The enzymic preparation of diphosphoinositides.

A procedure for the preparation of diphosphoinositides is described. Triphosphoinositides isolated from bovine brain are hydrolysed by the triphosphoinositide phosphatase (EC 3.1.3.36) from Crithidia fasciculata in the presence of MgC12 and cetyltrimethyl-ammonium bromide. The diphosphoinositides produced are not degraded further and can be recovered from the reaction mixture in greater than 80% yield. The product is chromatographically pure and has the same structure (1-phosphatidylinositol 4-phosphate) as naturally occurring diphosphoinositides.     

Canine Gaucher disease--the enzymic defect.

beta-Glucosidase activity was investigated in tissues from a case of canine Gaucher disease and from a normal dog. In the latter, enzyme activity showed two pH optima at pH 4-0-4-25 and pH 5-0-5-5. In Gaucher disease tissues, negligible activity could be measured at the mouse acidic pH.     

Complete enzymic digestion of acidic proteins.

Acidic proteins are usually resistant to complete enzymic hydrolysis. The increasing number of "unusual" amino acids, which are unstable to acid hydrolysis, makes it necessary to have a method of enzymic hydrolysis applicable to all proteins. The complete hydrolysis of four acidic proteins by subtilisin plus leucine amino-peptidase plus prolidase followed by carboxypeptidase C, is described. Recoveries of amino acids were in excellent agreement with the expected content from the known sequences.     

Semiautomated enzymic microassay for plasma L-alanine.

Plasma L-alanine is deaminated by bacterial alanine-dehydrogenase; the resulting ammonia is dialyzed out and measured by use of a continuous flow phenol-hypochlorite colorimetric microassay. Concentrations in the range of 10-1,000 mumol alanine/l can be determined in a 50-microliter sample. The optimal conditions for the assay are specified. Study of the analytical qualities of the technique shows high specificity, good reproducibility , and a detection limit of 6 mumol/l. Usual values in human plasma from arterial or venous blood are respectively 296 +/- 166 and 376 +/- 214 mumol alanine/l (x +/- 2 SD). The usual values in rats are close to those found in man.     

Cobalt cytochrome c: enzymic assays.

An improved synthesis for cobalt-cytochrome $\text{c}$ has been developed; its half reduction potential is ?140 ± 20mV. Reduced ^Cocyt-$\text{c}$³ is oxidized by bovine heart cytochrome $\text{c}$ oxidase at a rate ～45% that of the native cytochrome $\text{c}$. It is not reduced by mitochondrial NADH or succinate cytochrome $\text{c}$ reductase nor by microsomal NADH or NADPH cytochrome $\text{c}$ reductase.     

Mammalian protein methylesterase. Physical and enzymic properties.

Protein methylesterase (PME) amino acid composition and substrate specificity towards methylated normal and deamidated protein substrates were investigated. The enzyme contained 23% acidic and 5% basic residues. These values are consistent with a pI of 4.45. The product formed from methylated protein by PME was confirmed as methanol by h.p.l.c. The kcat. and Km values for several methylated protein substrates ranged from 20 x 10(-6) to 560 x 10(-6) s-1 and from 0.5 to 64 microM respectively. However, the kcat./Km ratios ranged within one order of magnitude from 11 to 52 M-1.s-1. Results with the irreversible cysteine-proteinase inhibitor E-64 suggested that these low values were in part due to the fact that only one out of 25 molecules in the PME preparations was enzymically active. When PME was incubated with methylated normal and deamidated calmodulin, the enzyme hydrolysed the latter substrate at a higher rate. The Km and kcat. for methylated normal calmodulin were 0.9 microM and 31 x 10(-6) s-1, whereas for methylated deamidated calmodulin values of 1.6 microM and 188 x 10(-6) s-1 were obtained. The kcat./Km ratios for methylated normal and deamidated calmodulin were 34 and 118 M-1.s-1 respectively. By contrast, results with methylated adrenocorticotropic hormone (ACTH) substrates indicated that the main difference between native and deamidated substrates resides in the Km rather than the kcat. The Km for methylated deamidated ACTH was 5-fold lower than that for methylated native ACTH. The kcat./Km ratios for methylated normal and deamidated ACTH were 43 and 185 M-1.s-1 respectively. These results indicate that PME recognizes native and deamidated methylated substrates as two different entities. This suggests that the methyl groups on native calmodulin and ACTH substrates may not be on the same amino acid residues as those on deamidated calmodulin and ACTH substrates.     

A vector method of representation of enzymic reactions: 1. Parametric classification of types of enzymic reaction

A new “parametric” classification of the types of single-substrate enzymic reactions is proposed that includes 15 different types: seven inhibited, seven activated and one initial (uninhibited, i=0 and nonactivated, a=0). The new classification takes into account both the “parametricity” of these reactions and the mechanisms of their action. It unites all the types in an original symmetric system which vividly demonstrates the interconnection between separate (strictly definite) types of inhibited and activated enzymic reactions. The proposed classification permits the revision and some corrections to traditional “competitive” terminology as well as the application of these ideas and mathematical approaches pertinent to the calculation of reactions of enzyme inhibition for data analysis of enzyme activation.