Polyacrylamide Gel Electrophoresis In Presence Research Articles

Properties of Two Molecular Forms of β‐Glucuronidase from the Mollusc Littorina littorea L.

The occurrence of the two molecular forms, I and II, in the beta-glucuronidase of the liver (hepatopancreas) from the marine mollusc Littorina littorea L. has been demonstrated for the first time. The two forms have been purified 355-fold and 1262-fold, respectively. Form I and II of beta-glucuronidase behave differently on DEAE-cellulose chromatography, polyacrylamide gel disc electrophoresis, isoelectric focusing (pH 5.5 and 4.2, respectively), optimum pH (4.4 and 3.4--4.1, respectively), thermal stability, Km (1.2 mM and 0.5 mM with p-nitrophenyl beta-D-glucuronide, 0.3 mM and 0.15 mM with phenolphthalein beta-D-glucuronide as substrates for form I and II, respectively) and V. Their molecular weight, estimated by gel filtration through Sephadex G-200, was about 250000 for both forms. Several subunits were separated by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate. This beta-glucuronidase is a glycoprotein, but sialic acid(s) were not detected. The enzyme was very active on synthetic substrates and also on hexasaccharides and tetrasaccharides containing glucuronic acid residues with beta 1 leads to 3 linkages; it had practially no activity on certain glycosaminoglycans. Hg2+ and glucaro-1,4-lactone were very effective inhibitors of this enzyme; the latter by a competitive mechanism.

Membrane ATPase of Bacillus subtilis. I. Purification and properties

The membrane ATPase (EC 3.6.1.3) of Bacillus subtilis can be solubilized by a shock-wash process. Two procedures for purifying the solubilized enzyme are reported. A protease inhibitor, phenylmethane sulfonylfluoride, was introduced in the solubilization and purification step. The resultant ATPase purified by density gradient centrifugation has a molecular weight of 315 000, an s 20,w of 13,4 and an ámino acid composition very similar to bacterial ATPases already studied. After exposure to polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate (SDS), or 8 M urea or SDS-urea, the purified ATPase can be dissociated in two non-identical subunits of molecular weights 59 000 (α) and 57 000 (β) with different charges. Kinetic studies showed that Ca 2+ or Zn 2+ are required for ATPase activity, although Mg 2+ was uneffective. At optimal Ca 2+ concentration, the Mg 2+ has an inhibitory effect. The K m for ATP is 1.3 mM. Inhibitors of the oxydative phosphorylation, of the mitochondrial ATPase and of the (Na + + K +)-ATPase are studied.

Effect of massive proximal small bowel resection on intestinal brush border membrane proteins in the dog.

Dog enterocyte brush border proteins have been studied after a 75% proximal resection of the small bowel. This study was carried on microvillar membrane preparations purified from ileal mucosa sampled before and after regeneration on neighbouring intestinal segments, each animal acting as its own control. After six weeks of regeneration a statistically significant decrease of the following enzyme specific activities was observed: lactase, cellobiase, maltase, sucrase, palatinase, dextranase, trehalase, alkaline phosphatase, aminopeptidase and gamma-glutamyl transferase. Analysis of brush border proteins by polyacrylamide gel electrophoresis in presence of sodium dodecyl sulphate have shown after regeneration a decreased rate for the proteins with a molecular weight higher than 100,000 daltons. Modifications of electrophoretic patterns seem to be related to the specific activity decreases observed for brush border enzymes after regeneration, since the molecular weight of these enzymes were found between 116,000 and 285,000 daltons, after gel filtration.

Non-allelic variants of histones 2a, 2b and 3 in mammals.

THE genome of eukaryotes is organised in nucleoprotein fibres formed by the interaction of the DNA with small basic proteins, the histones, and by specific interactions between certain histones1. To gain more insight into the molecular organisation of chromosomes it is necessary to determine in detail the complexity and variability of the histones. The chromosomes of eukaryotes contain five major types of histones. One of these, the very lysine-rich H1, consists of a small number of poly-peptides which differ slightly in primary structure and which are present in different relative amounts in different tissues2. The other four histones, which are involved in histone–histone interactions, were considered homogeneous and invariable until it became possible to resolve H2a, H2b and H3 into variants which exhibit tissue-specific variation by polyacrylamide gel electrophoresis in presence of non-ionic detergents3 (Fig. 1). We report here on the primary structure of the variants of mammalian histones 2a, 2b and 3.

Urea-extractable protein from human epidermis

The major protein fraction extracted from human epidermis by urea has the following characteristics: 1. (1) A molecular weight of approximately 600 000. 2. (2) A copper content of 0.03% giving a minimum molecular weight of 212 000. 3. (3) A non-protein moiety indicated by 1. (a) protein/dry weight ratio 2. (b) anomalous ultraviolet absorption spectrum 3. (c) high-voltage electrophoresis after mild alkaline or acid treatment 4. (d) an excess of imidazole groups by Pauly reaction compared with the number of histidine residues by amino acid analysis. The non-protein component is not urocanic acid or RNA as has been reported (Bernstein, I.A. (1970), J. Soc. Cosmet. Chem. 21, 583–594 and Sibrack, L.A., Gray, R.H. and Bernstein, I.A. (1974), J. Invest. Dermatol. 62, 394–405). 1. (4) The presence of interchain disulphide linkages indicated by polyacrylamide gel electrophoresis in sodium dodecyl sulphate with and without β-mercaptoethanol. 2. (5) Polyacrylamide gel electrophoresis in presence and absence of 6 M urea suggests that aggregates are broken down in urea.

Gramicidin S-synthetase electrophoretic characterization of the multienzyme

1. 1. A method characterizing the fully active gramicidin S-synthetase (EC.6 6.3.2.−) multienzyme in protein mixtures by a combination of sedimentation and polyacrylamide gel electrophoretic mobility data has been described. 2. 2. The molecular weight of 280 000 has been reevaluated by gradient centrifugation, gel filtration, and polyacrylamide gel electrophoresis in presence of sodium dodecyl sulfate. The size of the multienzyme is not changed by sodium dodecyl sulfate treatment. 3. 3. In polyacrylamide gel electrophoresis dimerisation occurs in Tris, while two bands, which may represent monomer and dimer, are observed in phosphate. 4. 4. Reliability of molecular weight determinations of sodium dodecyl sulfate-protein complexes of sizes up to 300 000 daltons has been determined, correlating either mobilities or retardation coefficients.

The effect of thiols on Saccharomyces fragilis.

Treatment of cell suspensions of Saccharomyces fragilis with 0.01 M beta-mercaptoethanol or dithiothreitol released a variety of substances of high and low molecular weight. Twenty-two high-molecular-weight glycoproteins were separated by a combination of chromatography on DEAE cellulose and polyacrylamide gel electrophoresis in presence of sodium dodecylsulphate. The carbohydrate components consisted of at least 95% mannose and the protein components had threonine and serine as the major amino acids. Only very small amounts of phosphorus were associated with the high-molecular-weight components. The low-molecular-weight substances werr probably released from the internal cell pool and uracil and hypoxanthine were identified as components of this fraction. It is suggested that in addition to breaking disulphide bridges in the cell wall the thiols may also render the plasmalemma permeable to certain low-molecular-weight substances. Such effects are not lethal since the yeast can be trained to grow in presence of 0.01 M mercaptoethanol.

Molecular weight of microsomal glucose-6-phosphatase of rat and human liver

Salt-washed and unwashed and aluminum oxide treated microsomes of human liver, and washed microsomes of liver of normally fed, 1 day fasted and alloxan diabetic rats were subjected to polyacrylamide gel electrophoresis in presence of sodium dodecylsulfate and urea. Unwashed and aluminum oxide treated microsomes showed several protein bands, but salt-washed microsomes gave only one major protein band. This major protein band which appeared in all the preparations showed glucose-6- P activity on incubation of the electrophoresed gel with the substrate and subsequent staining with ammonium sulfide. Molecular weight of this protein was estimated to be 63,000 ± 6,800. Glucose-6- P and pyrophosphatase activities were measured after subjecting lyophilized microsomes, aluminum oxide treated supernatant and residue fractions of human and rat liver to ionizing radiation in vacuo at different doses (Mrads). There was no significant difference between either the surviving fraction of glucose-6- P and pyrophosphatase activities or among the different preparations of human and rat liver at a given radiation dose. Computer analysis of the pooled data gave a value of 9.16 Mrads for 37% survival of the activity which corresponds to a molecular weight of 70,000.