Sedimentation enzyme molecular-weight determination

Sedimentation, enzyme molecular-weight determination by, 287 Sedimentation coefiBcients of a-amylases, 309 of /3-amylases, 333 of phosphorylases, 348 Selenium, in hemiacetal rings of monosaccharides, 206 Seleno sugars, 232 Septanoses, 228... 

A possibility of using the viscosimetiy and sedimentation and diffusion methods in studying the process of enzyme chitosan hydrolysis is discussed in the chapter. It is shown that a change in the intrinsic viscosity of chitosan may be determined by both the hydrolysis process in the glycoside bonds and transformation of the supramolecule stmcture of the polymer. Thus, for setting the hydrolysis process it is necessary to use an absolute method for molecular weight determination. 

For the determination of the sulfhydryl groups, the method of Habeed was used. Disulfide bridges were determinated by the same method with urea and DTT. The protein content was estimated by the method of Bradford with BSA as a standard. The molecular weight determinations were effected by molecular sieving over Sephadex G-200 and by sedimentation equilibrium. Analytical centrifugations experiments were performed with a Beckman model E ultracentrifuge equipped with an interferometric system for Mr determination. For the active enzyme sedimentation constant determination, we have used the method of Cohen and Mire (CEA method). 

The sedimentation coefficient is 3.0 S. The approximate molecular weight of the enzyme was determined by gel filtration (117) to be about... 

Some of the physicochemical parameters of the enzyme, as determined on the Kunitz preparation (S), are summarized in Table I. Sedimentation equilibrium analysis of molecular weight carried out on the enzyme crystallized from ammonium sulfate gave a value of 71,000 (5), in fair agreement with the value of 63,000 obtained by sedimentation velocity measurements on the Kunitz preparation (10). The enzyme has been reported to dissociate into subunits in the presence of sodium dodecyl... 

The heat stability of glutaminase-asparaginase modified with glu-taraldehyde-activated glycopeptides was slightly greater than that of the native enzyme, as was its susceptibility to trypsin.108 This modification decreased the pi by several pH units, and altered the association behavior the glycosylated enzyme had a much wider distribution of molecular weights as determined by sedimentation equilibrium. 

Early discrepancies in determinations of the molecular weight of rabbit muscle and yeast GAPDH s 91-93) were resolved by the definitive sedimentation equilibrium studies of Harrington and Karr (33) in conjunction with the chemical investigations of Harris and Perham 31). These established that the enzyme is a tetramer with a molecular weight of... 

The molecular weight of cytochrome c peroxidase has been determined to be 34,100 on the basis of a sedimentation constant of 3.55 S, a diffusion constant of 9.44 F, and a partial specific volume of 0.733 ml/g (4 )-The enzyme exists as a monodisperse monomer containing one ferric protoporphyrin IX, which is noncovalently bound (/, , 14). No other transition metal is detected in crystalline preparations of the enzyme (22). The apoenzyme moiety is an acidic protein with an isoelectric point at pH... 

The liver acid phosphatase III thus isolated had a molecular weight of 14,000 daltons as determined by filtration through a column of Sephadex G-75 that had been calibrated with markers of known molecular weiglit, and a molecular weight of 16,500 daltons on the basis of sedimentation equilibrium analysis. With p-nitrophenyl phosphate as substrate, the pH optimum was 5.5 and the Michaelis constant was 0.75 mM. The stability of the enzyme at 25° was dependent on pH and... 

Many physical characteristics have been determined for the mushroom tyrosinases. These include diffusion coefficients, sedimentation coefficients, frictional ratios, Stoke s radii, isoelectric points, and apparent molecular weights (17-20). Recent evidence suggests that tyrosinase is composed of two heavy chain components (H) of approximately 43-45 kd each and two light chain components (L) of approximately 13 kd each (18-20). The heavy chains contain the catalytic sites. Podila and Flurkey (21) have suggested that the heavy chains were synthesized as smaller molecular weight precursors to the native enzyme. Different isoenzyme forms of tyrosinase apparently contain different H chains (7,17). No role for the L chain subunits have been reported. 

Price and Radda (338) found that N-acetylimidazole could acetylate up to six tyrosine residues without loss of activity or alteration of Km for substrate however, reaction of about one tyrosine per subunit results in desensitization toward GTP, but the response to ADP is not abolished even by extensive 0-acetylation. Essentially the same results are observed upon nitration with tetranitromethane (TNM). Acetylation does not grossly alter the molecular weight, as measured by sedimentation velocity, or the conformation, as determined by ORD. The GTP site is not protected by NADH alone, but is partially protected (25-50%) by GTP and is at least 75% protected by inclusion of both GTP and NADH in the reaction mixture. Piszkiewicz et al. (339) confirmed these findings by modification with TNM. The reaction is biphasic with initial rapid formation of one residue of 3-nitrotyrosine per subunit. The primary site of reaction is tyrosine-406 in the linear sequence (340). Later (338) the same effect was obtained with chicken GDH with both enzymes there is no influence on activation by ADP. Further, the pH optima of the enzymes are not influenced by the degree of nitration or the inhibition by GTP or activation by ADP (338). 

In contrast the enzyme purified from lupin root nodule cytosol is reported to consist of a single polypeptide chain of molecular weight 235,000 (Boland and Benny, 1977). No information is available at present regarding the possible subunit structure of other eukaryote glutamate synthases although the ferredoxin-dependent enzyme from V. faba has a molecular weight of 150,000, as determined by gel filtration (Wallsgrove et al., 1977) and the pyridine nucleotide-dependent enzyme of Saccharomyces cerevisiae has a sedimentation coefficient of 14.6 S (Roon et al., 1974). 

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