Polymeric thiols as enzyme activators of serum creatine phosphokinase.

Several hydrophilic polymeric thiols were prepared from amino-activated polymeric supports by reaction with N-acetylhomocystein ethiolactone. Supports include agaroses, cellulose, Glycophase controlled-pore glass, and Matrex acrylic beads. Thiol content in these polymers was 3-72 mumol SH/g dry polymer. Several were effective solid-phase activators of the sulfhydryl-dependent enzyme creatine phosphokinase at concentrations comparable to that of monomeric thiol required for enzyme activation. The kinetic activation curves for the polymeric and the monomeric (thioglucose) activators were similar, suggesting unhindered interaction of the enzyme with the polymeric activator.

Purification and properties of creatinine iminohydrolase from Flavobacterium filamentosum.

Creatinine iminohydrolase (EC 3.5.4.21), which catalyzes hydrolysis of creatinine to N-methylhydantoin and ammonia, was purified from Flavobacterium filamentosum. The average molecular weight of the purified enzyme was 272,480, and the subunit molecular weight was 44,300. Extensive specificity studies indicated that the enzyme utilized cytosine (Km, 0.62 mM; Vm, 20.1 units/mg) as well as creatinine (Km, 5.00 mM; Vm, 40.4 units/mg) as a substrate. Each was a competitive inhibitor toward hydrolysis of the other compound. Dialysis of creatinine iminohydrolase in the presence of 0.01 M Tris phosphate buffer, pH 7.5, containing 1,10-phenanthroline decreased activity by 98%. Reactivation was accomplished by incubating the apoenzyme in the presence of certain divalent metal chlorides, listed in decreasing order of effectiveness: iron(II), zinc, cobalt(II), cadmium, and nickel. The extent of reactivation depended on the substrate and on which metal ion was added to the apoenzyme. Creatinine to cytosine activity ratios varied from 1:3.75 (iron(II) chloride), to 1:0.9 (zinc chloride), to 1:0.06 (nickel chloride). For different preparations of the holoenzyme that ratio ranged from 1:0.45 to 1:1.10. Variable but significant quantities of zinc and iron were present in all preparations of the purified enzyme.

An enzymic creatinine assay and a direct ammonia assay in coated thin films.

We developed a thin-film enzymic assay for creatinine that makes use of creatinine iminohydrolase (EC 3.5.4.21) to convert creatinine to N-methylhydantoin and ammonia. The ammonia diffuses through a semipermeable layer and is quantitated by reaction with bromphenol blue. A paired analysis of the sample on a separate coating without the enzymic reaction measures endogenous ammonia and, for samples with normal concentrations of ammonia, allows accurate determination of serum creatinine to 150 mg/L without dilution. Results of this assay (y) compare well with those by a liquid-chromatographic comparison assay (x) by linear regression (slope = 0.935, intercept = 1.13 mg/L, r2 = 0.995). It is insensitive to many substances, such as ketones and keto acids, that interfere with conventional assays. Results of the ammonia assay (y) correlate well with those by a semi-automated enzymic assay (x) based on glutamate dehydrogenase (slope = 1.068, intercept = 17.3 mumol/L, r2 = 0.985).

Purification and properties of L-alpha-glycerophosphate oxidase from Streptococcus faecium ATCC 12755.

A procedure was developed to purify the Streptococcus faecium ATCC 12755 L-alpha-glycerophosphate oxidase. The molecular weight of the purified enzyme was 131,000 and the subunit molecular weight was 72,000. Two moles of FAD were bound/mol of enzyme. Apo-L-alpha-glycerophosphate oxidase displayed physical properties similar to the holoenzyme as judged by electrophoresis in 10% buffer gels at pH 8.5 and by centrifugation in a 5 to 20% linear sucrose gradient. The apoenzyme was completely reactivated by incubation with FAD. L-alpha-Glycerophosphate oxidase was specific for L-alpha-glycerophosphate when compared with several other pohsphorylated glycerol and sugar derivatives. Oxygen was the preferred electron acceptor. At 10 mM DL-alpha-glycerophosphate (below the Km of 26 mM for L-alpha-glycerophosphate), activity was increased from 2.6- to 10-fold by increasing the buffer concentration from 0.01 to 0.1 m. This buffer effect was observed with potassium phosphate and other anionic buffers. In 0.001 m potassium phosphate buffer, pH 7.0, activity was increased by several divalent metal ions, including 10 mM CaCl2 (7.7-fold activation) and 10 mM MgCl, (6.8-fold activation). Fructose 6-phosphate and fructose1-phosphate were inhibitors of the L-alpha-glycerophosphate oxidase.

Multilayer film elements for clinical analysis: applications to representative chemical determinations.

Using the general concept of a dry multilayer analytical element, we can change chemical procedures and configurations to assay several blood components. In the assay of serum urea nitrogen, urease in the reagent layer catalyzes the hydrolysis of urea. A semipermeable membrane excludes aqueous base, but allows ammonia to diffuse to an underlying indicator layer. For the amylase determination, the enzyme hydrolyzes a dyed-starch substrate coated on top of the spreading layer; this produces small fragments, which diffuse to a registration layer. The increase of absorbance at 540 nm is correlated with amylase activity. Bilirubin complexes with a cationic polymer at the interface between the spreading and reagent layers. The direct reading at 460 nm allows determination of total bilirubin in the range 1 to 500 mg/liter. Tirglycerides are hydrolyzed in the spreading layer, and the resulting soluble glycerol readily diffuses into the reagent layer, where it is phosphorylated and subsequently oxidized by glycerophosphate oxidase to yield dihydroxyacetone phosphate and hydrogen peroxide. Peroxidase catalyzes production of a color commensurate with the hydrogen peroxide produced.

Characterization and in vivo production of three glycolipids from Candida Bogoriensis: 13-glucopyranosylglucopyranosyloxydocosanoic acid and its mono- and diacetylated derivatives.

Three glycosides of 13-hydroxydocosanoic acid isolated from Candida bogoriensis were characterized by quantitating the amount of carbohydrate, acetate, and hydroxy acid in each, and by gas-liquid chromatography and mass spectrometry of their methyl ester, trimethylsilyl ether derivatives. One of the glycosides was the diacetylated derivative of 13-glucosylglucosyloxydocosanoic acid previously characterized by Tulloch, Spencer, and Deinema (Can. J. Chem., 46: 345 [1968]), in which the disaccharide had the beta(1 --> 2) sophorose linkage and the acetyl groups were attached to the 6' and 6" positions of the glucose residues. The other two glycosides were 13-glucosylglucosyloxydocosanoic acid and its monoacetylated derivative. A comparison of the mass spectra of derivatives indicates that the acetyl group of the monoacetyl lipid is on the internal glucose. Methyl 13-glucosyloxydocosanoate was produced by acid hydrolysis of the methyl ester of the unacetylated glycolipid and was characterized by the same techniques as the other glycolipids. Time course of production of the three glycolipids is consistent with the diacetylated derivative being the first extra-cellular product and the other two glycolipids being formed by deacetylation. 13-Hydroxy[13-(3)H]docosanoic acid, methyl 13-hydroxy[13-(3)H]docosanoate, and 9-hydroxy[11,12-(3)H]-stearic acid were each incorporated into the glycolipid fraction.