Microheterogeneity and sialic acid in human plasma angiotensinogens in various physiological states

1978 ◽  
Vol 56 (9) ◽  
pp. 892-899 ◽  
Author(s):  
A. A. Faiers ◽  
A. Y. Loh ◽  
D. H. Osmond
Keyword(s):  
Sialic Acid ◽  
Rat Liver ◽  
Isoelectric Focusing ◽  
Structural Characteristics ◽  
Gel Filtration ◽  
Angiotensin I ◽  
Third Trimester ◽  
Human Renin ◽  
Isoelectric Ph ◽  
Normal Men

Pooled or individual plasmas from normal men, women, pregnant women (third trimester), anephric women, and rat liver perfusates were used as sources of angiotensinogen. The plasmas were fractionated and desalted by Sephadex gel filtration, then subjected to isoelectric focusing in a pH 4 to 6 gradient on 40 × 4 cm slabs of polyacrylamide gel. The gels were cut transversely into 0.5-cm-wide strips, the pH measured, and their angiotensinogen concentrations determined by incubation with excess human renin and radioimmunoassay of the product, angiotensin I. This revealed several peaks of angiotensinogen concentration indicative of microheterogeneity in all cases. Contrary to other claims, the isoelectric pH profiles of angiotensinogens in the various physiological states were substantially alike. Major peaks were found at pH 4.75 to 4.85 and 4.9 to 5.0 and minor peaks at pH 4.5 to 4.7 and 5.0 to 5.2; this resolution was greater than that achieved with rat liver angiotensinogens. Incubation of human angiotensinogens with neuraminidase for 3 or 16 h raised their isoelectric pH by about 0.5 U, probably due to removal of sialic acid. Since microheterogeneity persisted after desialylation, it is probably determined by structural characteristics other than sialic acid composition.

Studies on the purification of Entamoeba histolytica antigens by gel filtration. I. Some physicochemical properties of the isolated fractions

1970 ◽  
Vol 16 (6) ◽  
pp. 485-492 ◽  
Author(s):  
Z. Ali Khan ◽  
E. Meerovitch
Keyword(s):  
Physicochemical Properties ◽  
Entamoeba Histolytica ◽  
Isoelectric Focusing ◽  
Phosphate Buffer ◽  
Gel Filtration ◽  
Disc Electrophoresis ◽  
Aqueous Extracts ◽  
Molecular Weights ◽  
Isoelectric Ph ◽  
Considerable Loss

Aqueous extracts, prepared from Entamoeba histolytica (DKB strain) grown at 37 °C in TTY medium with Crithidia sp., were chromatographed on Sephadex G-200 and G-100 gels equilibrated with 0.15 M phosphate buffer, pH 7.2. Seven fractions were obtained, the molecular weights of which ranged from 650 000 to [Formula: see text]. The absorption spectra of either the whole amoebic extract or the first two fractions from the G-200 column were in the region of 280 mμ. The protein to carbohydrate ratio of the whole undialyzed amoebic extract was 2.6:1, which changed to 17:1 after dialysis, indicating a considerable loss of carbohydrate. The whole amoebic extract when subjected to disc electrophoresis resolved into 26 protein bands while the fractions, in addition to having contaminants from the preceding peaks, also contained their own characteristic protein bands. The first two fractions contained, as determined by disc electrophoresis and isoelectric focusing, a variety of different anionic proteins. The isoelectric pH of the first fraction was 5.8 to 6.0 and that of the second ranged between 4.5 and 6.4.

Stimulation of β-(l → 6)-glucanase production by oxidized pustulan

1972 ◽  
Vol 18 (10) ◽  
pp. 1543-1550 ◽  
Author(s):  
Robert G. Brown
Keyword(s):  
Enzyme Activity ◽  
Isoelectric Focusing ◽  
Gel Filtration ◽  
Tween 80 ◽  
Sole Source ◽  
Cellulase Production ◽  
Low Degree ◽  
Degree Of Oxidation ◽  
High Degree ◽  
Stimulation Of

A strain of Penicillium lilacinum, isolated from soil, produced pustulanase, β-(1 → 3)-glucanase, (EC. 3.2.1.6) and cellulase (EC.3.2.1.4) when cultivated on a medium containing pustulan as the sole source of carbon. If pustulan was replaced by ketopustulan, the production of pustulanase was stimulated about 10-fold although the amount of stimulation was dependent on the degree of oxidation of pustulan. β-(1 → 3)-Glucanase production was stimulated slightly by ketopustulan; however, the degree of oxidation did not affect significantly the yield of this enzyme. Cellulase production was either unaffected by the oxidized polymer, or at higher degrees of oxidation, decreased. Tween 80 stimulated the production of the three enzymes in media containing ketopustulan with a low degree of oxidation but was inhibitory to pustulanase and cellulase production in media containing ketopustulan with a high degree of oxidation. A combination of gel filtration and isoelectric focusing revealed that each enzyme activity was attributable to at least two proteins.

scholarly journals Properties of subunits of the multicatalytic proteinase complex revealed by the use of subunit-specific antibodies

1991 ◽  
Vol 278 (1) ◽  
pp. 171-177 ◽  
Author(s):  
A J Rivett ◽  
S T Sweeney
Keyword(s):  
Molecular Mass ◽  
Rat Liver ◽  
Gel Filtration ◽  
Immunoblot Analysis ◽  
Rat Tissues ◽  
Specific Antibodies ◽  
Multicatalytic Proteinase ◽  
Cell Extracts ◽  
Liver And Kidney ◽  
Lysosomal Proteinase

The multicatalytic proteinase (MCP) is a high-molecular-mass non-lysosomal proteinase that gives rise to a characteristic pattern of bands of molecular mass 22-34 kDa on SDS/PAGE gels. Isoelectric-focusing gels of the enzyme purified from rat liver show 16 bands with isoelectric points in the range of pH 5-8.5. Two-dimensional PAGE gels reveal that there are more than the previously reported 13 polypeptides associated with the MCP from rat liver and show a pattern of 15-20 major spots and several minor ones, similar to that of MCP isolated from some other sources. Possible relationships between the different polypeptides were investigated by immunoblot analysis of electrophoretically purified proteinase subunits with affinity-purified subunit-specific antibodies as well as antibodies raised against individual denatured subunits of the complex. The results demonstrate that many of the major polypeptide components of the MCP complex are antigenically distinct. Moreover comparison of immunoreactive material in crude cell extracts with that in purified MCP preparations has shown that the polypeptides are not derived from a smaller number of higher-molecular-mass subunits. Also, individual subunits have the same apparent molecular mass in a variety of rat tissues, suggesting close similarity between MCPs of different tissues. The highest concentrations of MCP subunits occur in liver and kidney. Gel-filtration analysis of crude extracts has demonstrated that MCP polypeptides are also associated with a higher-molecular-mass complex, which may be the 26 S proteinase that has been implicated in the degradation of ubiquitin-protein conjugates.

scholarly journals Rat liver alcohol dehydrogenase. Purification and properties

1971 ◽  
Vol 125 (4) ◽  
pp. 1039-1047 ◽  
Author(s):  
M J Arslanian ◽  
E Pascoe ◽  
J G Reinhold
Keyword(s):  
Molecular Weight ◽  
Alcohol Dehydrogenase ◽  
Rat Liver ◽  
Gel Filtration ◽  
Rabbit Antiserum ◽  
Minor Component ◽  
Disc Electrophoresis ◽  
Supernatant Fraction ◽  
Liver Alcohol Dehydrogenase ◽  
A Minor

Alcohol dehydrogenase (EC 1.1.1.1) from the rat liver supernatant fraction has been purified 200-fold and partially characterized. The isolation procedure involved ammonium sulphate fractionation, DEAE-Sephadex chromatography and gel filtration. The purified enzyme behaved as a homogeneous preparation as evaluated by cellulose acetate and polyacrylamide-gel disc electrophoresis. Sulphoethyl-Sephadex chromatography and immunoelectrophoresis with rabbit antiserum indicated the presence of a minor component. Rat liver alcohol dehydrogenase appears to contain 4mol of zinc/mol, has an estimated molecular weight of 65000 and consists of two subunits of similar molecular weight. Heavy-metal ions, thiol-blocking reagents, urea at concentrations below 8m, low pH (5.5) and chelating agents deactivate the enzyme but do not dissociate it into subunits. Deactivated enzyme could not be reactivated. The enzyme is strictly specific for NAD+ and has a broad specificity for alcohols, which are bound at a hydrophobic site. Inhibition occurred with the enzyme equilibrated with Zn2+ at concentrations above 0.1mm.

scholarly journals Effects of pronase and neuraminidase treatment on a myelin-associated glycoprotein in developing brain

1976 ◽  
Vol 156 (1) ◽  
pp. 143-150 ◽  
Author(s):  
R H Quarles
Keyword(s):  
Molecular Weight ◽  
Sialic Acid ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Apparent Molecular Weight ◽  
Weight Class ◽  
Developmental Difference ◽  
Adult Rats ◽  
Neuraminidase Treatment

Rats (14 days old) were injected with [14c]fucose and young adult rats with [3H]fucose in order to label the myelin-associated glycoproteins. As previously reported, the major [14C]fucose-labelled glycoprotein in the immature myelin had a higher apparent molecular weight on sodium dodecyl sulphate/polyacrylamide gels that the [3H]fucose-labelled glycoprotein in mature myelin. This predominant doubly labelled glycoprotein component was partially purified by preparative gel electrophoresis and converted to glycopeptides by extensive Pronase digestion. Gel filtration on Sephadex G-50 separated the glycopeptides into several clases, which were designted A,B, C AND D, from high to low molecular weight. The 14C-labelled glycopeptides from immature myeline were enriched in the highest-molecular-weight class A relative to the 3H-labelled glycopeptides from mature myelin. Neuraminidase treatment of the glycoprotein before Pronase digestion greatly decreased the proportion of glycopeptides fractionating in the higher-molecular-weight classes and largely eliminated the developmental differences that were apparent by gel filtration. However, neuraminidase treatment did not decrease the magnitude of the developmental difference revealed by electrophoresing the intact glycoprotein on sodium dodecyl sulphate gels, although it did decrease the apparent molecular weight of the glycoprotein from both the 15-day-old and adult rats by an amount comparable in magnitude to that developmental difference. The results from gel filtration of glycopeptides indicate that there is a higher content of large molecular weight, sialic acid-rich oligosaccharide units in the glycoprotein of immature myelin. However, the higher apparent molecular weight for the glycoprotein from 15-day-old rats on sodium dodcyl sulphate gels is not due primarily to its higher sialic acid content.

Purification of p-hydroxyphenylpyruvate hydroxylase from rat liver—requirements for cofactors

1976 ◽  
Vol 54 (5) ◽  
pp. 423-431 ◽  
Author(s):  
Kun-Tsan Lin ◽  
John C. Crawhall
Keyword(s):  
Molecular Weight ◽  
Enzyme Activity ◽  
Rat Liver ◽  
Gel Filtration ◽  
Protein Band ◽  
Final Concentration ◽  
Assay Method ◽  
Enzyme Extract ◽  
Crude Enzyme Extract ◽  
Single Protein

Theenzyme p-hydroxyphenylpyruvate hydroxylase (EC 1.13.11.27)from rat liver was studied with the assay method which measures the release of 14CO2 from p-hydroxyphenyl [carboxyl-,14C]pyruvate. Extensive dialysis of the crude enzyme extract against Tris buffer or purification involving ammonium sulfate, gel filtration, and ion exchange results in loss of enzyme activity that can be reactivated by Fe2+, dichlorophenolindophenol, and various other agents. The effect of these activators depends critically on their final concentration in the assay media.A 70-fold purification of the enzyme fraction yielded a preparation which behaved as a single protein band in Sephadex G-150. It had an isoelectric point at 5.85 and molecular weight of 63 000. The enzyme obtained appears to be different in some respects from those described by other workers from the liver of dog, human, chicken, and frog.

Lack of inhibition of human renin by human des-angiotensin I renin substrate

1981 ◽  
Vol 30 (18) ◽  
pp. 2630-2631 ◽  
Author(s):  
Kunio Hiwada ◽  
Yukimi Sogo ◽  
Yasuharu Takada ◽  
Tatsuo Kokubu
Keyword(s):  
Angiotensin I ◽  
Renin Substrate ◽  
Human Renin

Stability of buffalo casein micelles

1979 ◽  
Vol 46 (2) ◽  
pp. 401-405 ◽  
Author(s):  
Nripendra C. Ganguli
Keyword(s):  
Sialic Acid ◽  
Gel Filtration ◽  
Skim Milk ◽  
Heat Stability ◽  
Casein Micelles ◽  
Heat Stable ◽  
Micellar Casein ◽  
Acid Release ◽  
The Stability ◽  
Better Than

SUMMARYBuffalo skim-milk is less heat stable than cow skim-milk. Interchanging ultracentrifugal whey (UCW) and milk diffusate with micellar casein caused significant changes in the heat stability of buffalo casein micelles (BCM) and cow casein micelles (CCM). Buffalo UCW dramatically destabilized COM, whereas buffalo diffu-sate with CCM exhibited the highest heat stability.Cow κ-casein stabilizes αs-casein against precipitation by Ca better than buffalo º-casein. About 90% of αs-casein could be stabilized by κ: αs ratios of 0·20 and 0·231 for cow and buffalo, respectively.Sialic acid release from micellar κ-casein by rennet was higher than from acid κ-casein in both buffalo and cow caseins, the release being slower in buffalo. The released macropeptide from buffalo κ-casein was smaller than that from cow κ-casein as revealed by Sephadex gel filtration.Sub-units of BCM have less sialic acid (1·57mg/g) than whole micelles (2·70mg/g). On rennet action, 47% of bound sialic acid was released from sub-units as against 85% from whole micelles. The sub-micelles are less heat stable than whole micelles. Among ions tested, added Ca reduced heat stability more dramatically in whole micelles, whereas added phosphate improved the stability of micelles and, more strikingly, of sub-micelles. Citrate also improved the heat stability of sub-micelles but not of whole micelles.

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