Enzymatic activity of basidiomycetes

In mycelial acetone powder of the basidiomyceteOudemansiella mucida, the presence of enzymatic systems of NADH₂-oxidase and-dehydrogenase type and of NAD⁺-dependent alcoholdehydrogenase (using ethanol as substrate) has been detected. NADH₂-oxidase has its optimal pH value in the region of 6,0 and is not too active. NADH₂-dehydrogenase which reduces 2,6-dichlorophenolindophenol is very active, may be eluted from the mycelial preparation with water and its adequate pH is in the region of 6,2. On the other hand, NADH₂-dehydrogenase reducing triphenyltetrazolium chloride is non-eluable with water, its activity depends on the presence of menadione and its adequate pH lies in the region of 7,4.     

Enzymatic activity of the second component of complement.

Isolated C2 and C2i preparations were able to hydrolyze a number of synthetic esters containing basic amino acids, among which N-alpha-acetylglycyl-L-lysine methyl ester (AcGlyLysOMe) was most susceptible. The cleaving activity was a property of the C2 molecule, since it correlated with the presence of C2 on analyses of C2 preparations by ultracentrifugation in sucrose gradients, filtration through Sephadex G-200 columns, and on electrophoresis in acrylamide gels. Furthermore, acrylamide gel electrophoretic studies showed a shift in hydrolytic activity from the position occupied by C2 to that characteristic of C2i after incubation of C2 with C1s. The action was enzymatically mediated as evidenced by a bell-shaped pH activity curve, a linear dependence on C2 concentration, and the presence of Michaelis-Menten kinetics. The Michaelis constant for cleavage of AcGlyLysOMe by C2 was 1.8 X 10(-2) mol. Cleavage of C2 by C1s increased C2 enzymatic activity, yet chemical oxidation of the molecule, although enhancing hemolytic acitivity, failed to increase C2 hydrolytic activity. The observed enzymatic activity of C2 was found to be relevant to the function of C2 in the C42 complex, since AcGlyLysOMe competitively inhibited the C42 mediated cleavage of C3 in free solution and the C42 dependent binding of C3 to cells.     

Enzymatic activity in basidiomycetes

The oxidation-reduction activity of the basidiomyceteOudemansiella mucida was studied in relation to its growth in a laboratory fermentor. Cytochromes were detected in the mycelium destroyed by sonication, flavin diaphorase, polyphenoloxidase, peroxidase and catalase were found to be present in the mycelial homogenate from Waring-blendor. The obtained values of the enzymatic activity were dependent on the method of preparation of the mycelium. Homogenization in a Waring-blendor was the most appropriate. Certain interrelationships between the appearance and activity of the enzymes followed and the phase of submerged growth of the fungus were shown. The results document the succession of cytochromes, flavins and polyphenoloxidase and the specific time differences of the activity of catalase and that of peroxidase.     

Enzymatic activity of precursors of Bacillus megaterium spore protease.

The protease that initiates rapid proteolysis during germination of Bacillus megaterium spores is synthesized during sporulation as a 46,000-molecular-weight polypeptide (P46) and is processed later in sporulation to a 41,000-molecular-weight polypeptide (P41), which is converted to a 40,000-molecular-weight polypeptide (P40) early in spore germination. P40 is known to be both tetrameric and enzymatically active. In this work, we show that P46 and P41 are both tetrameric, but that only P41 is enzymatically active. The identification of a zymogen form (P46) of this protease explains in part the regulation of the activity of this enzyme.     

Activation of glutaraldehyde-crosslinked chymotrypsinogen-A. Enzymatic activity and circular dichroism studies

Phenylhydrazine does not inactivate papain or glyceraldehyde-3-phosphate dehydrogenase under anaerobic conditions. The inactivation of papain and glyceralde-hyde-3-phosphate dehydrogenase under aerobic conditions is ascribed to the oxidation of phenylhydrazine by O₂ which generates phenyldiimide and H₂O₂, both of which react with the essential sulfhydryl groups and inactivate the enzymes. Phenyldiimide generated from methyl phenylazoformate inactivates both of the sulfhydryl enzymes under anaerobic conditions. The inactivation of papain and GPD with aerobic, aqueous solutions of [¹⁴C]phenylhydrazine introduces a small amount of radioactivity into the enzymes which is discharged by dithiothreitol. The amount of radioactivity bound to papain is increased when cyanide is present in the inactivation mixture.When the β-[¹⁴C]thiocyanoalanine derivative of papain is treated with phenylhydrazine the radioactivity is discharged from the enzyme. Cyanide evidently reacts with the sulfenic acid derivative of papain to form a thiocyanate derivative. Phenylhydrazine presumably displaces cyanide from the thiocyanate derivative to form a sulfenyl hydrazide derivative to account for the increased incorporation of [¹⁴C]phenylhydrazine when papain is inactivated with aerobic solutions of [¹⁴C]-phenylhydrazine in the presence of cyanide. When the sulfhydryl group of papain is oxidized to a sulfenic acid with H₂O₂ and then treated with [¹⁴C]phenylhydrazine, ¹⁴C is not incorporated into the enzyme. These experiments suggest that the H₂O₂ in the aerobic solutions of phenylhydrazine oxidizes the sulfhydryl group at the active site of papain to a sulfenic acid. The [¹⁴C]phenyldiimide in these solutions reacts to some extent with the active sulfhydryl group to form a sulfenyl hydrazide derivative.     

Enzymatic activity of immobilized yeast phosphoglycerate kinase

This work reports the first evidence that recombinant yeast phosphoglycerate kinase (PGK) is still significantly active when immobilized on glass and muscovite mica. Using previous work to improve the sensitivity of the existing setup, Tapping Mode atomic force microscopy (AFM) was used in a liquid environment to determine the surface enzyme coverage of derivatized mica and glass slides. When associated to spectrophotometric measurements, the AFM data allows assessing the catalytic constant of surface enzymes and comparing it to bulk values. The validity of the Michaelis-Menten model for surface reactions is discussed, supported by spectroscopic measurements of the surface consumption of 1,3-bis-phosphoglycerate (1,3-BPG). Only a few percent of the enzyme material maintains its initial bulk activity. This value could constitute a guideline for biosensors made with the method used here whenever a rapid assessment of the remaining surface activity is needed.     

Enzymatic activity of dystrophic chicken sarcoplasmic reticulum

We have isolated sarcoplasmic reticulum from normal and dystrophic chicken muscle, using an improved isolation procedure. Dystrophic sarcoplasmic reticulum has a reduced level of calcium-sensitive ATPase activity, phosphoenzyme formation, and steady-state calcium transport. Anion-stimulated calcium transport by dystrophic sarcoplasmic reticulum is also reduced when measured under the proper conditions, and dystrophic sarcoplasmic reticulum shows no alteration in calcium efflux rate. Active calcium phosphate loading of the normal and dystrophic sarcoplasmic reticulum preparations indicates that a reduced percentage jof the dystrophic vesicles are capable of active calcium transport. The loaded dystrophic sarcoplasmic reticulum vesicles exhibit the same relative reductions in enzymatic activity as the starting sarcoplasmic reticulum preparations. However, the enzyme activities of normal and dystrophic sarcoplasmic reticulum are similar in the presence of detergent and exogenous phospholipid. On the basis of these results, we suggest that the lipid microenvironment of the dystrophic enzyme is altered.