scholarly journals Enzyme activity in partly dissociated fragments of rat intestinal maltase/glucoamylase

1979 ◽  
Vol 177 (2) ◽  
pp. 487-492 ◽  
Author(s):  
P R Flanagan ◽  
G G Forstner
Keyword(s):  
Enzyme Activity ◽  
Sodium Dodecyl Sulphate ◽  
Protein Pattern ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Band Pattern ◽  
Ph Values ◽  
Major Species ◽  
Complete Dissociation ◽  
Alpha Glucosidase

Pure rat intestinal maltase/glucoamylase was partially inactivated in 1% sodium dodecyl sulphage by heating at 40–70 degree C for 5 min at pH 7.5, or by lowering the pH to 5.4–6.6 at 24 degree C. When partially active preparations were electrophoresed in the presence of sodium dodecyl sulphate, a complicated protein band pattern of incompletely dissociated fragments of the enzyme was observed. Complete dissociation of the enzyme in sodium dodecyl sulphate, induced by boiling or by pH values below 5.4, was accompanied by total loss of enzyme activity and simplification of the protein pattern to five major species. Although the original enzyme band was absent from some partially dissociated preparations, enzyme activity was present and was associated with several transient protein bands on the gels. Maltase and alpha-glucosidase activities were detected in these bands, but glucoamylase activity was absent.

scholarly journals Cytochrome P450 102A2 Catalyzes Efficient Oxidation of Sodium Dodecyl Sulphate: A Molecular Tool for Remediation

2010 ◽  
Vol 2010 ◽  
pp. 1-7 ◽  
Author(s):  
Irene Axarli ◽  
Ariadne Prigipaki ◽  
Nikolaos E. Labrou
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Reaction Rate ◽  
Sodium Dodecyl ◽  
Cytochrome P450s ◽  
Ph Values ◽  
Maximum Value ◽  
Endogenous Compounds ◽  
Ph Range ◽  
Bell Shaped Curve

Bacterial cytochrome P450s (CYPs) constitute an important family of monooxygenase enzymes that carry out essential roles in the metabolism of endogenous compounds and foreign chemicals. In the present work we report the characterization of CYP102A2 from B. subtilis with a focus on its substrate specificity. CYP102A2 is more active in oxidation of sodium dodecyl sulphate (SDS) than any other characterized CYP. The effect of SDS and NADPH concentration on reaction rate showed nonhyperbolic and hyperbolic dependence, respectively. The enzyme was found to exhibit a bell-shaped curve for plots of activity versus pH, over pH values 5.9–8.5. The rate of SDS oxidation reached the maximum value approximately at pH 7.2 and the pH transition observed controlled by two ps in the acidic and basic pH range. The results are discussed in relation to the future biotechnology applications of CYPs.

The Effect of Leucocyte Elastase on the Immunoelectrophoretic Behaviour of α1-Antitrypsin

1984 ◽  
Vol 66 (2) ◽  
pp. 217-224 ◽  
Author(s):  
R. A. Stockley ◽  
S. C. Afford
Keyword(s):  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Enzyme Inhibitor ◽  
Sodium Dodecyl ◽  
Limited Proteolysis ◽  
Two Dimensional ◽  
Ph Values ◽  
Inhibitory Capacity ◽  
Leucocyte Elastase ◽  
Enzyme Complexes

1. Two-dimensional immunoelectrophoresis and conventional sodium dodecyl sulphate-polyacryl-amide gel electrophoresis was performed on various mixtures of purified α1-antitrypsin (α1AT) and leucocyte elastase (LE). 2. The results confirm that α1AT inhibits LE by the formation of enzyme-inhibitor complexes demonstrable by both techniques. 3. The complexes break down with time and are not affected by pH in the presence of excess α1AT. However, the breakdown is more rapid in the presence of excess enzyme only at pH values where LE remains active. The resultant products of the complex breakdown include inactivated LE and α1AT that has undergone limited proteolysis. 4. It is concluded that the presence or absence of α1AT-enzyme complexes as demonstrated by two-dimensional immunoelectrophoresis must be interpreted with caution when studying α1AT function in lung secretions. The absence of such complexes does not mean that previous interaction with enzyme has not occurred, thereby accounting for a reduction in α1AT inhibitory capacity.

scholarly journals Purification and characterization of mouse α-lactalbumin and preparation of its antibody

1980 ◽  
Vol 185 (1) ◽  
pp. 227-237 ◽  
Author(s):  
Y Nagamatsu ◽  
T Oka
Keyword(s):  
Concanavalin A ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Deae Cellulose ◽  
Polyacrylamide Gel ◽  
Single Band ◽  
Major Species ◽  
Alpha Lactalbumin

alpha-Lactalbumin was purified to apparent homogeneity from mouse milk by combined use of gel filtration, chromatography on DEAE-cellulose and hydroxyapatite, and concanavalin A-Sepharose affinity chromatography. Mouse alpha-lactalbumin exists in several species with different charges and in two molecular-size forms. The smaller form, which constituted over 90% of total alpha-lactalbumin, included two major and two minor species, each of which showed different electrophoretic mobility on polyacrylamide-gel electrophoresis, but gave the same single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis in two different buffer systems and over the range 10-15% acrylamide concentrations. The molecular weight was estimated as 14 100. The two major species of the smaller form had the same amino acid composition and contained no significant amount of carbohydrate. The larger form of alpha-lactalbumin, consisting of two species with different charges, was present in a small amount (less than 10%) in the milk and was isolated by its ability to interact with concanavalin A-Sepharose. Each of the two species also gave the same single band of apparent mol.w.t 18 500 on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. However, this value may be anomalous, since this larger form appears to be glycosylated, and glycoproteins can behave anomalously on sodium dodecyl sulphate/polyacrylamide gels by binding less sodium dodecyl sulphate. All species of mouse alpha-lactalbumin from milk were active in the lactose synthase reaction and showed identical immunological properties, as determined by the mono-specific antibody prepared against the small major species. The presence of both the larger and the smaller forms, each in a percentage concentration similar to that found in milk, was also demonstrated in alpha-lactalbumin induced by hormones in organ cultureof pregnant-mouse mammary gland.

scholarly journals Phosphoenolpyruvate carboxykinase from guinea-pig liver mitochondria. Immunological evidence for increase in enzyme amount during neonatal development

1984 ◽  
Vol 221 (1) ◽  
pp. 105-111 ◽  
Author(s):  
S Wicheanvonagoon ◽  
I J Arinze
Keyword(s):  
Guinea Pig ◽  
Sodium Dodecyl Sulphate ◽  
Phosphoenolpyruvate Carboxykinase ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Liver Mitochondria ◽  
Polyacrylamide Gels ◽  
Neonatal Development ◽  
Pig Liver ◽  
Guinea Pig Liver

Phosphoenolpyruvate carboxykinase was purified from mitochondria of guinea-pig liver by affinity chromatography on GMP-Sepharose. The enzyme was purified 100-fold to a high degree of electrophoretic homogeneity as judged by detection of a single protein band on sodium dodecyl sulphate/polyacrylamide gels. The yield was about 16%. The Mr of the purified enzyme was estimated to be 68500 +/- 680 by analysis on sodium dodecyl sulphate/polyacrylamide gels. Antibodies raised in rabbits against the purified enzyme were highly specific for mitochondrial phosphoenolpyruvate carboxykinase and did not precipitate the cytosolic form of this enzyme from either rat or guinea-pig liver cytosol. The use of this antibody showed that starvation does not increase the amount of the enzyme. However, neonatal-development-dependent increase in its activity is shown to be mediated by accumulation of phosphoenol pyruvate carboxykinase-specific protein.

scholarly journals Purification of rat intestinal maltase/glucoamylase and its anomalous dissociation either by heat or by low pH

1978 ◽  
Vol 173 (2) ◽  
pp. 553-563 ◽  
Author(s):  
P R Flanagan ◽  
G G Forstner
Keyword(s):  
New Species ◽  
Sialic Acid ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Low Ph ◽  
Precipitin Line ◽  
Molecular Weights ◽  
Ph Values

Maltase-glucoamylase, a microvillous membrane ectoenzyme, was solubilized from rat intestinal mucosa by digestion with papain and subsequently purified to homogeneity with an overall yield of 10–20%. An antibody to the purified enzyme formed a single precipitin line in immunodiffusion experiments with an intestinal homogenate. The enzyme was shown to be an acidic glycoprotein (20% sugar by weight) which contained low amounts of cysteine and no sialic acid. At pH3–6, maltase activity was slowly lost, but the enzyme was re-activated by re-adjustment of the pH to neutrality. However, in the presence of sodium dodecyl sulphate, acid pH values inactivated maltase irreversibly, and at the same time converted the enzyme (mol.wt. 500000 approx.) into five new species with apparent molecular weights ranging from 134000 to 480000 as judged by polyacrylamide-gel electrophoresis. The same five fragments were also formed by boiling the enzyme for brief periods in the presence of sodium dodecyl sulphate or urea either with or without reducing agents. The dissociated species were stable on re-electrophoresis, and amino acid analysis showed them to be very similar to each other and to the original enzyme. The bands migrated anomalously on polyacrylamide gels of different concentration, thereby preventing the assignment of precise molecular weights. It is possible that the five species may represent stable aggregates of a common monomer of the enzyme.

The interaction of bovine milk caseins with the detergent sodium dodecyl sulphate. I. The relationship between the composition and the size of the protein–detergent aggregate

1970 ◽  
Vol 37 (2) ◽  
pp. 245-257 ◽  
Author(s):  
G. C. Cheeseman ◽  
Joan Jeffcoat
Keyword(s):  
Molecular Weight ◽  
High Molecular Weight ◽  
Sodium Dodecyl Sulphate ◽  
Sodium Dodecyl ◽  
Bovine Milk ◽  
Maximum Amount ◽  
Protein Molecules ◽  
The Individual ◽  
The Relationship ◽  
Complete Dissociation

SummaryStudy of the dissociation of high-molecular-weight aggregates of preparations of αs1-, β-, κ-, and para-κ-casein by the detergent, sodium dodecyl sulphate (SDS), showed that there are differences in the aggregation properties of the individual caseins. Binding of detergent led first to the dissociation of casein aggregates and then to further interaction with the casein molecules. The amounts of detergent required to give the minimum sized protein-detergent aggregate when expressed as mg/mg casein were similar for κ-, para-κ- and αs1-casein but much less for β-casein. However, expressed as mole/mole the requirement for κ- and αs1-casein was similar but was twice that found for para-κ- and β-casein. The maximum amount of SDS bound was about twice that required for complete dissociation of the aggregates for κ-, para-κ- and αs1-casein but was 13 times greater for β-casein.Complete dissociation of κ-casein aggregates by SDS alone was not possible due to the presence of aggregates formed by disulphide linkages. These aggregates, which consisted of 3±1 protein molecules, accounted for about one-third of the κ-casein in the preparations examined.

scholarly journals Co-purification of galactosyltransferases from chick-embryo liver

1985 ◽  
Vol 227 (2) ◽  
pp. 573-582 ◽  
Author(s):  
K Furukawa ◽  
S Roth
Keyword(s):  
Chick Embryo ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Protein Band ◽  
Polyacrylamide Gel ◽  
Cellulose Acetate Electrophoresis ◽  
Km Value ◽  
Alpha Lactalbumin

Two galactosyltransferases with nearly identical Mr values were purified 5000-7000-fold from microsomal membranes of chick-embryo livers by using several affinity columns. One enzyme transfers galactose from UDP-galactose to form a β-(1→4)-linkage to GlcNAc (N-acetylglucosamine) or AsAgAGP [asialo-agalacto-(alpha 1-acid glycoprotein)]. The other enzyme forms a β-(1→3)-linkage to AsOSM [asialo-(ovine submaxillary mucin)]. Both enzymes were solubilized (85%) from a microsomal pellet by using 1% Triton X-100 in 0.1 M-NaCl. The supernatant activities were subjected to DEAE-Sepharose chromatography and four affinity columns: UDP-hexanolamine-Sepharose, alpha-lactalbumin-Sepharose, GlcNAc-Sepharose and either AsAgAGP-Sepharose or AsOSM-Sepharose. The AsAgAGP enzyme [(1→4)-transferase] and the AsOSM enzyme [(1→3)-transferase] behave identically on the DEAE-Sepharose and UDP-hexanolamine-Sepharose columns, and similarly on the alpha-lactalbumin-Sepharose column. Final separation of the two enzymes, however, could only be achieved on affinity columns of their immobilized respective acceptors. Both purified enzymes migrate as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after silver staining, and both have an apparent Mr of 68 000. The enzymes were radioiodinated and again subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioautographic analyses showed only one, intensely radioactive, band. Activity stains performed for both transferases after cellulose acetate electrophoresis indicate that, with this system too, both activities have identical mobilities, and co-migrate, as well, with the major, silver-stained, protein band. Kinetic studies with the purified enzymes show that the Km value for GlcNAc, for the (1→4)-transferase, is 4mM; for the (1→3)-transferase the Km value for AsOSM is 5mM, in terms of GalNAc (N-acetylgalactosamine) equivalents. Both enzymes have a Km value of 25 microM for UDP-galactose.

Stepwise isolation and properties of unstable Chinese hamster cell variants that overproduce adenylate deaminase

1982 ◽  
Vol 2 (11) ◽  
pp. 1346-1353
Author(s):  
M Debatisse ◽  
M Berry ◽  
G Buttin
Keyword(s):  
Enzyme Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Chinese Hamster ◽  
Protein Band ◽  
Chinese Hamster Cell ◽  
Selective Medium ◽  
Striking Similarity ◽  
Cell Extracts ◽  
Adenylate Deaminase

Addition of coformycin (0.5 microgram/ml) to a culture medium containing adenine causes in Chinese hamster fibroblasts a lethal depletion of IMP. Resistant variants have been recovered, some of which exhibit increased adenylate deaminase activity. (Debatisse et al., J. Cell. Physiol., 106:1-11, 1981). The selective medium was made more specific for the isolation of this class of variants by supplementation with azaserine. The hyperactive variants remained sensitive to coformycin concentrations above that used for their selection and were unstable. Their frequency was not increased by ethyl methane sulfonate mutagenesis. The resistant phenotype and the increased activity of adenylate deaminase behaved as semidominant traits in hybrids. No change was detected in the Km for AMP, the cofactor requirement, or the chromatographic properties of adenylate deaminase in the variants. Through stepwise selection in media supplemented with increasing coformycin concentrations, unstable clones with adenylate deaminase activity up to 150-fold the wild-type level were isolated; from an unstable clone, a stable subclone with reduced resistance and enzyme activity was recovered. Evidence that increased adenylate deaminase activity is the manifestation of overaccumulation of the enzyme protein was supplied by the correlation of enzyme activity with the intensity of a protein band comigrating with purified adenylate deaminase during sodium dodecyl sulfate-polyacrylamide gel electrophoresis of cell extracts. Several unidentified additional bands showed comparable quantitative changes. The striking similarity between the adenylate deaminase-overproducing lines and unstable dihydrofolate reductase-overproducing lines generated by gene amplification strongly suggests that the coformycin-resistant variants also resulted from amplification of an adenylate deaminase gene.

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