embryo extract by a procedure consisting of affinity chromatography on denatured collagen linked to agarose, elution with ethylene glycol and gel filtration. The molecular weight of the purified enzyme is about 160000 by gel filtration The enzyme is probably a glycoprotein, since (a) its activity is inhibited by concanavalin A, and (b) the enzyme is bound to columns of this lectin coupled to agarose and can be eluted with a buffer containing methyl alpha-D-mannoside. The Km values for Fe2+, 2-oxoglutarate, O2 and ascorbate in the prolyl 3-hydroxylase reaction were found to be very similar to those previously reported for these co-substrates in the prolyl 4-hydroxylase and lysyl hydroxylase reactions.

The Nature of Inactive Renin in Human Plasma and Amniotic Fluid

1978 ◽  
Vol 55 (1) ◽  
pp. 41-50 ◽  
Author(s):  
A. A. Shulkes ◽  
R. R. Gibson ◽  
S. L. Skinner
Keyword(s):  
Molecular Weight ◽  
Amniotic Fluid ◽  
Human Plasma ◽  
Gel Filtration ◽  
Dilution Effect ◽  
Exchange Chromatography ◽  
Acid Activation ◽  
Temperature Dependent ◽  
Inactive Renin ◽  
Full Activation

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.

Two Different Types of Inactive Renin in Human Plasma: Molecular and Kinetic Properties

1984 ◽  
Vol 67 (4) ◽  
pp. 421-425
Author(s):  
Yoichi Imamura ◽  
Kunio Hiwada ◽  
Tatsuo Kokubu
Keyword(s):  
Molecular Weight ◽  
Human Plasma ◽  
Kinetic Properties ◽  
Carbohydrate Composition ◽  
Ph Profile ◽  
Isoelectric Points ◽  
Different Types ◽  
Km Value ◽  
Inactive Renin ◽  
Fresh Plasma

1. We separated inactive renin in human plasma into two types, adsorbed and non-adsorbed, by chromatography on a concanavalin A-Sepharose column. About 75% of fresh plasma inactive renin was adsorbed to the column, and the rest passed through it. Non-adsorbed and adsorbed inactive renins were partially purified. 2. Non-adsorbed inactive renin had a molecular weight of 48000 and an isoelectric point of 5.44. Adsorbed inactive renin had a molecular weight of 46000 and isoelectric points of 5.56 and 5.80. 3. After activation with trypsin, both activated inactive renins were similar with respect to molecular weight (45000), thermostability, Km value (0.56 μmol/l) and pH profile. But pI values of both activated inactive renins differed. 4. These results indicate that there exist in human plasma two different types of inactive renin which differ in carbohydrate composition.

scholarly journals Evidence that functional subunits of antihemophilic factor (Factor VIII) are linked by noncovalent bonds

Blood ◽  
1976 ◽  
Vol 48 (1) ◽  
pp. 87-94 ◽  
Author(s):  
MC Poon ◽  
OD Ratnoff
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Protease Inhibitors ◽  
Proteolytic Enzymes ◽  
Gel Filtration ◽  
Factor Ix ◽  
Low Molecular Weight ◽  
Native State ◽  
Noncovalent Bonds ◽  
Antihemophilic Factor

Abstract Partially purified human antihemophilic factor (AHF, factor VIII), when treated with high concentrations of salt, has been shown to dissociate into two components: one, of relatively low molecular weight, possesses procoagulant activity, and the other, of higher molecular weight, forms precipitates with heterologous antiserum against AHF and supports ristocetin-induced platelet aggregation. The ease of separation suggests that the two components in the native state might be held together by noncovalent bonds. Earlier observations do not exclude the possibility that the subunits may be covalently bonded in nature but might be severed by plasma proteolytic enzymes during laboratory manipulation. The issue was examined by preparing partially purified AHF from fresh human plasma in the presence of protease inhibitors, including benzamidine, soybean trypsin inhibitor, epsilon-aminocaproic acid, heparin, and hirudin. Under these conditons, gel filtration in the presence of 0.25 M calcium chloride and 0.001 M benzamidine resulted in its separation into two components, having properties identical to those separated in the absence of these protease inhibitors. The inhibitor mixture blocked generation and action of streptokinase- and kaolin-activated plasmin from plasma, and protected both plasma AHF and partially purified AHF from the action of thrombin. Surface-induced activation of PTA (factor XI) was partially inhibited, and that of Christmas factor (factor IX) was completely inhibited. This observation provides further evidence that in the native state the high- and low-molecular-weight components of preparations of antihemophilic factor are held together by noncovalent bonds.

Affinity Chromatography of Human Factor VIII Using Human and Rabbit Antibodies to Factor VIII

1979 ◽  
Vol 42 (04) ◽  
pp. 1306-1315 ◽  
Author(s):  
Janet L Lane ◽  
H Ekert ◽  
A Vafiadis
Keyword(s):  
Molecular Weight ◽  
Factor Viii ◽  
Affinity Chromatography ◽  
Protein Concentration ◽  
Gel Filtration ◽  
Subunit Structure ◽  
Molecular Weights ◽  
Human Factor Viii ◽  
Sds Page ◽  
Normal Human

SummaryFactor VIII, purified by gel filtration on Sepharose 2B, has an 8 band multiple subunit structure, with molecular weights ranging from 30,000 to 230,000, on reduction and SDS-PAGE at a protein concentration of 400 μg/gel. Affinity chromatography of this factor VIII preparation with insolubilized haemophilic antibody to factor VIII showed that 45-81% VIII:C and 0-33% VIILRag were attached to the column. Elution of the column with 0.25 M CaCl2 did not show VIII:C or VIILRag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed that 95 % of the protein bound by haemophilic antibody had a molecular weight similar to the low molecular weight subunits of the reduced factor VIII.In control experiments with normal Human IgG, 3% of VIII:C and 5% of VIILRag were attached to the column. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the protein, showed 2 faint bands with molecular weight consistent with heavy and light chains of IgG.Similar experiments with antibody to factor VIII showed that 67-83% of VIILC and 61-76% of VIII:Rag were attached to the column. Elution of the column with 0.25 M CaCl2 showed 10% of the applied VIII:C, but no VIII:Rag in the eluate. NH4SCN dissociation of the column, followed by reduction and SDS-PAGE of the dissociated protein, showed an 8 band subunit structure similar to the reduced factor VIII.

Isolation and Properties of Elastase-Like Enzymes from Human Platelets and Pig Aorta

1975 ◽  
Author(s):  
W. Hornebeck ◽  
Y. Legrand ◽  
J. P. Caen ◽  
L. Robert
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Affinity Chromatography ◽  
Amino Acid Composition ◽  
Proteolytic Activity ◽  
Gel Filtration ◽  
Inhibitory Effect ◽  
Human Platelets ◽  
Purification Procedure ◽  
Pancreatic Elastase

An elastase-like enzyme has been isolated from human platelets. Its purification using precipitations with ammonium sulphate, gel filtration and affinity chromatography on Agarose-elastin, is described. The acrylamide gel of the affinity peak reveals only one band corresponding to a molecular weight of about 25,000 daltons. The amino acid composition is similar to pancreatic elastase. Using the same kind of purification procedure an aortic elastase-like enzyme has also been isolated and characterized. These two enzymes possess comparable proteolytic activity on various synthetic and natural substrates considered as specific for elastases. The ratio of their activity on these substrates differs however from that of pancreatic elastase. The inhibitory effect of α1, antitrypsine and α2 macroglobuline were also studied and shown to differ quantitatively from those on pancreatic elastase. These elastase like enzymes may be responsible for the degradation of elastin occuring in ageing and arteriosclerosis.

scholarly journals The cellulase of Fusarium solani. Purification and specificity of the β-(1→4)-glucanase and the β-d-glucosidase components

1971 ◽  
Vol 121 (3) ◽  
pp. 353-362 ◽  
Author(s):  
T. M. Wood
Keyword(s):  
Heat Treatment ◽  
Molecular Weight ◽  
Phosphoric Acid ◽  
Isoelectric Focusing ◽  
Fusarium Solani ◽  
Reducing Sugars ◽  
Activity Ratio ◽  
Gel Filtration ◽  
Cellulase Activity ◽  
Isoelectric Points

1. Cell-free culture filtrates of the fungus Fusarium solani were examined for homogeneity with respect to β-d-glucosidase and Cx activities. 2. o-Nitrophenyl β-d-glucoside and cellobiose were both used as substrates for β-d-glucosidase activity. 3. No evidence for the non-identity of nitrophenyl β-d-glucosidase and cellobiase activities could be found, either by heat treatment, gel filtration on Sephadex G-100 or by isoelectric focusing. 4. The β-d-glucosidase component was also a feeble exo-β-glucanase: it had a molecular weight of approx. 400000. 5. The fall in viscosity of a solution of CM-cellulose, the formation of reducing sugars in a solution of CM-cellulose and the solubilization of phosphoric acid-swollen cellulose (Walseth cellulose), were all used for the measurement of Cx activity. 6. The ratio of the two types of CM-cellulase activity was not changed after gel filtration on Sephadex G-100 or after chromatography on DEAE-Sephadex. 7. Three peaks of Cx activity were obtained after electrofocusing, but all three possessed the same ratio of the two types of CM-cellulase activity as well as the same CM-cellulase/Walseth activity ratio, as the unfractionated enzyme; all three isoenzymes (isoelectric points, 4.75, 4.80–4.85 and 5.15) acted in synergism with a mixture of the C1 and the β-d-glucosidase components to the same extent in the solubilization of cotton fibre. 8. The molecular weight of the Cx component was approx. 37000.

Characteristics of a Renin-Binding Substance for the Conversion of Renin into a Higher-Molecular-Weight Form in the Dog

1979 ◽  
Vol 57 (4) ◽  
pp. 345-350 ◽  
Author(s):  
Minoru Kawamura ◽  
Fumihiko Ikemoto ◽  
Susumu Funakawa ◽  
Kenjiro Yamamoto
Keyword(s):  
Molecular Weight ◽  
Gel Filtration ◽  
Filtration Fraction ◽  
Acid Ph ◽  
Binding Substance ◽  
Molecular Weight Form

1. Renal cortical homogenates of the dog were subjected to sieve separation, a Nucleopore Filter being used to separate the renin granules. 2. The molecular weight of renin in the granules was estimated to be about 40 000 by gel filtration. Renin was converted into a higher-molecular-weight form (60 000) by mixing with cytosol in the presence of sodium tetrathionate, a thiol inhibitor. 3. When cytosol was pretreated with acid (pH 30) or heating (100°C), the molecular-weight conversion did not occur. 4. Cytosol was separated into three parts by gel filtration. Fraction A included substances with a molecular weight of over 47 000, fraction B from 47 000 to 32 000, and fraction C from 32 000 to 15 000. The mixture of renin in the granules with fraction A and sodium tetrathionate resulted in the formation of a higher-molecular-weight form of the enzyme, but no change in molecular weight was detected when renin was mixed with fractions B or C and sodium tetrathionate.

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