Enzymes sedimentation velocities

Urease activity in soils has been found to reflect the bacterial count and content of organic matter. The urease isolated from an Australian forest soil (87) was crystallized and found to have a specific activity of 75 Sumner units (S.U.) per mg. The molecular weight species were estimated (sedimentation velocity) to be 42, 131, and 217 X 103. That urease activity persists in soils is shown by the finding that enzymic activities, including urease, could be demonstrated in soil samples over 8000 years old (88). 

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 dissociation of AMDH was also followed by sedimentation velocity measurements (Pundak et al, 1981) and neutron scattering (Zaccai et al, 1986b). Samples of AMDH were incubated at various NaCl concentrations at 20°C and aliquots were withdrawn at various times and analyzed by ultracentrifugation. Dissociation of the enzyme was visualized by the appearance of a slower sedimenting boundary (Fig. 1). The relative amounts of the protein in both boundaries could be measured and the dissociation rates could be calculated. These... 

Physical measurements support a molecular weight of approximately 200,000. This value is also in accord with gel exclusion studies on Sephadex G-200. Thus the enzyme contains 1 mole of flavin and 4 g-atoms of nonheme iron per mole.. . . The sedimentation velocity of the beef heart enzyme at 10-15 mg protein/ml is 6.5 S. . . This preparation could oxidize succinate in the presence of ferricyanide or phenazine methosulfate (PMS) as electron acceptor but was unable to transfer elec-to be unable to interact with the respiratory chain. 

Figure 10.4. Ultraceutrifugatiou Studies of ATCase. Sedimentation velocity patterns of (A) native ATCase and (B) the enzyme after treatment withp-hydroxymercuribenzoate show that the enzyme can be dissociated into regulatory and catalytic subunits. [After J. C. Gerhart and H. K. Schachman. Biochemistry 4(1965) 1054.]...

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). 

The catalytic activity responsible for the conversion of p-coumarlc acid Into p-hydroxybenzolc acid was sedimentable at 15 OOO g and could be further purified by sedimentation velocity centrifugation. By means of marker enzymes It was demonstrated that mitochondria are the organelles housing the respective enzymes, and that the mitochondrial Inner membrane is the site where the formation of p-hydroxybenzolc acid and then, most probably, further conversion into ubiquinone takes place (Table 1). 

The above sixfigures pertain to ultracentrtfuge sedimentation velocity runs. Sedimentation is from the meniscus on the left to the bottom of the tube on the right. When no PMB is present, the enzyme sediments as a single pe dc. As the concentration of PMB increases, gradually the enzyme splits into two peaks with sedimentation coeftidents of 2.8 and 5.6S.M14X 10 M.the enzyme seems to have ftdly dissociated into its subunits. 

FIGURE 35.1. Representative profiles of the activity of the AChE molecular forms in soleus, EDL, and hemidiaphragm muscles. Profiles at the top of each column are from untreated muscles followed by profiles of activity of AChE molecular forms of muscles 24 h and 7 days, respectively, after receiving an acute dose of soman (100 pg/kg, s.c.). The AChE activity scale is in arbitrary units based on the pmole substrate hydrolyzed/min by the enzyme activity in each fraction. The sedimentation values of the AChE molecular forms are given in the profiles of untreated muscles above the associated peaks. Sedimentation values were determined by the location of the added sedimentation standards, P-galactosidase (16.0 S), catalase (11.1 S), and alkaline phosphatase (6.1 S), following velocity sedimentation of the gradients. 

Sedimentation coefficients—Sjow—representing the velocity of sedimentation in unit gravitational field in water at 20°C, may be used, together with diffusion constants, to calculate the relative molecular masses of purified enzyme preparations. Reported values of S20W for cholinesterase isolated from human or equine serum are given in Table 7. Furthermore, the Sjow value for highly purified butyrylcholine esterase of porcine parotid gland is 9.7 units (T9) and it is 2.5 units for the enzyme from P. polycolor (N2). 

After dissociation of the native enzyme complex at elevated pH values, the / subunits form high molecular weight aggregates. These aggregates form a complex mixture of different molecular species, which sediment at velocities of about 48 and 70 S. Electron micrographs show hollow, spherical vesicles with diameters of about... 

The a-amylase from human urine has been crystallized. The crystalline enzyme (mol. wt. 4.5 x 10 ) was homogeneous on examination by velocity sedimentation and gel filtration, etc., although electrophoresis revealed the presence of, at least, five isoenzymes. 

The extracellular cellulase from a Myxobacter species has been purified until it was homogeneous on electrophoresis and by velocity sedimentation. Although L-valine was detected at the A-terminus, the enzyme also possesses chitosanase activity these enzymic activities decreased at the same rate during denaturation. 

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