Preliminary Evidence for the Conversion of Dog Renin into a Higher—Molecular—Weight Form by Cold Storage

1980 ◽  
Vol 58 (6) ◽  
pp. 451-456 ◽  
Author(s):  
Minoru Kawamura ◽  
Fumihiko Ikemoto ◽  
Kenjiro Yamamoto
Keyword(s):  
Molecular Weight ◽  
Cold Storage ◽  
Soluble Fraction ◽  
Gel Filtration ◽  
Molecular Size ◽  
Preliminary Evidence ◽  
Concomitant Increase ◽  
Binding Substance ◽  
Dog Kidney ◽  
Molecular Weight Form

1. A soluble fraction of renal cortical homogenate (cytosol) and renin extracted from isolated renin granules of the dog kidney were kept at 0°C. 2. Although the molecular weight of the renin in the cytosol on day 1 was estimated to be about 40 000 by gel filtration, the renin was completely converted into a higher—molecular—weight form (60 000) by day 7. The renin in the granules kept its molecular size of 40 000 during cold storage. 3. This type of molecular—weight conversion could be performed without protease inhibitors. 4. Dithiothreitol neither inhibited the conversion into the higher—molecular—weight form of renin during cold storage nor led to a reduction in the molecular weight of renin, although the oxidation of thiol groups has been proposed as the mechanism for the molecular—weight conversion of renin. 5. Keeping a mixture of renin from the granules and crude renin—binding substance at 0°C for 7 days resulted in the conversion of the renin into the higher—molecular—weight form, indicating that the renin—binding substance we have previously described is required for the conversion during cold storage. 6. Acidification caused the higher—molecular—weight form of renin formed in the cytosol to change to the lower—molecular—weight form, with a concomitant increase in activity of about 50%.

Sulphydryl Oxidation in the Mechanism of Molecular-Weight Conversion of Renin in Dog Kidney

1982 ◽  
Vol 62 (2) ◽  
pp. 157-162 ◽  
Author(s):  
Fumihiko Ikemoto ◽  
Kazuo Takaori ◽  
Hiroshi Iwao ◽  
Kenjiro Yamamoto
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Renal Cortex ◽  
Soluble Fraction ◽  
Low Molecular Weight ◽  
Oxidizing Agent ◽  
Nitrobenzoic Acid ◽  
Binding Substance ◽  
Sh Group ◽  
Dog Kidney

1. A high-molecular-weight renin (Mr 60 000) was formed by the reaction of a low-molecular-weight renin (Mr 40 000) with a renin-binding substance in canine renal cortical extract in the presence of the sulphydryl (SH) group oxidizing agent potassium tetrathionate; thus the reaction required SH oxidation. 2. Renin extracted from isolated renin granules was adsorbed on to thiopropyl Sepharose 6B, and then liberated with dithiothreitol (50 mmol/l), indicating that it possessed on SH moiety(s). 3. However, the renin was capable of reaction with the renin-binding substance even after its SH moiety (or moieties) was protected with 5,5′-dithiobis-(2-nitrobenzoic acid). 4. The high-molecular-weight renin was converted into the low-molecular-weight renin by incubation (37°C, 15 min) with cytosol (soluble fraction) of renal cortex and liver. Such converting ability was diminished after the cytosol was treated with perchloric acid or potassium tetrathionate. 5. These results suggest that the reaction of renin with the renin-binding substance does not require disulphide bond(s) and that an enzymelike substance which is sensitive to SH oxidation is involved in the conversion from the high-molecular-weight renin into the low-molecular weight renin.

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.

Subcellular Distribution of Low- and High Molecular-Weight Renin and its Relation to a Renin Inhibitor in Pig Renal Cortex

1980 ◽  
Vol 59 (5) ◽  
pp. 337-345 ◽  
Author(s):  
G. A. Sagnella ◽  
P. R. B. Caldwell ◽  
W. S. Peart
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Subcellular Distribution ◽  
Renal Cortex ◽  
Soluble Fraction ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Main Peak ◽  
Low Molecular Weight ◽  
Molecular Weight Form

1. The subcellular distribution of low-molecular-weight and high-molecular weight forms of pig renin has been investigated. 2. Renin, in aqueous extracts of a ‘renin granular fraction’ prepared by differential centrifugation, after gel filtration on Sephadex G-100 displayed an apparent molecular weight of 40 000 and was not activated by acidification to pH 2.8. 3. Renin in the soluble fraction separated on Sephadex G-100 at neutral pH displayed a main peak of activity with an apparent molecular weight of 40000. When eluates were acidified to pH 2.8 (2°C, 60 min) a marked increase in renin activity was observed in the region corresponding to an apparent molecular weight of 50 000. 4. A renin inhibitory material was isolated from the soluble fraction by DEAE chromatography. This material displayed an apparent molecular weight of 50000 and it was destroyed by acidification to pH 2.8. 5. The presence of the proteolytic inhibitor N-ethylmaleimide yielded an apparently high-molecular-weight form of renin (60000–70000) from the soluble fraction, but this was not found in the granular fraction. 6. We conclude that pig renal renin is stored within membrane-bounded subcellular organelles as the low-molecular-weight form. High-molecular-weight renin and renin inhibitory activity are localized to the cortical soluble fraction. In addition, the soluble fraction contains a material which in the presence of N-ethylmaleimide results in the formation of an apparently high-molecular-weight renin.

Interconversion between High- and Low-Molecular-Weight Forms of Renin in Dog Kidney

1980 ◽  
Vol 59 (s6) ◽  
pp. 25s-27s ◽  
Author(s):  
K. Yamamoto ◽  
F. Ikemoto ◽  
M. Kawamura ◽  
K. Takaori
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Apparent Molecular Weight ◽  
Kidney Cortex ◽  
Low Molecular Weight ◽  
Cytosol Fraction ◽  
High Molecular Weight Form ◽  
Binding Substance ◽  
Dog Kidney ◽  
Molecular Weight Form

1. The low-molecular-weight (40 000) form of renin was converted into the high-molecular-weight (60 000) form of renin with sulphydryl oxidation, and the high-molecular-weight form of renin was re-converted into the low-molecular-weight form with a reduction of disulphide bonds in the renal cortical homogenate of the dog. Therefore, the low- and high-molecular-weight forms of renin were interconvertible. 2. The formation of high-molecular-weight form of renin required a renin binding substance which was found to be included in the cytosol fraction of kidney cortex of the dog. 3. The renin binding substance of the dog was unstable to heat and low pH, but vitally resistant to Triton X-100 and chloroform. It did not bind to concanavalin A Sepharose 4B. 4. The renin binding substance was eluted in the molecular-weight region between 156 000 and 60 000 on Sephadex G-200, and such apparent molecular weight was not altered by urea at 4 mol/l; thus molecular weight greater than the theoretically expected value of 20 000 was indicated.

scholarly journals Calcium Binding by Particle-Free Supernatants of Homogenates of Skeletal Muscle

1965 ◽  
Vol 49 (1) ◽  
pp. 131-149 ◽  
Author(s):  
F. Norman Briggs ◽  
Martin Fleishman
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Stability Constant ◽  
Calcium Binding ◽  
Weight Fraction ◽  
Preliminary Evidence ◽  
High Molecular Weight Fraction ◽  
Total Calcium ◽  
Chelex 100 ◽  
Binding Substance

A high molecular weight fraction of a soluble Marsh muscle-relaxing preparation has been shown to contain a calcium-complexing substance. By examining the nature of the competition between this fraction and chelex-100 for calcium at various total calcium concentrations it has been possible to calculate the concentration and calcium stability constant of this calcium-complexing substance. Taking into account dilutions which occur during the preparation of fractions containing this substance its concentration may be estimated at about 2·10-4 in muscle and its calcium stability constant was found to be about 1.5·105 M-1. Preliminary evidence suggests that the calcium-binding substance is a protein.

Effect of extractant and molecular size on the optical and chemical properties of soil humic acids

Soil Research ◽  
1969 ◽  
Vol 7 (3) ◽  
pp. 229 ◽  
Author(s):  
JHA Butler ◽  
JN Ladd
Keyword(s):  
Molecular Weight ◽  
Amino Acid ◽  
Humic Acids ◽  
Gel Filtration ◽  
Chemical Properties ◽  
Molecular Size ◽  
Carboxyl Content ◽  
Peptide Bonds ◽  
Molecular Weights ◽  
Amino Acid Contents

Humic acids extracted from soil with sodium pyrophosphate have greater proportions of lower molecular weight material, less acid-hydrolysable amino acid nitrogen contents, but greater carboxyl contents and extinction values (260 and 450 nm) than humic acids extracted subsequently from the same sample with alkali. Humic acids extracted with alkali from fresh soil samples have intermediate values. Extinction values at 260 nm are directly correlated with carboxyl contents for a given soil. Different crop histories have no significant effect on the measured properties of the extracted humic acids. An alkali-extracted humic acid has been fractionated by gel filtration into seven fractions of different nominal molecular weight ranges. As the molecular weights of the fractions increase, both aliphatic C-H (based on infrared absorption at 2900 cm-1) and acid-hydrolysable amino acid contents increase, whereas extinction values at 260 nm and carboxyl contents decrease. The infrared spectra of the high molecular weight fractions have peaks at 1650 and 1510 cm-1 which correlate with acid-hydrolysable amino acid contents and which correspond to amide I and II bands of peptide bonds. Alkaline hydrolysis to split peptide bonds eliminates both these peaks. The spectra also have peaks at 1720 and 1210 cm-1 which correlate with the carboxyl content.

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

1972 ◽  
Vol 130 (1) ◽  
pp. 211-219 ◽  
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.

scholarly journals Characterization of proteins from the cytoskeleton of Giardia lamblia

1983 ◽  
Vol 59 (1) ◽  
pp. 81-103 ◽  
Author(s):  
R. Crossley ◽  
D.V. Holberton
Keyword(s):  
Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Giardia Lamblia ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Molecular Size ◽  
Molecular Weights

Proteins from the axonemes and disc cytoskeleton of Giardia lamblia have been examined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. In addition to tubulin and the 30 X 10(3) molecular weight disc protein, at least 18 minor components copurify with the two major proteins in Triton-insoluble structures. The most prominent minor bands have the apparent molecular weights of 110 X 10(3), 95 X 10(3) and 81 X 10(3). Protein of 30 X 10(3) molecular weight accounts for about 20% of organelle protein on gels. In continuous 25 mM-Tris-glycine buffer it migrates mostly as a close-spaced doublet of polypeptides, which are here given the name giardins. Giardia tubulin and giardin have been purified by gel filtration chromatography in the presence of sodium dodecyl sulphate. Well-separated fractions were obtained that could be further characterized. Both proteins are heterogeneous when examined by isoelectric focusing. Five tubulin chains were detected by PAGE Blue 83 dye-binding after focusing in a broad-range ampholyte gel. Giardin is slightly less acidic than tubulin. On gels it splits into four major and four minor chains with isoelectric points in the pI range from 5.8 to 6.2. The amino acid composition of the giardin fraction has been determined, and compared to Giardia tubulin and a rat brain tubulin standard. Giardins are rich in helix-forming residues, particularly leucine. They have a low content of proline and glycine; therefore they may have extensive alpha-helical regions and be rod-shaped. As integral proteins of disc microribbons, giardins in vivo associate closely with tubulin. The properties of giardins indicate that in a number of respects - molecular size, charge, stoichiometry - their structural interaction with tubulin assemblies will be different from other tubulin-accessory protein copolymers studied in vitro.

scholarly journals High Molecular Weight Factor VIII Coagulant Activity in Cryoprecipitate and Polyethylene Glycol Precipitates

1977 ◽  
Author(s):  
K. A. Rickard ◽  
T. Exner ◽  
H. Kronenberg
Keyword(s):  
Molecular Weight ◽  
Polyethylene Glycol ◽  
Factor Viii ◽  
High Molecular Weight ◽  
Gel Filtration ◽  
Lower Molecular Weight ◽  
Human Antibody ◽  
High Molecular Weight Material ◽  
Coagulant Activity ◽  
Molecular Weight Form

Gel filtration of human plasma cryoprecipitate on Sepharose 2B indicated the molecular weight of factor VIII coagulant activity (VIIIc) to be significantly greater than that found in antihaemophilic concentrate. Polyethylene glycol at 3% concentration precipitated approximately half of the VIIIc from cryoprecipitate. This activity eluted as high molecular weight material on gel filtration. The addition of more polyethylene glycol to a concentration of 8% precipitated most of the remaining VIIIc from cryoprecipitate. This activity appeared to be of significantly lower molecular weight, approximately corresponding in elution volume to that observed for antihaemophilic concentrate. The possibility that an antibody to VIIIc generated in a patient treated with cryoprecipitate might be directed against the higher molecular weight form of factor VIII was investigated. However, no significant differences between the higher and lower molecular weight forms of factor VIII either in stability or in reactivity with human antibody to factor VIII were found.

Immunoaffinity Chromatography of Canine High-Molecular-Weight Renin: Partial Purification and Characterization

1983 ◽  
Vol 65 (2) ◽  
pp. 117-120 ◽  
Author(s):  
Fumihiko Ikemoto ◽  
Victor J. Dzau ◽  
Edgar Haber ◽  
Kazuo Takaori ◽  
Kenjiro Yamamoto
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Specific Activity ◽  
Immunoaffinity Chromatography ◽  
Partial Purification ◽  
Low Molecular Weight ◽  
Specific Method ◽  
Disulphide Bonds ◽  
Binding Substance ◽  
Molecular Weight Form

1. Canine high-molecular-weight renin (mol. wt. 60 000) is believed to be a complex of renin (low-molecular-weight form, mol. wt. 40 000) and renin-binding substance. The immunocross-reactivity of high-molecular-weight renin and low-molecular-weight renin was demonstrated by using antibodies specific to low-molecular-weight renin. 2. Immunoaffinity chromatography with renin-specific antibodies coupled to Sepharose provided a simple and specific method for isolation of high-molecular-weight renin. High-molecular-weight renin with a specific activity of 137 600 ng of ANG I h−1 mg−1 of protein (19.6 Goldblatt units/mg of protein) was obtained. 3. This high-molecular-weight renin was stable in dithiothreitol (25 mmol/l), suggesting that disulphide bonds may not be involved in the binding mechanism between low-molecular-weight renin and renin-binding substance. 4. However, exposure to low pH (3.0) resulted in conversion of high-molecular-weight renin into the low-molecular-weight form.

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