We describe a novel method for determining serum triglycerides, in which an enzymatic hydrolysis replaces the more commonly used saponification procedure. Under the conditions of the assay, the enzymatic hydrolysis can be completed in less than 10 min by the combined action of a microbial lipase and a protease. We have been able to demonstrate complete hydrolysis of triglycerides by thin-layer chromatography of the reaction products, by recovery of glycerol from sera of known triglycerides content, and by comparison of triglyceride assays on a number of sera assayed by our method vs. the AutoAnalyzer procedure. The hydrolysis is directly coupled to the enzymatic determination of glycerol, and is followed through absorbance changes at 340 nm. The assay is simple, rapid, and requires only 50 µl or less of sample. Because the enzymes used do not release glycerol from other compounds in serum, the hydrolysis can be considered specific for triglycerides.
Two new sulfur-containing pyrimidine nucleotides have been isolated from hydrolyzates of Escherichia coli transfer RNA. The structures, 2-thiocytosine and 5-methylaminomethyl-2-thiouracil, have been assigned to the bases as a result of study of ultraviolet and mass spectra. An acid-degradation product, S-methylamino-methyluracil, has been synthesized and is identical to that derived from the natural product.
The cytokinin, N(6)-(Delta(2)-isopentenyl) adenosine occurs in the soluble RNA of yeast and mammalian tissue and has now been detected in plant soluble RNA. A hydroxylated derivative of this cytokinin 6-(cis-4-hydroxy-3-methylbut-2-enylamino)-9-,beta-D-ribofuranosylpurine has also been identified as a constituent of plant soluble RNA.
We describe a direct colorimetric assay for alpha-amylase, with 2-chloro-4-nitrophenyl-alpha-maltotrioside as substrate. Both human pancreatic and salivary amylase split this substrate without the use of helper enzymes, yielding free 2-chloro-4-nitrophenol, which is monitored at 405 nm. The performance of this reagent compares well with that of Pantrak Amylase (Behring Diagnostics) for both serum and urine samples. The reagent is very stable in dry powder form and is stable for one month at 2 to 8 degrees C after reconstitution. Because of the rapid color development and linear kinetics (less than 30 s), the assay is easily automated. Results can be obtained in less than 5 min.
Kinetic and "high-pressure" liquid-chromatographic studies were performed on the reaction of human pancreatic and salivary alpha-amylase (EC 3.2.1.1) with p-nitrophenyloligosaccharides containing four to seven glucose units. The kinetic studies indicate that, as the temperature increases from 25 to 37 degrees C, the apparent Km of the enzyme for a given substrate increases slightly. However, as the number of glucose units in the substrate increases, the apparent Km decreases. The apparent Vmax increases as the temperature increases, and decreases as the number of glucose units in the substrate increases. In contrast, chromatographic data indicate that the relative rate of hydrolysis by alpha-amylase is pG7 greater than pG6 greater than pG5 greater than pG4. The differences between the kinetic and the chromatographic results are discussed. p-Nitrophenyl-alpha-maltopentaoside and p-nitrophenyl-alpha-maltohexaoside are superior to the other p-nitrophenyloligosaccharides as substrates for determining alpha-amylase activity in an alpha-glucosidase-coupled assay system.
Pantrak E.K. (endpoint and kinetic) Amylase reagent (Calbiochem-Behring) is the first commercially available alpha-amylase reagent in which p-nitrophenyl-d-glycosides are used as the substrate. We describe the effect of reagent composition on reagent performance. The reagent performance compares well with that of Amylochrome reagent (Hoffmann-La Roche), Du Pont aca, and Beckman D.S. amylase reagents in assays of sera and urines. We detected no interference from increased concentrations of glucose or pyruvate in the sample. The reagent can be used in either a manual fixed-time or an automatable kinetic assay.
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