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Results for “Enzyme Assays”

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At least 19 recordsLinked to original sources
PubMed1976

The metabolism of 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid into cholic: an enzyme assay using homogenates of human liver.

An enzyme assay was developed to measure the conversion of the bile acid precursor, 3alpha, 7alpha, 12alpha-trihydroxy-5beta-cholestan-26-oic acid (THCA), into cholic acid using homogenates of human liver biopsies. The average rate of metabolism of THCA into cholic acid was found to be 3.9 +/- 0.5 (+/- 1 SD) pmoles of cholic acid formed/mg liver/minute in twelve normal liver biopsies. This assay system can be used to determine if the syndrome of neonatal cholestasis associated with a metabolic block in the conversion of THCA into cholic acid is transmitted as a genetic trait.

Adolescent↗
PubMed1979

Urinary enzyme assays in toxicological studies in the rat and marmoset.

The relative merits of the automated, fluorimetric assay of urinary enzymes and cell exfoliation were compared with other commonly used tests for renal damage. Two types of nephrotoxic agent were used, causing crystal nephropathy and acute tubular necrosis respectively. Groups of marmosets were given one of two drugs known to cause crystal nephropathy. One agent caused intermittent increases in urinary enzyme excretion and an early increase in cell excretion which was not sustained. The second agent in contrast caused elevated cell and enzyme excretion, increasing throughout the period of administration. A nephrotoxic anti-tumour agent also caused increases in cell and enzyme excretion when given to marmosets. The early changes produced by this agent were studied using catheterised rats. Hourly samples of urine were collected and urinary beta-glycosidase excretion was found to give an early indication of renal damage, which correlated with albuminuria and glycosuria. The fluorimetric assay of urinary enzymes provides a sensitive, non-invasive test of nephrotoxicity.

Acetylglucosaminidase↗
PubMed1976

Fluoride inhibition of inorganic pyrophosphatase. I. Kinetic studies in a Mg2+-PPi system using a new continuous enzyme assay.

Reversible inhibition of bakers' yeast inorganic pyrophosphatase (EC 3.6.1.1) by fluoride has been studied as a function of substrate, metal-ion activator and inhibitor concentrations and pH using a new continuous enzyme assay with an automatic phosphate analyzer. The inhibition was shown to be the result of tight binding of fluoride by two catalytically active enzyme-substrate complexes. The reaction between pyrophosphatase and fluoride is relatively slow, so that the rate constants for the binding and release of the inhibitor were derived from phosphate formation curves measured on the time scale of enzyme assays. The pH-dependence of the inhibition reaction in the alkaline medium indicates that both the fluoride-enzyme interaction and the catalytic step of the pyrophosphatase reaction are controlled by the same group on the protein. In the acidic medium, the inhibition is considerably enhanced, presumably because of the protonation of another enzyme group.

Autoanalysis↗
PubMed1977

Enzymic assays for isomers of 2,6-diaminopimelic acid in walls of Bacillus cereus and Bacillus megaterium.

An enzymic assay for individual isomers (meso-, LL- and DD-) of 2,6-diaminopimelate was developed. The enzyme 2,6-diaminopimelate decarboxylase specifically attacked meso-diaminopimelate and was used to measure this isomer manometrically. The meso- and LL-isomers were measured together manometrically in a coupled assay with diaminopimelate decarboxylase and diaminopimelate epimerase (which converts LL-diaminopimelate into meso-diaminopimelate). The DD-isomer was not attacked by either enzyme and was measured, as residual diaminopimelate after the coupled assay, by a colorimetric method, which was also used to measure total diaminopimelate before enzymic treatments. The coupled enzymes were also used to prepare pure DD-isomer from chemically synthesized diaminopimelate. A mixture of diaminopimelate isomers was present in walls of four strains of Bacillus megaterium [in each about 75% (w/w) meso-, 18% LL- and 7% DD-] and in walls of two strains of Bacillus cereus (about 85% meso-, 8% LL- and 7% DD-). One strain of B. cereus contained at least 95% meso-diaminopimelate, with only traces of LL- and DD-isomers. Peptidoglycan from Escherichia coli was assayed as containing at least 95% meso-isomer. The proportion of isomers in the wall of a strain of B. megaterium remained constant after growth in a variety of different media.

Amino Acid Isomerases↗
PubMed1979

Enzymic assay of total cholesterol involving chemical or enzymic hydrolysis--a comparison of methods.

Manual procedures for estimating serum total cholesterol by use of cholesterol oxidase (EC 1.1.3.6) and the phenol--aminophenazone--peroxidase chromogenic system are described, in which cholesteryl esters are hydrolyzed either by use of pancreatic cholesterol ester hydrolase (EC 3.1.1.13) or saponification by ethanolic potassium hydroxide. Both methods are linear up to a cholesterol concentration of 12 mmol/L and are reproducible (between-run CV, about 1.1%). The chemical hydrolysis method yields results that are about 10% lower than those obtained by enzymic hydrolysis, because of incomplete removal of interfering thiols generated during the saponification of serum. The chemical hydrolysis procedure is much less susceptible to interferences, particularly by bilirubin, but the enzymic hydrolysis system is simpler to perform and therefore has a greater potential for mechanization.

Cholesterol↗
PubMed1979

Enzymic assay of plasma cholesterol: a comparison of analytical variations found using the Greiner GSA II and the Technicon SMA 12/60 and SMA II.

Cholesterol was assayed on the Greiner GSA II and the Technicon SMA 12/60 and SMA II autoanalyzers, with Allain's entirely enzymic method (Allain et al. (1974), Clin. Chem. 20, 470--475). The coefficients of variation in the results over a period of one month were less than 6% with the GSA II and the SMA II, but up to 9% on the SMA 12/60. Although the accuracy of the enzymic method depends on the specificity of the cholesterol oxidase and cholesterol esterase, the results were in good agreement with those obtained by Abell's method (Abell et al. (1952), J. Biol. Chem. 195, 357--366) on fresh or freeze dried human plasma. The cholesterol concentration was stable at 4 degrees C, or in frozen or freeze dried plasma. None of the drugs tested interfered with the enzymic assay.

Autoanalysis↗
PubMed1975

Effect of enzymic assay conditions on sulfite reduction catalysed by desulfoviridin from Desulfovibrio gigas.

The type and the amount of end products resulting from sulfite reduction catalysed by a single partially purified desulfoviridin preparation from Desulfovibrio gigas were shown to depend upon the enzymic assay conditions employed. Both manometric and spectrophotometric assays were used, with reduced methyl viologen serving as the electron donor in each system. Trithionate, thiosulfate, tetrathionate and sulfide were identified as possible end products. In the manometric assays, sulfide production was favoured by high reduced methyl viologen concentrations, low sulfite concentrations and a pH value of 7.0 as opposed to 6.0. In the spectrophotometric assays, results approaching the stoichiometric conversion of sulfite to sulfide were obtained only at high initial reduced methyl viologen concentrations.

Desulfovibrio↗
PubMed1976

A new look at the measurement and interpretation of enzyme assays.

Some methodological problems in clinical enzymology, including instability of enzymes in the incubation mixture and requirements for optimal reaction conditions, are highlighted. The importance of a knowledge of fundamental enzyme biochemistry and physiology as the basis for their diagnostic application is stressed, and the different behaviour of some hepatic enzymes--namely, GOT, GPT, gamma-GT, and OCT, in various pathological conditions is traced back to their characteristic biochemical and physiological properties. In the field of urinary enzymes a knowledge of the ideal requirements for the enzyme investigation of the various renal functions and of the properties of potentially valuable enzymes permits a critical selection of the really useful ones.

Alanine Transaminase↗
PubMed1977

The conjugation of testosterone with horseradish peroxidase and a sensitive enzyme assay for the conjugate.

The formation of a horseradish peroxidase-testosterone conjugate for the enzyme-linked immunoassay of testosterone was investigated, using tritiated testosterone to follow the reaction. The formation of testosterone-3-(carboxymethyl) oxime-peroxidase by the mixed anhydride method was found to give a conjugate of high enzymatic activity and with three molecules of testosterone per molecule of peroxidase. The optimum conditions for the assay of peroxidase activity were studied and an assay capable of measuring 1 to 5 ng of the conjugate developed; the standard curve being virtually linear. The stability of the conjugate in solution and the effect of lyophilisation on enzymatic activity are also described. The peroxidase-testosterone conjugate was suitable for enzyme-linked immunoassay and the quantities measurable with the peroxidase assay covered the range necessary for a plasma testosterone assay. The stability of the conjugate was such that no particular precautions were necessary for its storage.

Aminosalicylic Acids↗
PubMed1977

Differential stabilities of soil enzymes. Assay and properties of phosphatase and arylsulphatase.

Methods have been refined for the assay of phosphatase and arylsulphatase activities in soil, based on the chromogenic p-nitrophenyl ester substrates. Basic assay conditions have been defined, and pH optima and kinetic parameters have been determined. The enzymes follow Michaelis-Menten kinetics; this conclusion is based on three methods of analysis of data determined over a wide range of substrate concentrations. The enzyme activities are very stable to storage of wet soil for up to 4 weeks at soil temperatures and above. For example, phosphatase had a half-life of approximately 2 weeks at 50 degrees C; arylsulphatase was rather less stable. Both enzymes retained 80% of activity after incubation with pronase for 1 week at 25 degrees C. On the basis of this work and studies on other soil enzymes, it is concluded that remarkable stability is a general feature of soil enzymes.

Arylsulfatases↗
PubMed1975

Radio-enzymic assay of oxo steroids by their reduction with the tritiated form of reduced nicotinamide--adenine dinucleotide.

A method of analysis is proposed based on the enzyme-catalysed transfer of tritium from [3H]NADH to suitable substrates. Its practicability is demonstrated on the examples of oestrone and progesterone with the respective use of the 3 beta, 17 beta- and 3 alpha, 20 beta-hydroxysteroid oxidoreductase. Specificity is tested by application to the analysis of the plasma of pregnant women and measurement of the 3H/14C ratios on purification of the enzymic reduction products.

Estrone↗
PubMed1975

Hormone and enzyme assays in pregnancy. V. A rapid method for measuring the placental cystine-aminopeptidase using 1-cystine-bis-1-cystine-bis-p-nitroanilide-nitroanilide as substrate.

A rapid and simple method for determination of the placental cystine-aminopeptidase (P-CAP) activity in plasma is presented. The enzyme-catalysed hydrolysis of the substrate, 1-cystine-bis-p-nitroanilide, is followed in a spectrophotometer by reading the absorbance of the product p-nitroaniline at 380 nm. The absorbance increases linearily with time after a lag-period of 30 seconds to 5 min. The reaction is followed for about 10 min and the increase in absorbtion per min is calculated from the linear part of the absorbtion curve. The test could also be performed by taking 2 readings at about 5 min intervals. In the studies of the enzyme kinetics a competitive inhibitor of the reaction was found in plasma. The amount of the inhibitory substances seemed to be nearly constant in the plasma samples studied from normal pregnant women as well as in plasma samples with high enzyme activity (twin-pregnancy) and low enzyme activity (severe pre-eclampsia). The normal enzyme activity pattern showed an increase from about 210 days of pregnancy and towards the term. A coefficient of correlation of plus 0.83 with the more time consuming method using 1-cystine-di-beta-naphtylamide as substrate was found. It was concluded that the present method could replace the method described by Babuna & Yenen (1966) at least from about 210 days of pregnancy and where a rapid answer is necessary.

Adult↗