Release Of P-nitroaniline Research Articles

A New Method to Assay Des-γ-carboxyprothrombin: Results Obtained in 75 Cases of Hepatocellular Carcinoma

A new method for assaying the activity of des-gamma-carboxyprothrombin (DCP), using staphylocoagulase on undiluted adsorbed plasma, is described. The thrombin-coagulase formed is measured on a chromogenic substrate, and the results are expressed in milliunits per milliliter of increment of the optical density following the release of p-nitroaniline. Levels of DCP in 96 normal subjects were under 10 mU/ml (mean, 3.58 mU/ml). Of 70 nonhepatectomized patients with hepatocellular carcinomas, 74% had increased DCP levels of between 20 and 420 mU/ml (most of the values were between 20 and 100 mU/ml). Des-carboxyprothrombin and alpha-fetoprotein measurements gave complementary information, one marker or the other being positive in 87% of hepatocellular carcinoma. Fourteen of 15 patients with metastatic carcinoma of the liver had normal DCP levels, as did 95 patients with liver cirrhosis and 13 patients with chronic hepatitis. When the level of "total factor II" is below 40%, it is recommended that a second determination of DCP be performed 5 days after the injection of vitamin K, to exclude any vitamin K deficiency (in the case of hepatocellular carcinoma the DCP level will remain elevated). The DCP assay appears more sensitive and more specific than the alpha-fetoprotein assay for the diagnosis of hepatocellular carcinoma; furthermore, both tests are complementary.

1109 BLOOD ENDOTOXIN DETERMINATIONS IN THE EVALUATION OF FEBRILE NEUTROPENIC PATIENTS

An increasing number of febrile episodes in neutropenic patients are never explained etiologically. As a result many such episodes are managed by purely empiric therapy. We wished to explore the hypothesis that some of these episodes might be explained by endotoxemia in the absence of bacteremia. To test this possibility we employed a highly sensitive endo toxin assay which is based on the chromogenic release of p-nitroaniline from a peptide-linked form in the presence of endotoxin (ET) and limulus lysate. This assay, as developed by Seikagaku Kogyo Co, Tokyo, is capable of detecting circulating ET in pg/ml concentrations. We found that circulating ET was not detectable in normal patients. By contrast circulating ET in concentrations ranging from 20–200 pg/ml was found in neutropenic patients suffering febrile episodes. Endotoxemia was found in the absence of bacteremia and, on occasion, the detection of endotoxemia preceeded the onset of demonstrable gram negative septicemia by several days. We believe that the sensitive detection of circulating ET can be a valuable adjunct in the assessment of the febrile neutropenic patient, and we are currently extending our studies.

Stability in rat plasma and serum of lysosomally degradable oligopeptide sequences in N-(2-hydroxypropyl) methacrylamide copolymers

Soluble copolymers of -(2-hydroxypropyl)methacrylamide (HPMA) were prepared containing either oligopeptide side chains terminating in p-nitroaniline, or oligopeptide sequences forming crosslinks between polymer chains. Such copolymers have potential as targetable drug carriers and already it has been shown that oligopeptide side chains and oligopeptide crosslinks are degraded intracellularly by lysosomal enzymes. The susceptibility of these oligopeptide sequences to degradation on incubation with rat plasma or rat serum was evaluated by monitoring either the liberation of p-nitroaniline or, with the crosslinked polymers, the change in molecular weight distribution. Release of p-nitroaniline from some of the polymers was not detectable, and from others proceeded very slowly, the maximum rate being from the side chain Gly-Gly-Phe-Leu-Glv-Phe-NAp where 5.1% of the bound p-nitroaniline was released by rat serum over a 5 h incubation period. No cleavage of crosslinked HPMA copolymers by plasma or serum was detectable even after a 24 h incubation period.

Purification and properties of bovine liver gamma-glutamyltransferase.

gamma-Glutamyltransferase [EC 2.3.2.2] (gamma-GTP) was purified to a homogeneous state from bovine liver. It had an apparent molecular weight of about 110,000, as judged by polyacrylamide gel electrophoresis, and consisted of two non-identical glycopeptides with molecular weights of 68,000 and 27,000. The amino acid composition of the bovine liver enzyme was similar to those of other gamma-GTPs. The enzyme contained large amounts of neutral sugars, amino sugars and sialic acid, although the sialic acid content varied in different preparations. The Michaelis constant of the enzyme was estimated to be 0.8 mM for gamma-L-glutamyl-p-nitroanilide in the presence of glycylglycine and 1.25 mM in the absence of glycylglycine. Glutathione competitively inhibited the release of p-nitroaniline from gamma-L-glutamyl-p-nitroanilide with a Ki value of 0.3 mM. The specific activities of the enzyme for gamma-L-glutamyl-p-nitroanilide in the presence of glycylglycine (pH 8.6) and for glutathione, a natural substrate (pH 7.4), were comparable to those reported for gamma-GTPs from other mammalian sources. The bovine liver enzyme showed the same gamma-glutamyl group acceptor specificity as other gamma-GTPs from normal mammalian tissues. The phosphate-independent glutaminase activity of the enzyme was much lower than that of the rat kidney enzyme both in the presence and absence of maleate.

Chromogenic substrate assays: mechanism, specificity and application in blood coagulation and fibrinolysis.

SummaryA new generation of specific and sensitive substrates for enzymes has been developed; they consist of a peptide moiety and a chromophoric leaving group, usually p-nitroanilide. The rate of release of p-nitroaniline from this type of substrate is proportional to the enzymatic activity, and can be measured spectrophotometrically from the increase in the absorbancy at 405 nm per unit of time. The specificity of these chromogenic substrates is determined by the nature and the sequence of their amino acids. Possible applications of these substrates in the field of blood coagulation and fibrinolysis include the monitoring of heparin therapy, oral anticoagulant therapy and thrombolytic therapy. The clinical value of a number of these chromogenic substrate assays is still under investigation.

Activated Clotting Factors in Factor IX Concentrates

AbstractThe precise quantitation of activated factors in human factor IX concentrates has been accomplished with the use of recently developed, specific assays for factors IXa, Xa, and thrombin. The assay for factor IXa, which measures the initial rate of 3H-factor-X activation, was shown to be specific for factor IXa in the concentrates. Activated factor IX concentrates contained 1.0–2.3 microgram/ml of factor IXa; whereas the assays of unactivated concentrates were negative (less than 0.2 microgram/ml). The assays of factor Xa and thrombin, which measure the initial rate of p-nitroaniline release from S-2222 and S-2238, respectively, showed similar small amounts of factor Xa (4–34 ng/ml) and thrombin (12–76 ng/ml) in the activated and unactivated concentrates. The nonactivated partial thromboplastin time of the concentrates correlated significantly with the factor IXa content, but not with factor Xa or thrombin. Antithrombin III antigen in 3 of 4 concentrates was several-fold higher than antithrombin III activity, suggesting the presence of antithrombin III complexed with activated factors. These results support the hypothesis that the degree of activation of factor IX concentrates is related primarily to the concentration of factor IXa, which may be responsible for the thrombogenicity of these concentrates in some clinical settings.

Activated clotting factors in factor IX concentrates

The precise quantitation of activated factors in human factor IX concentrates has been accomplished with the use of recently developed, specific assays for factors IXa, Xa, and thrombin. The assay for factor IXa, which measures the initial rate of 3H-factor-X activation, was shown to be specific for factor IXa in the concentrates. Activated factor IX concentrates contained 1.0-2.3 microgram/ml of factor IXa; whereas the assays of unactivated concentrates were negative (less than 0.2 microgram/ml). The assays of factor Xa and thrombin, which measure the initial rate of p-nitroaniline release from S-2222 and S-2238, respectively, showed similar small amounts of factor Xa (4-34 ng/ml) and thrombin (12-76 ng/ml) in the activated and unactivated concentrates. The nonactivated partial thromboplastin time of the concentrates correlated significantly with the factor IXa content, but not with factor Xa or thrombin. Antithrombin III antigen in 3 of 4 concentrates was several-fold higher than antithrombin III activity, suggesting the presence of antithrombin III complexed with activated factors. These results support the hypothesis that the degree of activation of factor IX concentrates is related primarily to the concentration of factor IXa, which may be responsible for the thrombogenicity of these concentrates in some clinical settings.

Interaction of glutathione analogues with Hydra attenuata gamma-glutamyltransferase.

gamma-Glutamyltransferase activity was studied in extracts of the cnidarian Hydra attenuata. The binding of gamma-glutamyl peptide analogues to the enzyme was studied by observing their effects on heat denaturation and their inhibition of p-nitroaniline release from gamma-glutamyl p-nitroanilide. Neither position-1 analogues, in which the gamma-glutamyl moiety was changed to a beta-aspartyl (beta-Asp-Abu-Gly) or an alpha-glutamyl (Glu-Abu-Gly) linkage, nor glutamate protected the enzyme against inactivation at 58 degrees C. GSH (reduced glutathione), gamma-Glu-Abu-Gly and gamma-Glu-Met on the other hand did prevent heat denaturation. GSH and analogues of GSH were competitive inhibitors of p-nitroaniline release, but those analogues in which glycine was replaced by 2-aminoisobutyrate, phenylalanine, leucine or tyrosine had Ki values that were approximately five times those of analogues with the cysteine residue replaced.

Gamma-Glutamyl Transpeptidase in Rat Ascites Tumor Cell LY-5. Lack of Functional Correlation of Its Catalytic Activity with the Amino Acid Transport

gamma-Glutamyl transpeptidase activity was detected in rat ascites tumor cells (LY-5) suspended in Hanks' balanced saline solution using L-gamma-glutamyl-p-nitroanilide as a substrate. Whole-cell suspension of the tumor cells exhibited full activity of the enzyme without detectable cell disruption under the conditions examined. Various amino acids, transported through specific membrane carriers, did not affect the accessibility of substrate for the enzyme. An inhibitor of sodium-dependent transport systems of amino acids caused no significant change in the rate of enzyme catalysis. Like glutathione or S-methylglutathione, S-acetyldextran (mol. wt 215000) derivative of glutathione, which is believed to be unable to penetrate into intact cells, caused marked inhibition of the rate of p-nitroaniline release from the synthetic substrate by the tumor cells. These data indicated that the active site of the enzyme faced to the outer surface of the cells. gamma-Glutamyl transpeptidase of the tumor cells was successfully affinity-labeled by 6-diazo-5-oxo-L-norleucine, a glutamine analog, without causing detectable change in the viability of the cells under the conditions examined. The rate of transport of alanine, leucine, glycine and glutamine into cells was not affected by the inactivation of this enzyme with the affinity label. Thus, the activity of gamma-glutamyl transpeptidase located on the outer surface of tumor cell membrane does not seem to be requisite for the transport process of amino acids.

γ-Glutamyl transpeptidase in hydra

γ-Glutamyl transpeptidase (EC 2.3.2.2) activity is described in the coelenterate, Hydra attenuata , using the substrate γ-glutamyl-p-nitroanilide. The properties of the γ-glutamyl donor required for binding to the transpeptidase were investigated by measuring the ability of GSH analogs to inhibit the release of p-nitroaniline. Whereas no binding was observed when the γ-glutamyl moiety was altered, analogs with substitution in the Cys residue were capable of binding to the enzyme. A specificity for the Gly residue was indicated because analogs containing Leu or Tyr in place of Gly exhibited decreased binding capacities for the hydra transpeptidase. A comparison of these data with those obtained using the same analogs in the GSH induced feeding response bioassay shows that γ-glutamyl transpeptidase activity and the GSH receptor for the hydra feeding response have different specificities.

Role of γ-Glutamyl Transpeptidase Activity in the Diagnosis of Hepatobiliary Disease

The enzyme gamma-glutamyl transpeptidase is widely distributed throughout the body, notably kidney, seminal vesicles, pancreas, liver, spleen and brain. Being one of the enzymes of the gamma-glutamyl cycle, it is involved in aminoacid transport, catalysing a transpeptidation reaction between gamma-glutamyl peptides and most common amino acids. Methods of assay of the enzyme are based on its ability also to act on synthetic amides of glutamic acid; kinetic methods monitoring the release of p-nitroaniline from the substrate L-gamma-glutamyl p-nitroanilide are the most satisfactory. In diseases of the liver, the highest levels occur in association with cirrhosis, alcoholism, hepatic secondaries and cholestasis. As the enzyme is present in the endoplasmic reticulum of the hepatocyte, its activity is increased in situations leading to microsomal enzyme induction. Raised levels can also occur in pancreatitis, diabetes, myocardial infarction, congestive cardiac failure, chronic renal failure, cerebrovascular accidents, cerebral tumours and chronic obstructive pulmonary disease. Although the lack of specificity must be recognised, the estimation can be useful in the elucidation of some clearly defined problems arising during investigation of patients with suspected hepatic disease, especially where performed as part of a biochemical profile.

Properties of .GAMMA.-glutamyltransferase from Lentinus edodes.

An enzyme preparation catalyzing p-nitroaniline release from γ-glutamyl-p-nitroanilide was obtained in a 200-fold purified state from fruit bodies of an edible mushroom, Lentinus edodes. Analysis of the final preparation by differential centrifugation revealed that the enzyme was still bound with subcellular particles. The enzyme catalyzed both the hydrolysis and transfer of the γ-glutamyl moiety from γ-glutamyl-p-nitroanilide, but exhibited essentially no activity of glutaminase, glutamine aminotransferase, glutamine synthetase or γ-glutamyl cyclotransferase. With γ-glutamyl-p-nitroanilide the activity was maximal at about pH 7.6. The enzyme activity increased with an increasing concentration of Tris-HCl buffer, but not with phosphate buffer which was inhibitory. An apparent Michaelis constant of 4 mm was obtained in 0.5 m Tris-HCl buffer at pH 7.6. S-Alkylcysteine sulfoxide served as the best glutamyl acceptor. A serine-borate mixture, pCMB, Cu2+, Hg2+ and Zn2+ were potent inhibitors. All the experiment...

Enzymatic determination of zinc below one part per billion

A stable and very sensitive reagent for the determination of zinc is obtained by removing zinc from pig kidney aminopeptidase, a commercially available metalloenzyme. Up to a given limit, the enzymatic activity of the reagent is strictly proportional to the concentration of zinc ions in the assay system. Aminopeptidase activity is determined by measuring the rate of release of p-nitroaniline from the chromogenic substrate L-leucine- p-nitroanilide. Thus, with a simple recording photometer, rapid and accurate determinations of free zinc ions in concentrations ranging from 5 pg-10 μg ml -1 can be achieved. The selectivity of the method is such that 0.1 ng Zn ml -1 can be assayed in the presence of a 10–100-fold molar amounts of a wide array of other cations with a relative error below 10%. The analytical procedure has been extended to the assay of Cu 2+, Co 2+ and Ni 2+ in the ng ml -1 range.