Showing papers on "Enzyme assay published in 1981"

Induction of pulmonary indoleamine 2,3-dioxygenase by interferon

Abstract: Pulmonary indoleamine 2,3-dioxygenase [indoleamine: oxygen 2,3-oxidoreductase(decyclizing)] has been found to be induced (30- to 100-fold) in the mouse after a single intraperitoneal administration of bacterial endotoxin [Yoshida, R. & Hayaishi, O. (1978) Proc. Natl. Acad. Sci. USA 75, 3998-4000] or during in vivo virus infection [Yoshida, R., Urade, Y., Tokuda M. & Hayaishi, O. (1979) Proc. Natl. Acad. Sci. USA 76, 4084-4086]. In the present study, an in vitro system with mouse lung slices was developed in which bacterial endotoxin (5 micrograms/ml)produced an induction (approximately 10-fold) of indoleamine 2,3-dioxygenase. The endotoxin was substituted by interferon from mouse L cells or mouse brain. The pulmonary enzyme activity increased almost linearly for 48 hr after addition of mouse interferon (10(4) units/ml) to lung slices. Interferon from mouse L cells or mouse brain produced a 10- to 15-fold increase in the enzyme activity, whereas that from human leukocytes was all but ineffective. The effect also was observed using highly purified L-cell interferon, prepared by poly(U) affinity column chromatography. When interferon was treated either by heat, alpha-chymotrypsin, or anti-interferon serum, such increase in the enzyme activity was diminished essentially to the same extent as seen in the antiviral activity. The increase in the enzyme activity was blocked when actinomycin D or cycloheximide was added to the slices before interferon treatment. These results suggest that the enzyme induction was produced by interferon and not by possible contaminants in the interferon preparations.

Regulation of T cell differentiation: in vitro induction of 20 alpha-hydroxysteroid dehydrogenase in splenic lymphocytes from athymic mice by a unique lymphokine.

Abstract: The enzyme 20 alpha-hydroxysteroid dehydrogenase (20 alpha SDH) has previously been shown to be a specific enzyme marker of mature T cells. In vivo, the expression of 20 alpha SDH is thymus dependent, in that splenic lymphocytes from athymic mice have only low levels of activity, although the levels of enzyme activity increase gradually with age. In vitro, 20 alpha SDH can be induced in splenic lymphocytes from nu/nu mice by conditioned media from mitogen- or alloantigen-stimulated normal lymphocytes. Induction is rapid in vitro. Beginning after a lag period of approximately 6 hr, enzyme activity increases linearly for approximately 20 to 30 hr resulting in a 5- to 10-fold increase in activity. Induction is blocked by mitomycin C, suggesting a requirement for cell proliferation. The phenotype of both the precursor and the induced lymphocyte populations is Thy 1.2-, Lyt 1-, 2-, suggesting that induction of 20 alpha SDH expression is an early step in T cell differentiation. The factor responsible for 20 alpha SDH induction has been partially purified and is distinct from other known lymphokines in both its biochemical and functional properties. The term interleukin 3 is proposed for this factor.

D-fructose dehydrogenase of Gluconobacter industrius: purification, characterization, and application to enzymatic microdetermination of D-fructose.

Abstract: D-Fructose dehydrogenase was solubilized and purified from the membrane fraction of glycerol-grown Gluconobacter industrius IFO 3260 by a procedure involving solubilization of the enzyme with Triton X-100 and subsequent fractionation on diethylaminoethyl-cellulose and hydroxylapatite columns. The purified enzyme was tightly bound to a c-type cytochrome and another peptide existing as a dehydrogenase-cytochrome complex. The purified enzyme was deemed pure by analytical ultracentrifugation as well as by gel filtration on a Sephadex G-200 column. The molecular weight of the enzyme complex was determined to be about 140,000, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of three components having molecular weights of 67,000 (dehydrogenase), 50,800 (cytochrome c), and 19,700 (unknown function). Only D-fructose was readily oxidized by the enzyme in the presence of dyes such as ferricyanide, 2,6-dichlorophenolindophenol, or phenazine methosulfate. Nicotinamide adenine dinucleotide, nicotinamide adenine dinucleotide phosphate, and oxygen did not function as electron acceptors. The optimum pH of D-fructose oxidation was 4.0. The enzyme was stable at pH 4.5 to 6.0 Stability of the purified enzyme was much enhanced by the presence of detergent in the enzyme solution. Removal of detergent from the enzyme solution facilitated the aggregation of the enzyme and caused its inactivation. An apparent Michaelis constant for D-fructose was observed to be 10(-2) M with the purified enzyme. D-Fructose dehydrogenase was shown to be a satisfactory reagent for microdetermination of D-fructose.

Widespread occurrence of calcium-activated, phospholipid-dependent protein kinase in mammalian tissues.

Abstract: Ca2+-activated, phospholipid-dependent multifunctional protein kinase originally found in rat brain occurs in a variety of mammalian tissues. In most tissues the enzyme activity is comparable to that of cyclic AMP-dependent protein kinase when assayed with calf thymus H1 histone as phosphate acceptor. In some tissues such as platelets, brain, and lymphocytes the enzyme far exceeds the cyclic AMP-dependent enzyme. This species of protein kinase found in various tissues shows very similar physical, kinetic, and catalytic properties, and does not appear to show tissue and species specificities. It is conceivable that this protein kinase plays roles in transmembrane control of protein phosphorylation by a large number of extracellular messengers which induce phosphatidylinositol turnover in their target tissues.

The protochlorophyllide holochrome of barley (Hordeum vulgare L.). The effect of light on the NADPH:protochlorophyllide oxidoreductase.

Abstract: During the illumination of etiolated barley plants a rapid decline of the NADPH: protochlorophyllide oxidoreductase is observed. Within the first 5 min of continuous light approximately 90% of the enzyme activity present in dark-grown barley plants disappears and, at the same time, the amount of enzyme protein is diminished by more than 60%. No stable polypeptide fragments have been found which might be formed during the light-induced degradation of the enzyme protein. The rate of enzyme protein synthesis is not drastically affected at the beginning of the illumination period. During the subsequent light-dependent chloroplast development a phytochrome-induced decline in the rate of protein synthesis, concomittant with a continuous light-dependent degradation of the enzyme protein, leads to a progressive decrease of the concentration of the enzyme. After 6 h of continuous light, when the rate of chlorophyll accumulation is at its greatest, only traces of the enzyme protein are visible and the enzyme activity is no longer detectable within the plants. In contrast to previous concepts of chlorophyll biosynthesis in higher plants, our results present evidence that the NADPH: protochlorophyllide oxidoreductase functions only for a short time period after the onset of light.

Purification and characterisation of a membrane-bound substance-P-degrading enzyme from human brain.

Abstract: A membrane-bound enzyme which degrades substance P (an undecapeptide) has been purified from human brain. The properties of this enzyme suggest that it may be involved in the physiological inactivation of the peptide by neural tissues. Enzyme activity was extracted from a membrane fraction of human diencephalon with a non-ionic detergent, Brij 35, and activity was monitored by measuring the disappearance of added substance P using radioimmunoassay, bioassay or radiochemical assay. The enzyme was purified about 1000-fold by chromatography on DEAE-cellulose, hydroxyapatite and Sephadex gel filtration columns. To identify the cleavage sites in substance P, the peptide was incubated with the purified enzyme and the breakdown products were separated by reverse-phase high-performance liquid chromatography and identified by amino acid analysis. The results suggested that the enzyme preparation was functionally homogeneous and it cleaved substance P between Gln6–Phe7, Phe7–Phe8 and Phe8-Gly9, with no exopeptidase action. The enzyme had a pH optimum in the range 7–9 and was strongly inhibited by metal-chelating agents, but not affected by most other peptidase inhibitors; it can thus be classified as a neutral metallo-endopeptidase. The enzyme was thermolabile and had a molecular weight of 40000–50000 as estimated by gel filtration, density-gradient ultracentrifugation and sodium dodecylsulphate gel electrophoresis. The highly purified substance-P-degrading enzyme could be distinguished from previously described peptidases for which substance P is a substrate. An important feature was that substance P was the preferred substrate among various other neuropeptides tested.

Spectrophotometric assay for urinary N-acetyl-beta-D-glucosaminidase activity.

Abstract: An improved assay for N-acetyl-beta-D-glucosaminidase activity in urine is described that involves (a) gel filtration to separate the enzyme from inhibitors in urine, (b) enzymic hydrolysis of p-nitrophenyl-N-acetyl-beta-D-glucosaminide at pH 4.4, and (c) spectrophotometry of the liberated p-nitrophenylate. Measurements of activity of the enzyme in 58 urine specimens correlated closely (r = 0.9998) with results by an established procedure. The within-run coefficient of variation (CV) was 3.7%; the between-run CV averaged 6.8%. Reference values for the activity were established by assays of urine specimens from 135 healthy persons, age two weeks to 52 years. Efficacy of the assay for detection of nephrotoxicity was demonstrated in rats after experimental induction of reversible renal insufficiency by intraperitoneal injection of nickel chloride. Clinical application of the assay in approximately 1000 patients corroborated its utility for detection and monitoring of renal disorders.

Photoregulation of the Carotenoid Biosynthetic Pathway in Albino and White Collar Mutants of Neurospora crassa

Abstract: The conversion of isopentenyl pyrophosphate to phytoene in Neurospora crassa requires both a soluble and a particulate fraction. Soluble and particulate enzyme fractions obtained from light-treated and dark-grown wild type, albino-1, albino-2, albino-3, and white collar-1 strains were mixed in various combinations, and the activity for conversion of [1-(14)C]isopentenyl pyrophosphate to phytoene was assayed. From such experiments it can be concluded that: (a) albino-3 is defective in the soluble fraction; (b) albino-2 is defective in the particulate fraction; (c) the in vivo light treatment increases the enzyme activity in the particulate fraction; (d) this light effect occurs in wild type, albino-1, and albino-3 strains; and (e) enzyme activity is present in the particulate fraction obtained from the white collar-1 mutant, but the in vivo light treatment does not cause an increase in this activity. To measure directly the level of particulate enzyme activity, [(14)C]geranylgeranyl pyrophosphate was used as a substrate. This compound, which is not available commercially, was synthesized enzymically using extracts of pea cotyledons. Particulate enzyme fractions obtained from wild type, albino-1, and albino-3 strains incorporate [(14)C]geranylgeranyl pyrophosphate into phytoene, and this activity is higher in extracts obtained from light-treated cultures. The particulate fraction obtained from the white collar-1 mutant also incorporates [(14)C]geranylgeranyl pyrophosphate into phytoene, but the in vivo light treatment does not cause an increase in this activity. No incorporation occurs when particulate fractions obtained from either dark-grown or light-treated albino-2 cultures are assayed. The soluble enzyme fraction obtained from the albino-3 mutant was shown to be almost totally defective in enzyme activity required for the biosynthesis of [(14)C]geranylgeranyl pyrophosphate from [1-(14)C]isopentenyl pyrophosphate. An in vivo light treatment increases the level of this activity in wild type, albino-1, albino-2, and albino-3 strains, but not in the white collar-1 mutant. A model is presented to account for all of the results obtained in this investigation. It is proposed that the white collar-1 strain is a regulatory mutant blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains are each defective for a different enzyme in the carotenoid biosynthetic pathway.

Production of l-Phenylalanine from trans-Cinnamic Acid with Rhodotorula glutinis Containing l-Phenylalanine Ammonia-Lyase Activity

Abstract: An enzymatic method using l-phenylalanine ammonia-lyase (EC 4.3.1.5) for the rapid conversion of trans-cinnamic acid to l-phenylalanine has been investigated. With Rhodotorula glutinis, enzyme activity as high as 0.3 U/ml of culture broth was obtained. The enzyme activity was kept stable for a relatively long time during cultivation by the addition of l-isoleucine. Optimization of the parameters of the conversion reaction resulted in accumulation of 18 mg of l-phenylalanine per ml of reaction mixture. The conversion yield from trans-cinnamic acid was about 70%. The method may provide a rapid and practical way to produce l-phenylalanine useful as an essential amino acid.

[9] Methods for the preparation of enzyme-antibody conjugates for use in enzyme immunoassay

Abstract: Publisher Summary This chapter describes the methods for the preparation of enzyme–antibody conjugates for use in enzyme immunoassay. Enzyme-linked immunoadsorbent assay type assays are becoming established as the method of choice for screening for antigens derived from, and antibodies directed against, microorganisms. The effect of the cross-linking reaction on the antibody and enzyme activity can be determined by measuring both the activities before and after the conjugation reaction. Methods that have been used to measure the antibody activity include: radial immunodiffusion, reverse-rocket immunoelectrophoresis, and radioimmunoassay. The efficiency of the coupling reaction can be determined by separating the conjugated from the nonconjugated material and measuring the proportion of antibody and enzyme activity in the conjugate. The one-step glutaraldehyde method is a widely used and technically undemanding procedure and many enzyme immunoassays have been successfully developed, using labels prepared by it. Nonlabeled antibody should be removed from the conjugate, as it lowers the sensitivity of the assay. It is found that for those assays, where the enzyme activity is measured on the solid phase, removal of free enzyme from the conjugate is not essential.

Effect of a new angiotensin converting enzyme inhibitor MK 421 and its lysine analogue on the components of the renin system in healthy subjects.

Abstract: 1 MK 421 and its lysine analogue are two new inhibitors of angiotensin converting enzyme. Ten mg of both compounds were each given p.o. to 12 normotensive volunteers to determine their effect on the various components of the renin angiotensin aldosterone system. 2 Plasma converting enzyme activity decreased to very low levels within 3 to 4 h to recover only slowly over the next 72 h. Plasma angiotensin II and aldosterone also fell but returned to baseline within 24 h, whereas plasma renin activity rose reflecting the low angiotensin II levels. 3 There was a close correlation between both angiotensin II and aldosterone levels and the logarithm of plasma converting enzyme activity demonstrating that angiotensin II and aldosterone fell only when converting enzyme activity was reduced to very low levels. 4 Mean hourly urinary sodium excretion increased markedly 6 to 10 h post-drug, while blood pressure decreased slightly. Both drugs were well tolerated. 5 Thus 10 mg of MK 421 or its lysine analogue given orally are effective and long acting angiotensin converting enzyme inhibitors.

Hypoxanthine-guanine phosphoribosyltransferase variants: correlation of clinical phenotype with enzyme activity.

Abstract: A deficiency of the enzyme hypoxanthine-guanine phosphoribosyltransferase(HGPRT) is associated with a varying clinical picture which may include hyperuricaemia, neurological abnormalities and bizarre self-mutilating behaviour. Due to technical problems with the usual in vitro enzyme assays, it has not been possible to establish a correlation between the degree of the enzyme deficiency and the severity of the clinical manifestations. In this study, the HGPRT activity of 12 patients with various clinical features was measured by quantitative analysis of the incorporation of radioactive precursors into purine compounds in intact fibroblasts. The results demonstrate that a correlation between the severity of the clinical symptoms and the degree of the enzyme deficiency as measured in intact fibroblasts does in fact exist.

The Bile-Salt-Stimulated Lipase in Human Milk Purification and Characterization

Abstract: The bile-salt-stimulated lipase was purified from human whey by chromatography on heparin-Sepharose and Affi-Gel blue. The purified enzyme gave a single band with a molecular weight of 90000 on dodecylsulphate/polyacrylamide gels and this band accounted for at least 98% of the protein on the gel. An antiserum to the purified lipase completely inhibited the enzyme activity and gave a single precipitate against human whey and purified lipase. The bile-salt-stimulated lipase was inhibited by diisopropylfluorophosphate, which bound to the purified enzyme in a molar ratio of 0.85 mol/mol. The lipase is a glycoprotein with a high content of acidic amino acid residues and an isoelectric point of around 4. Proline constitutes more than 10% of the total amino acid residues. The purified lipase has a turnover number of around 150 s−1.

Enzymatic hydrolysis of cellulose: Evaluation of cellulase culture filtrates under use conditions

Abstract: Culture filtrates from three mutant strains of Trichoderma reesei grown on lactose and on cellulose were compared under use conditions on four cellulose substrates. Cellulose culture filtrates contained five to six times as much cellulase as lactose culture filtrates. Unconcentrated cellulose culture filtrates produced up to 10% sugar solutions from 15% cellulose in 24 hours. Specific activity in enzyme assays and efficiency in saccharification tests were low for enzymes from all the mutants. Over a wide range the percent saccharification of a substrate in a given time was directly proportional to the logarithm of the ratio of initial concentrations of enzyme and substrate. As a result of this, dilute enzyme is more efficient than concentrated enzyme, but if high sugar concentrations are desired, very large quantities of enzyme are required. Since the slopes of these plots varied, the relative activity of cellulase on different substrates may be affected by enzyme concentration. (Refs. 28).

D-Glucose Dehydrogenase of Gluconobacter suboxydans: Solubilization, Purification and Characterization

Abstract: D-Glucose dehydrogenase was purified to homogeneity from the membrane of Gluconobacter suboxydans IFO 12528. The enzyme was solubilized from the membrane with Triton X-100 and fractionated with SE-cellulose and hydroxyapatite columns. Other species of membrane-bound dehydrogenases which were cosolubilized with D-glucose dehydrogenase were eliminated by treating the enzyme at fairly acidic pH in the initial step of enzyme purification. The purified enzyme was homogeneous in analytical ultracentrifugation and sucrose density gradient centrifugation. The enzyme had an apparent sedimentation constant of 4.2 S and showed a molecular weight of 87,000 by urea-sodium dodecylsulfate gel electrophoresis. The spectral studies with the purified enzyme preparation showed the participation of pyrroloquinoline quinone, a new prosthetic group, in the enzyme. Phenazine methosulfate was found to be the best electron acceptor in D-glucose oxidation with the enzyme. The optimum pH of D-glucose oxidation was at pH 3.0 with po...

Expression of angiotensin-converting enzyme activity in cultured pulmonary artery endothelial cells

Abstract: Angiotensin-converting enzyme (EC 3.4.15.1) is a carboxyterminal dipeptidyl peptidase. The enzyme catalyzes the conversion of the decapeptide angiotensin I to the octapeptide angiotensin II. In addition, the enzyme catabolizes bradykinin. Because of these actions, the enzyme is of pivotal importance in blood pressure homeostasis. Numerous investigators have demonstrated the presence of the enzyme in association with endothelial cells but relatively little is known concerning the factors controlling the expression of enzyme activity by endothelial cells in culture. We have demonstrated that endothelial cells in culture do not express significant amounts of enzyme activity until several days after growth ceases due to high cell density. This is important because it demonstrates a change in function with stage of growth in culture and a possible difference in functional capabilities between nondividing endothelial cells and cells that are dividing in response to injury. Since density-dependent expression of differentiated traits does not appear to be unique to endothelial cells an understanding of the mechanisms underlying this phenomenon may provide a general explanation for the expression of differentiated traits by cultured cells.

Properties of peptidylarginine deiminase from the epidermis of newborn rats.

Abstract: An enzyme which catalyzes the coversion of arginyl residues to citrullyl residues in protein was obtained from the extract of the epidermis of newborn rats. The enzyme required Ca2+ for its activity. The enzyme activity was enhanced in the presence of DTT. The maximum activity was observed at pH 7.5 at 50 degrees C in the presence of 10 mm CaCl2 and 2 mM DTT. The activity was inhibited strongly by treatment of the enzyme with monoiodoacetate or PCMB, which suggests that the epidermal enzyme is an SH-enzyme. The molecular weight of the enzyme was calculated by gel filtration to be about 48,000. It was essential for the alpha-amino or alpha-carboxyl group of the L-arginine substrate to be involved in a peptide linkage. The enzyme showed marked activities towards N-substituted L-arginine derivatives such as BZ-L-Arg, BZ-L-Arg-NH2, and BZ-Gly-L-Arg, But the action of the enzyme on free L-arginine was negligible. The enzyme activity was affected by the nature of the residue neighboring the arginyl residue in proteins. The authors propose the name "peptidylarginine deiminase" for this enzyme. A considerable specificity of the enzyme for proteins from the epidermal cells in terminal differentiation was observed. The results suggest that citrullyl residues in membranous protein of horny cells of the epidermis of newborn rat are formed by the action of epidermal peptidylarginine deiminase.