Showing papers on "Enzyme assay published in 1980"

Purification and Characterization of Tyrosine Hydroxylase from a Clonal Pheochromocytoma Cell Line

Abstract: Tyrosine hydroxylase (TH) was purified from pheochromocytoma PC-12 cloned cells by a short and gentle procedure. The enzyme was isolated in pure form and has a molecular weight of approximately 210,000-220,000. SDS electrophoresis yields one single protein band with a molecular weight of approximately 62,000. Antiserum to rat pheochromocytoma TH was obtained in rabbits and immunotitration data show that the antiserum to rat TH reduces the activity of the homologous enzyme more effectively than the activity of the heterologous enzyme. The activity of purified TH is stimulated by a cAMP-dependent protein kinase phosphorylating system (PKP system), and the highest percentage of stimulation is obtained when the enzyme activity is measured at physiological pH’s. The stimulation of the purified enzyme by the PKP system results in a reduction of the apparent Km for the cofactor 6-MePH4 and in an increase of the Ki for dopamine. Incubation of purified TH with the PKP system and [32P]ATP, resulted in incorporation of radioactivity into the 62,000 subunit of the enzyme.

Oxidation of Methanol by the Yeast, Pichia pastoris. Purification and Properties of the Alcohol Oxidase

Abstract: The enzymes of methanol oxidation were investigated in a new yeast strain, Pichia pastoris IFP 206, with high yield (0.42 g cell per g of methanol). The following enzymes were detected in cell free extracts of P. pastoris: alcohol oxidase, catalase, formaldehyde and formate dehydrogenases. The alcohol oxidase was purified from cell free extracts of P. pastoris containing high amount of the enzyme (33%) with a good yield (55%). The preparation was homogenous by immunochemical methods. The enzyme had a molecular weight of 675,000 and was composed of eight identical subunits of M.W. 80,000. Each subunit contained one FAD. The N-terminal sequence was found to be: Ala-Ile-Pro-Glu-Glu-Phe-Asp-Ile-Leu-Val-Leu-Gly-The protein had 65 free −SH groups per molecule. The optimum temperature for the enzyme activity was 37°C and the activation energy was 11.1 kcal/mol. Optimum pH was 7.5 and the enzyme activity was unstable at acidic pH. The apparent Km for methanol were 1.4 and 3.1 mm at oxygen concentrations of 0.19 a...

A new one-step method for the cytochemical localization of ouabain-sensitive, potassium-dependent p-nitrophenylphosphatase activity.

Abstract: A new one-step method for the light and electron microscopic localization of the ouabain-sensitive, K-dependent p-nitrophenylphosphatase (K-NPPase) activity of the Na-K-ATPase complex is introduced. The incubation medium contains p-nitrophenylphosphate (NPP) as substrate, lead citrate as the capture reagent, and dimethylsulfoxide (DMSO) as an activator. It is usable at the optimal pH of the K-NPPase, which is about pH 9.0 in the presence of 25% of DMSO. The effects of fixation, lead concentration, and DMSO on the enzyme activity were studied using rat kidney as a test tissue. The fixation of tissues in a mixture of 2% paraformaldehyde and 0.5% glutaraldehyde for 60 min at 0 degrees--4 degrees C preserved 45% of the enzyme activity. In the absence of DMSO, lead citrate (4.0 mM) caused 82% inhibition of the enzyme activity in fixed tissue. However, the addition of DMSO (25%) caused about 3-fold activation of the remaining activity. Cytochemical demonstration of the ouabain-sensitive K-NPPase activity was successfully made by this method at both light and electron microscopic levels.

Serum angiotensin-converting enzyme activity in evaluating the clinical course of sarcoidosis.

Abstract: Thirty-five patients with sarcoidosis were serially monitored for serum angiotensin-converting enzyme activity by a simple radiochemical assay, and we analyzed the relation of such activit...

Allosteric inhibition of the Ca2+-activated hydrophilic channel of the mitochondrial inner membrane by nucleotides

Abstract: The control by nucleotides of the Ca2+ -activated channel which regulates the nonspecific permeability of the mitochondrial inner membrane has been investigated quantitatively. The cooperative binding of two molecules of ADP to the internal (matrix) side of the channel causes a mixed-type inhibition of channel activity. ATP, AMP, cAMP and GDP are all ineffective. NADH shows a pattern of inhibition similar to that of ADP, though the apparent KI is higher by a factor of 200. NADPH relieves the inhibition by NADH. NAD+ also inhibits, b,t its affinity is a factor of 10 less than that of NADH. When ADP and NADH are added together, they act synergistically to inhibit the Ca2+-activated channel. It is concluded that the concept of the modification of enzyme activity by the allosteric binding of nucleotides, which is well established for soluble enzyme systems, also has application to the regulation of channels that control membrane permeability.

Properties of Mitochondrial and Peroxisomal Enoyl-CoA Hydratases from Rat Liver

Abstract: Mitochondrial and peroxisomal enoyl-CoA hydratases were purified from rat liver. The mitochondrial enzyme, with a molecular weight of 161,000, was composed of 6 identical subunits. The molecular structure of the rat liver enzyme was very similar to that of the bovine liver enzyme. Acetoacetyl-CoA was a competitive inhibitor of the mitochondrial enzymes. The results of titration of the rat liver enzyme with acetoacetyl-CoA suggest that 3 subunits of the enzyme exhibit catalytic activity. The catalytic properties of the enzyme were studied. The peroxisomal enzyme was composed of one polypeptide with a molecular weight of 70,000-81,000. Some of the enzyme molecules were shown to be cleaved to two polypeptides in the cell by the following methods: amino acid analysis, peptide mapping and immunoprecipitin reaction. The catalytic properties of the peroxisomal enzyme were different from those of the mitochondrial enzyme. The peroxisomal enzyme is a bifunctional enzyme exhibiting 3-hydroxyacyl-CoA dehydrogenase activity. Studies on the titration with acetoacetyl-CoA, the effects of salts, SH titration and proteolytic inactivation suggest that the active centers for these two reactions are located at different sites.

Discrepancy between antihypertensive effect and angiotensin converting enzyme inhibition by captopril.

Abstract: Captopril, an inhibitor of angiotensin converting enzyme, was administered twice daily to 13 hypertensive patients for a mean period of 9 weeks. Continuous blood pressure control in the ambulatory patients was established with a portable blood pressure recorder. Notwithstanding, in eight patients with normal renal function, plasma converting enzyme was found to resume normal activity before administration of the morning dose of captopril. Only in 5 patients with impaired renal function did some blockade of plasma converting enzyme persist for more than 12 hours. Measured plasma converting enzyme activity seemed to reflect total conversion of angiotensin I, including conversion in the pulmonary vascular bed, since changes in its activity were closely paralled by changes in plasma aldosterone levels. Bradykinin accumulation seems unlikely when converting enzyme and thus, presumably, kininase II has resumed normal activity. Captopril administration does not seem to alter plasma epinephrine or norepinephrine levels. Blood pressure reduction in the face of normal angiotensin converting enzyme activity is probably due to hyporesponsiveness of the arterioles to pressor hormones, which may be due to specific renin-related and/or nonspecific effects of captopril.

Synthesis and biological activity of a ketomethylene analogue of a tripeptide inhibitor of angiotensin converting enzyme.

Abstract: An analogue of a tripeptide inhibitor of angiotensin converting enzyme, Bz-Phe-Gly-Pro, has been synthesized in which the amide bond connecting phenylalanine and glycine has been replaced by a ketomethylene group. This nonpeptide analogue, 20, shows more potent converting enzyme inhibiting activity, I50 = 0.07 microM, than Bz-Phe-Gly-Pro, I50 = 9.4 microM, or than the orally active D-3-mercapto-2-methylpropanoyl-L-proline (captopril, 1), I50 = 0.30 microM. Compound 20 has a Ki of 1.06 X 10(-7) and either competitive or noncompetitive enzyme kinetics depending on what substrate is used in the converting enzyme assay. In tests for inhibition of angiotensin I induced contractions in the guinea pig ileum, 20 has one-tenth the activity of 1.

Mitochondrial carbonic anhydrase

Abstract: We have assayed carbonic anhydrase activity (carbonate dehydratase, carbonate hydro-lyase, EC 4.2.1.1) and bicarbonate permeability in suspensions of broken and intact guinea pig mitochondria by monitoring the disappearance of C16O18O. We found significant activity in preparations from liver and skeletal muscle, but not in preparations from heart muscle, brain, and kidney. Intact mitochondria containing carbonic anhydrase produce a two-phase acceleration of the disappearance of the labeled CO2, which indicates that the enzyme is located in a region more accessible to CO2 than to HCO3-. Acetazolamide inhibits the enzyme activity instantly in broken mitochondria but only after a delay in intact mitochondria, indicating that the enzyme is in a region not immediately accessible to the inhibitor. Sonication of mitochondria containing carbonic anhydrase activity releases the enzyme, which remains in the supernatant after sedimentation of the submitochondrial particles. This shows that mitochondrial carbonic anhydrase is in the matrix compartment and not in, or bound to, the inner membrane. The activity of the enzyme increases markedly with increasing pH. The enzyme activity of intact mitochondria is greater than that of the broken mitochondria at the same pH of the suspending fluid, corresponding to an intramitochondrial pH that is 0.2-0.5 unit more alkaline.

Photorespiration mutants of Arabidopsis thaliana deficient in serine-glyoxylate aminotransferase activity.

Abstract: Three mutants of the crucifer Arabidopsis thaliana (Linnaeus) Heynhold were isolated that are completely lacking in activity catalyzed by serine-glyoxylate aminotransferase (EC 2.6.1.45), a peroxisomal enzyme involved in photorespiratory carbon metabolism. These mutants were viable and exhibited normal photosynthesis under conditions that suppressed photorespiration, but they were inviable and photosynthesized at greatly reduced rates under conditions that promoted photorespiration. Serine and glycine accumulated as end products of photosynthesis in the mutants, mostly at the expense of starch and sucrose. The mutants are allelic, and the segregation patterns of plant viability, photosynthetic activity, and enzyme activity in the F1 and F2 generations indicated that all the observed effects were caused by a single recessive nuclear mutation. This conclusion was confirmed by the isolation of seven revertants in which viability, photosynthetic capacity, and enzyme activity were simultaneously restored. Mutants of the type described here, in which photorespiration is changed from a merely wasteful process into one that is lethal, may permit the direct selection of secondary mutations that reduce photorespiration.

Changes in prolyl endopeptidase during maturation of rat brain and hydrolysis of substance P by the purified enzyme.

Abstract: We determined changes in prolyl endopeptidase activity in developing rat brain. A new and highly sensitive fluorogenic substrate, 7-(succinyl-Gly-Pro)-4-methylcoumarinamide, was used for determination of the enzyme activity. The enzyme activity per brain increased until 2 weeks of age, and then decreased during maturation. The enzyme was purified about 7800-fold from the brain of the rat at 2 or 3 weeks of age. The enzyme has a pH optimum of 5.8 to 6.5, and an approximate molecular weight of 70,000. The enzyme activity was completely inhibited by low concentrations of diisopropylfluorophosphate and partially inhibited by high concentrations of phenylmethanesul-phonylfluoride, which are potent serine protease inhibitors. Moreover, thiolblocking agents and some heavy metals also have a strong effect on the activity. Bacitracin was found to be a potent inhibitor, with an IC50 value of 2.5 × 10−6m at 0.5 mm of the substrate. The enzyme was proved to hydrolyze the NH2-terminal tetrapeptide, Arg1-Pro2-Lys3-Pro4, from substance P to produce the heptapeptide, Gln5-Gln6-Phe7-Phe8-Gly9-Leu10-Met11-CONH2. The Km value of the hydrolysis of substance P was 1.0 mm. This enzyme may be related to the regulation of substance P in the brain, and to the development of neurones by forming the tetrapeptide because the tetrapeptide has almost the same effect as substance P on the neurite extension of neuroblastoma.

Distribution and Properties of a Potassium-dependent Asparaginase Isolated from Developing Seeds of Pisum sativum and Other Plants

Abstract: Asparaginase (EC 3.5.1.1) was isolated from the developing seed of Pisum sativum. The enzyme is dependent upon the presence of K + for activity, although Na + and Rb + may substitute to a lesser extent. Maximum activity was obtained at K + concentrations above 20 millimolar. Potassium ions protected the enzyme against heat denaturation. The enzyme has a molecular weight of 68,300. Asparaginase activity developed initially in the testa, with maximum activity (3.6 micromoles per hour per seed) being present 13 days after flowering. Maximum activity (1.2 micromoles per hour per seed) did not develop in the cotyledon until 21 days after flowering. Glutamine synthetase and glutamate dehydrogenase were also present in the testae and cotyledons but maximum activity developed later than that of asparaginase. Potassium-dependent asparaginase activity was also detected in the developing seeds of Vicia faba, Phaseolus multiflorus, Zea mays, Hordeum vulgare, and two Lupinus varieties. No stimulation of activity was detected with the enzyme isolated from Lupinus polyphyllus, which has previously been shown to contain a K + -independent enzyme.

Steroid 5α-Reductase Deficiency in a 65-Year-Old Male Pseudohermaphrodite: The Natural History, Ultrastructure of the Testes, and Evidence for Inherited Enzyme Heterogeneity*

Abstract: We report a 65-yr-old male pseudohermaphrodite with steroid 5 alpha-reductase deficiency in whom there was no medical intervention before, during, or after puberty, enabling us to observe the natural history of this condition. The affected subject has an android build, with more facial and body hair than in previously described affected adults. Although the subject was raised as a girl, a male gender identity evolved with the events of puberty, but social factors have delayed the complete expression of a male gender role. Plasma levels of dihydrotestosterone and the in vivo conversion of radiolabeled testosterone to dihydrotestosterone were decreased. There was an elevated urinary etiocholanolone to androsterone ratio, typical of the syndrome. Characterization of 5 alpha-reductase enzyme activity in cultured genital skin fibroblasts demonstrated a pattern of enzyme activity distinctly different from three previously described families with this condition. There was decreased enzyme affinity for testosterone and NADPH. Also, the stability of the enzyme to elevated temperature was not protected by NADPH, resulting in rapid disappearance of enzyme activity after inhibition of protein synthesis with cycloheximide. Electron microscopic evaluation of the testes was carried out.

Degradation of Basement-Membrane Collagen by Neutral Proteases from Human Leukocytes

Abstract: A neutral extract from human leukocytes was shown to have proteolytic activity which could degrade triple-helical basement membrane collagen from bovine lens capsules into specific triple-helical sub-fragments. The enzyme responsible is not identical to the leukocyte collagenase active against interstitial collagen types I, II and III. After denaturation, the neutral protease reaction products showed three major peptides with molecular weights of 70000, 50000 and 30000, by dodecylsulphate gel electrophoresis analysis. Electron microscopic observation and viscosity measurements showed the initial twice-pepsinized collagen to be comprised of intact rod-like molecules about 300 nm long. The fragments produced by the protease were also triple-helical and were resistant to the action of trypsin at 20°C. The fragments were completely degraded by purified bacterial collagenase or by raising the reaction temperature above the melting temperature of the basement membrane collagen during incubation with leukocyte extract. Enzyme activity has its pH optimum between 7 and 9, EDTA, EGTA and 1, 10-phenanthroline (metal-chelating agents) completely inhibited enzyme activity, as did serum. Partial inhibition of the activity was obtained with phenylmethylsulfonyl fluoride and soybean trypsin inhibitor.

Purification and characterization of 3-deoxy-D-manno-octulosonate 8-phosphate synthetase from Escherichia coli.

Abstract: 3-Deoxy-D-manno-octulosonate (KDO)-8-phosphate synthetase has been purified 450-fold from frozen Escherichia coli B cells. The purified enzyme catalyzed the stoichiometric formation of KDO-8-phosphate and Pi from phosphoenolpyruvate (PEP) and D-arabinose-5-phosphate. The enzyme showed no metal requirement for activity and was inhibited by 1 mM Cd2+, Cu2+, Zn2+, and Hg2+. The inhibition by Hg2+ could be reversed by dithiothreitol. The optimum temperature for enzyme activity was determined to be 45 degrees C, and the energy of activation calculated by the Arrhenius equation was 15,000 calories (ca. 3,585 J) per mol. The enzyme activity was shown to be pH and buffer dependent, showing two pH optima, one at pH 4.0 to 6.0 in succinate buffer and one at pH 9.0 in glycine buffer. The isoelectric point of the enzyme was 5.1. KDO-8-phosphate synthetase had a molecular weight of 90,000 +/- 6,000 as determined by molecular sieving through G-200 Sephadex and by Ferguson analysis using polyacrylamide gels. Based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the 90,000-molecular-weight native enzyme was composed of three identical subunits, each with an apparent molecular weight of 32,000 +/- 4,000. The enzyme had an apparent Km for D-arabinose-5-phosphate of 2 X 10(-5) M and an apparent Km for PEP of 6 X 10(-6) M. No other sugar or sugar-phosphate could substitute for D-arabinose-5-phosphate. D-Ribose-5-phosphate was a competitive inhibitor of D-arabinose-5-phosphate, with an apparent Ki of 1 X 10(-3) M. The purified enzyme has been utilized to synthesize millimole quantities of pure KDO-8-phosphate.

Variant human phosphoribosylpyrophosphate synthetase altered in regulatory and catalytic functions.

Abstract: An inherited, structurally abnormal and superactive form of the enzyme 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) synthetase (EC 2.7.6.1) has been characterized in fibroblasts cultured from a 14-yr-old male (S.M.) with clinical manifestations of uric acid overproduction present since infancy. PP-ribose-P synthetase from the cells of this child showed four- to fivefold greater than normal resistance to purine nucleotide (ADP and GDP) feedback inhibition of enzyme activity and hyperbolic rather than sigmoidal inorganic phosphate (Pi) activation in incompletely dialyzed extracts. Excessive maximal velocity of the enzyme reaction catalyzed by the mutant enzyme was indicated by: enzyme activities twice those of normal at all concentrations of Pi in chromatographed fibroblast extracts; normal affinity constants for substrates and for the activator, Mg2+; and twofold greater than normal activity per immunoreactive enzyme molecule. The mutant enzyme thus possessed deficient regulatory and superactive catalytic properties, two mechanisms previously demonstrated individually to underlie the excessive PPRribose-P and uric acid synthesis of affected members of families with superactive PP-ribose-P synthetases. Increased PP-ribose-P concentration (4-fold) and generation (2.7-fold) and enhanced rates of PP-ribose-P dependent purine synthetic reactions, including purine synthesis de novo, in S.M. fibroblasts confirmed the functional significance of this patient's mutant enzyme. Diminished stability of the variant PP-ribose-P synthetase was manifested in vitro by increased thermal lability and in vivo by deficiency of enzyme activity at Pi concentrations greater than 0.3 mM in hemolysates and by an accelerated, age-related decrement in enzyme activity in lysates of erythrocytes separated by specific density. Despite the diminished amount of PP-ribose-P synthetase in the S.M. erythrocyte population, S.M. erythrocytes had increased PP-ribose-P concentration and increased rates of incorporation of [14C]adenine and hypoxanthine into acid-soluble nucleotides during incubation at 1 mM Pi. These findings provided further confirmation of the extent to which PP-ribose-P synthesis is modulated in the normal cell at physiological Pi concentration by purine nucleotide inhibition of PP-ribose-P synthetase. The activity and kinetic characteristics of PP-ribose-P synthetase from fibroblasts of the mother of patient S.M. indicated that this woman was a heterozygous carrier of the enzyme defect expressed in hemizygous manner by her son.

Study on aminopeptidase A

Abstract: A comparative biochemical and histochemical investigation of aminopeptidase A (APA, E.C. 3.4.11.7) was carried out. α-Glu-1NA, α-Glu-2NA, α-Glu-MNA and Asp-2NA were used as substrates, Fast Blue B (FBB), hexazotized new fuchsin (HNF) and hexazonium-p-rosaniline (HPR) as coupling agents. Biochemical determination of APA activity was performed in whole homogenates as well as in homogenized freeze-dried cryostat sections of many rat, guinea-pig and human organs. α-Glu-2NA proved to be the best substrate for the biochemical determination of APA activity. It displays favourable kinetic properties and abilities for spectrophotometric as well as fluorometric measurements.K m of rat kidney enzyme amounts to 0.15–0.43 mM, of rat jejunum enzyme 0.35 mM, of myocardial enzyme 0.2 mM and of rat brain enzyme 0.25 mM. The enzyme is activated by Ca2+ and inhibited by EDTA (1 mM) and 1,10-phenanthroline (1 mM). E600, DFP and PCMB (1 mM) did not influence APA activity. The activity was detected in every organ examined. Great organ and species differences were demonstrated. The highest APA activity resides in the kidney and in the small intestinal mucosa. The structural association of APA is not absolutely firm and up to 30% (depending on the organ and eventual pretreatment of sections with chloroform-acetone which impedes the leakage to some extent) escapes into the aqueous incubation solution. The highest values were recorded in phosphate and cacodylate buffers followed by citrate phosphate, citrate, Tris-HCl and acetate buffers. There were no great differences in activities measured at pH 6.5 and 7.2. For the histochemical demonstration of APA activity α-Glu-MNA is the substrate of choice.K m estimated biochemically amounts to 0.3–0.43 mM for the kidney enzyme, 0.35 mM for the rat jejunal enzyme and 0.5 mM for rat brain enzyme.K m estimated by microdensitometric measurements of the reaction product in the brush border of cells of proximal tubules and of jejunal enterocytes at the sites of villi amount to 0.6 and 0.4 mM respectively. The best results were obtained using 2–4 mM concentration of α-Glu-MNA, and satisfactory results using 0.36–0.72 mM concentration. FBB appears to be the best coupling agent in the routine. The most sensitive way to depict APA in sites with low and moderate activity is to use chloroform-acetone pretreated cryostat sections adherent to semipermeable membranes. Sites with high enzyme activity are best revealed upon incubation of chloroform-acetone pretreated cryostat sections adherent to slides. Of the unsubstituted naphthylamine derivative of α-Glu or Asp α-Glu-2NA is superior to α-Glu-1NA or Asp-2NA. In this case HPR or HNF are to be used as coupling agents in cryostat sections adherent to slides. Due to a higher decomposition rate and higher inhibitory influence of diazonium salts on APA activity in the alkaline pH range it is advisable to carry out the reaction at pH 6.5–7. The most important binding sites of APA are: The functional significance of APA is discussed in relation to the degradation of angiotensin II (endothelium, kidney) and to other brush border peptidases releasing amino acids which are absorbed (enterocytes). The histochemical demonstration is indispensable for a correct interpretation of biochemical data.