The properties of immobilized whole cell ofHumicola spp. with rifamycin oxidase activity

Summary The whole cell ofHumicola spp. ATCC 20620 with rifamycin oxidase activity was immobilized by copolymerization with acrylamide. The whole cell was defatted by treatment with acetone to reduce the diffusional resistance through the cell membrane. The recovery of enzyme activity after the immobilization step was about 50%. The acetone-defatted cell showed the maximum activity at pH 7.5 for both free and the immobilized forms. No appreciable activity loss could be detected when stored at 4 °C and pH 7.8 for one month, while the half life at 40 °C and pH 8 was decreased to about 8 days. The apparent Km values of rifamycin oxidase for the free and immobilized acetonedefatted cells were 0.3mM and 0.6mM, respectively. The enzyme demonstrated substrate inhibition, but the degree of substrate inhibition was different between two forms of the enzyme preparation. A complete substrate inhibition was observed for the immobilized cell, whereas the enzyme activity was partially inhibited at high substrate concentration in the acetone-defatted cells.     

Some parameters affecting the activity of monoamine oxidase in purified bovine brain mitochondria.

Abstract— Some parameters affecting the activity of monoamine oxidase (MAO) in purified beef brain mitochondria were investigated, and diversities in enzyme properties were found as a function of substrate. The deamination of the biogenic amines: serotonin, dopamine, tyramine, tryptamine, phenylethylamine and two non-physiological amines, kynuramine and m-iodobenzylamine, was studied. Anions in high concentrations inhibited enzyme activity with kynuramine being the substrate most affected. Among the biogenic amines, the activity with the indolalkylamines showed greater sensitivity to mono-valent anions such as chloride than to polyvalent ions such as phosphate whereas the opposite was true with the phenylalkylamines. However, pyrophosphate ion had little or no effect on MAO activity, regardless of substrate. The inhibition of kynuramine and serotonin deamination was non-competitive but mixed competitive inhibition was found with tyramine and phenylethylamine. The activity of MAO was markedly affected by pH, and it had been previously reported that the substrates showed different pH optima in their oxidation. The effect of pH on activity has been attributed in part to changes in the ionization of the substrate and the hypothesis that the true substrate is the non-protonated amine. This was reflected in kinetic studies showing high substrate inhibition with increased pH. It was calculated that phenylethylamine would have the highest percentage of un-ionized amine at pH 8.2 and 9.1. At these pHs, there was more pronounced inhibition with high substrate concentrations of phenylethylamine than with the other substrates. In contrast, there was little inhibition with high substrate concentrations of tyramine which was the most ionizable of the substrates tested. When K_m values obtained at pH 7.4, 8.2 and 9.1 were corrected for ionization of the substrate, the corrected K_m was lowest at pH 7.4 for all substrates. Less than 50% of MAO activity was lost when beef brain mitochondria was heated at 50°C for 20 min. However, there was only a slight variation with substrate in the thermal inactivation experiments. It is concluded that the mitochondrial membrane environment surrounding the enzyme imposes certain restrictions on the enzymatic activity with respect to the different substrates which, in turn, are also affected by such parameters as pH and ions. The results are discussed in terms of the relationship of these factors to the question of enzyme multiplicity.     

STUDIES ON THE NATURAL SUBSTRATE FOR PROTEIN METHYLASE II IN MAMMALIAN BRAIN AND BLOOD

—The activity of protein methylase II (S-adenosylmethionine:protein-carboxyl methyltransferase, EC 2.1.1.24), which methylates (esterifies) free carboxyl groups in the substrate protein, was measured in several mammalian organs in an effort to elucidate the nature of the natural substrate for the enzyme. The highest endogenous substrate activity was found in posterior and anterior pituitary glands, possibly in association with neurosecretory granules. In other parts of the brain endogenous substrates are lacking, although the cytosol fractions contain high activity of the enzyme which methylates exogenously added substrates. Rat whole blood also contains endogenous substrate protein. The protein precipitated by 50% (NH₄)₂SO₄ contained active substrate protein whereas blood protein methylase II is localized exclusively in the erythrocytes. Cohn fractions I, II and III are more active as substrate for protein methylase II than fraction V.     

Further characterization of peptidylglycine alpha-amidating monooxygenase from bovine neurointermediate pituitary

The ability of purified bovine neurointermediate pituitary peptidyl glycine alpha-amidating monooxygenase to catalyze the conversion of peptide substrates (D-Tyr-X-Gly) into amidated product peptides (D-Tyr-X-NH2) was evaluated. The pH optimum of the reaction was pH 8.5 when X was Val, Trp, or Pro, but 5.5 to 6.0 when X was Glu. Similar maximum velocity (Vmax) values were obtained for the Val, Trp, and Pro substrates while the Glu substrate had a substantially higher Vmax. The Michaelis-Menten constant (Km) of the enzyme for the peptide substrate increased in the order Trp less than Val less than Pro much less than Glu. Increasing levels of ascorbate brought about parallel increases in Km and Vmax, suggesting the presence of an irreversible step separating the interaction of the enzyme with the two substrates. The effect of copper on enzyme activity was dependent on the peptide substrate and the reaction pH. With the Val substrate, exogenous copper was required for optimal activity; no other metal ion tested could substitute for copper. With the Glu substrate, exogenous copper was not required for optimal activity; however, diethyldithiocarbamate, a copper chelator, inhibited activity and only copper could reverse this inhibitory effect. The ability of various cofactors to stimulate alpha-amidating monooxygenase activity was also dependent on assay conditions. With the Val or Glu substrate in the presence of exogenous copper, a variety of cofactors in addition to ascorbate were capable of supporting activity. With the Glu substrate in the absence of exogenous copper, the requirement of the enzyme for ascorbate was more strict. In keeping with the proposed reaction mechanism, nearly 1 mol ascorbate was consumed for each mole of D-Tyr-Glu-NH2 produced.     

Production of laccase from Trametes versicolor by solid-state fermentation using olive leaves as a phenolic substrate

The aim of the present study was to investigate whether olive leaves were feasible as a substrate for laccase production by the white-rot fungus Trametes versicolor FPRL 28A INI under solid-state fermentation conditions. Different experiments were conducted to select the variables that allow obtaining high levels of laccase activity. In particular, the effects of the initial moisture content, substrate particle size, supplementation with inorganic and organic nitrogen sources were evaluated. Highest laccase activity (276.62 ± 25.67 U/g dry substrate) was achieved with 80 % initial moisture content and 1.4–1.6 mm particle size of the substrate supplemented with yeast extract (1 % (w/w) nitrogen). Such a high activity was obtained without any addition of inducers.     

Biochemical characterization of prostasin, a channel activating protease

Human prostasin was recently identified as a potential regulator of epithelial sodium channel (ENaC) function. Through the use of positional scanning combinatorial substrate libraries, prostasin was shown to have a preference for poly-basic substrates: in position P4 preference was for arginine or lysine; in P3 preference was for histidine, lysine or arginine; in P2 preference was for basic or large hydrophobic amino acids; and in P1 preference was for arginine and lysine. P1', P2', and P3' displayed broad selectivity with the exception of a lack of activity for isoleucine, and P4' had a preference for small, unbranched, amino acids such as alanine and serine. A prostasin-preferred poly-basic cleavage site was found in the extracellular domains of the ENaC alpha- and beta-subunits, and may present a mechanism for prostasin activation. The absence of activity seen with substrates containing isoleucine in position P1' explains the inability of prostasin to autoactivate and suggests that prostasin proteolytic activity is regulated by an upstream protease. Prostasin activity was highly influenced by mono- and divalent metal ions which were potent inhibitors and substrate specific modulators of enzymatic activity. In the presence of sub-inhibitory concentrations of zinc, the activity of prostasin increased several-fold and its substrate specificity was significantly altered in favor of a strong preference for histidine in positions P3 or P4 of the substrate.     

亚热带砂岩发育森林土壤酚氧化酶活性测定方法优化

酚氧化酶在土壤有机质降解过程中起重要作用,然而,目前用于测定土壤酚氧化酶活性的方法尚未统一。本研究以亚热带地区砂岩发育的3种不同林分的森林土壤为对象,探讨底物类型、pH值、土壤储存条件、储存时间、底物浓度、水土比、培养时间和温度对土壤酚氧化酶活性的影响,以期建立统一、可比较的测定亚热带森林土壤酚氧化酶活性的方法。结果表明: 浸提液pH值显著影响土壤酚氧化酶活性,且与目前普遍使用的左旋多巴胺(L-DOPA)相比,2,2′-联氨-双(3-乙基苯并噻唑啉-6-磺酸)-二胺盐(ABTS)所测得的氧化酶活性更高、适用pH值范围更广,说明ABTS可能更适合作为测定亚热带森林酸性土壤酚氧化酶活性的底物。储存方式显著影响酚氧化酶活性,3种供试土壤样品酚氧化酶活性均随时间呈降低的趋势,降幅表现为风干> 4 ℃冷藏> -20 ℃冷冻> -80 ℃冷冻,表明在无法保证快速测定土壤酚氧化酶活性的情况下,冷冻保存方式更有利于维持土壤酚氧化酶活性。底物浓度、水土比以及培养时间和温度均影响土壤酚氧化酶活性。当土壤样品与浸提液比例为1∶100时,选择2 mmol·L^-1浓度的ABTS为底物,在25~30 ℃下培养4 h,测定酚氧化酶活性结果重复性好、灵敏度高,是测定亚热带森林酸性土壤酚氧化酶活性的最优条件。     

Fluorometric assay for tissue transglutaminase-mediated transamidation activity

Herein, we report the development of a direct discontinuous fluorometric transamidation assay for determining tissue transglutaminase (TG2) activity. In the assay reaction, TG2 catalyzes the formation of a biotin-fluorophore conjugate, using a fluorescent, high affinity γ-glutamyl donor substrate and a biotinylated amine as a γ-glutamyl acceptor substrate. After the reaction, the conjugate is fixed on streptavidin-coated beads and excess substrate is washed away, allowing the transamidation activity to be quantified by fluorescence measurement. This method was used to detect the activity of as little as 0.6 mU of purified TG2, and can be used for detection of activity from crude cellular lysates. Furthermore, this assay can be used for screening potential inhibitors and synthetic substrates, the latter of which was demonstrated herein.     

Characterization of Pea Chloroplast D-Enzyme (4-alpha-d-Glucanotransferase)

Pea (Pisum sativum L.) chloroplast D-enzyme (4-α-d-glucanotransferase, EC 2.4. 1.25) was purified greater than 750-fold and partially characterized. It is a dimer with a subunit M_r of ca. 50,000. Optimal activity is between pH 7.5 and 8.0 with maltotriose as substrate and the enzyme's K_m for maltotriose is 3.3 millimolar. Chloroplast D-enzyme converts maltotriose to maltopentaose and glucose via the exchange of α-1,4-glycosidic linkages. Maltotriose acts either as a donor or acceptor of a maltosyl group. The enzyme has highest activity with maltotriose as substrate. As initial substrate degree of polymerization is increased to maltoheptaose, D-enzyme activity drops to zero at 10 millimolar substrate concentrations and by 70% at 1 millimolar concentrations. The enzyme cannot use maltose as a substrate. Glucose was found to be a suitable acceptor substrate for this D-enzyme. Addition of glucose to incubation mixtures, or production of glucose by D-enzyme, prevents the synthesis of maltodextrins larger than maltopentaose. Removal of glucose produced by D-enzyme activity with maltotriose as substrate resulted in the synthesis of maltopentaose and maltodextrins with sufficient degrees of polymerization to be suitable substrates for pea chloroplast starch phosphorylase. The possible role of D-enzyme in pea chloroplast starch metabolism is discussed.     

Purification and properties of a glutathione-S-transferase from corn which conjugates s-triazine herbicides

A glutathione-S-transferase involved in atrazine conjugation was purified 43-fold from corn with a total yield of 36%. The purified enzyme has a MW of 45 000 as determined by gel filtration. The estimated activation energy of the enzyme is 6.4 kcal/mol and the optimum pH for activity between 8 and 8.5. Substrate specificity studies with s-triazines indicated that atrazine was the best substrate followed by simazine and propazine. The Cl group at the 2-position was essential for enzyme activity, and replacement by a SCH₃ group resulted in a total loss of activity. The absence of an alkyl group resulted in a reduction of conjugation and 2-chloro-4,6-bis-amino-s-triazine was the poorest substrate. With insecticidal substrates (organophosphates), conjugating activity was observed only with diazinon and little or no activity was observed with ethyl parathion, malathion and etrimfos. No activity was found using methyl iodide as a substrate. The purified enzyme has properties similar to those of an aryl-S-transferase. Quinones were inhibitors of this enzyme.     

Comparison between two methods of assaying relative microbial activity in marine environments.

Two methods for determining relative microbial activity in the marine environment were compared. In one method, a single concentration of a labeled substrate was used to calculate rates of substrate utilization; in the other, multiple concentrations of the same substrate (heterotrophic activity method) were used to calculate maximum potential substrate utilization rates. These studies were made on 232 seawater and 79 sediment samples taken from a variety of marine environments. The highest correlations between these two methods were seen in the sediment samples tested. The lowest correlation coerfficient seen in the sediment samples was 0.90, and the highest was 0.98. In seawater samples (six studies), the lowest correlation coefficient was 0.77 and the highest was 0.95. The correlation between these two methods was also substrate concentration dependent. Higher correlation coefficients were observed when higher substrate concentrations were used. Under certain conditions, these two methods appear to be comparable for estimating relative levels of microbial activity in the marine environment.     

Comparison between two methods of assaying relative microbial activity in marine environments.

Two methods for determining relative microbial activity in the marine environment were compared. In one method, a single concentration of a labeled substrate was used to calculate rates of substrate utilization; in the other, multiple concentrations of the same substrate (heterotrophic activity method) were used to calculate maximum potential substrate utilization rates. These studies were made on 232 seawater and 79 sediment samples taken from a variety of marine environments. The highest correlations between these two methods were seen in the sediment samples tested. The lowest correlation coerfficient seen in the sediment samples was 0.90, and the highest was 0.98. In seawater samples (six studies), the lowest correlation coefficient was 0.77 and the highest was 0.95. The correlation between these two methods was also substrate concentration dependent. Higher correlation coefficients were observed when higher substrate concentrations were used. Under certain conditions, these two methods appear to be comparable for estimating relative levels of microbial activity in the marine environment.     

A microreactor for the study of biotransformations by a cross-linked γ-lactamase enzyme

A (+)-γ-lactamase was precipitated, cross-linked and the resulting solid crushed prior to immobilisation within a capillary column microreactor. The microreactor was subsequently used to study enzyme stability, activity, kinetics and substrate specificity. The thermophilic (+)-γ-lactamase retained 100% of its initial activity at the assay temperature, 80°C, for 6 h and retained 52% activity after 10 h, indicating the advantage of immobilisation. This high stability of the immobilised enzyme provided the advantage that it could be utilised to screen many compounds in the microreactor system. This advantage overcame the fact that the immobilisation process affected enzyme kinetics and activity, which was reduced (by 70%) compared to the free enzyme. In general, the enzyme displayed similar substrate specificity to that found in a previous study for the free enzyme; however, enhanced activity was seen towards one substrate, acrylamide. The system developed correlates well with the free enzyme in batch assay and indicates the suitability of the system for enzyme substrate screening, allowing a significant reduction in cost, due to the reduced amounts of enzyme, substrates and other assay constituents required.     

Kinetic characterization of gyroxin,a serine protease from Crotalus durissus terrificus venom

This work describes for the first time the characterization of the enzymatic features of gyroxin, a serine protease from Crotalus durissus terrificus venom, capable to induce barrel rotation syndrome in rodents. Measuring the hydrolysis of the substrate ZFR-MCA, the optimal pH for proteolytic cleavage of gyroxin was found to be at pH 8.4. Increases in the hydrolytic activity were observed at temperatures from 25 °C to 45 °C, and increases of NaCl concentration up to 1 M led to activity decreases. The preference of gyroxin for Arg residues at the substrate P1 position was also demonstrated. Taken together, this work describes the characterization of substrate specificity of gyroxin, as well as the effects of salt and pH on its enzymatic activity.     

Role of arylalkylamine <Emphasis Type="Italic">N</Emphasis>-acetyltransferase in regulation of biogenic amines levels by gonadotropins in <Emphasis Type="Italic">Drosophila</Emphasis>

The effect of 20-hydroxyecdysone (20E) and the juvenile hormone (JH) on the activity of the arylalkylamine N-acetyltransferase (AANAT) was studied in young females of wild-type D. virilis and D. melanogaster. 20E feeding of the flies led to a decrease in AANAT activity in both species when dopamine (DA) was used as substrate, but did not affect the enzyme activity when octopamine (OA) was used as substrate. JH application increased AANAT activity with DA as substrate in both species, but did not change it with OA as substrate. AANAT activity was also measured in young females of a JH-deficient strain of D. melanogaster, apterous ^56f . A decrease in the enzyme activity was observed in the mutant females as compared to wild-type. Mechanisms of regulation of DA level by gonadotropins in Drosophila are discussed.     

MULTIPLE FORMS OF PROTEIN KINASES IN MYELIN AND MICROSOMAL FRACTIONS OF BOVINE BRAIN

Abstract— The activity profiles of the solubilized protein kinases from the microsomal and myelin fractions of bovine brain were examined by column chromatography and sucrose density gradient centrifugation. The main peak of adenosine 3',5'-monophosphate (cyclic AMP)-dependent activity with histone as substrate for each membrane enzyme was eluted with about 0.2 m -NaCl on a DEAE-cellulose column. A peak of activity stimulated with cyclic AMP was also eluted with about 0.1 m -NaCl for the microsomal enzyme. A peak with protamine and casein as substrate for the microsomal or myelin enzyme, respectively, was larger than that with histone as substrate for each enzyme. The first peak with histone as substrate on a DEAE–cellulose column appeared as two peaks on the Sepharose 6B column. The second peak with histone as substrate on DEAE–cellulose column was shown to be a holoenzyme consisting of regulatory and catalytic subunits. The holoenzyme and subunits were eluted at similar positions to each other between both membrane enzymes on Sepharose 6B column. The holoenzyme sedimented as two peaks of activity on sucrose density gradient centrifugation, both of which were stimulated with cyclic AMP. The preincubation of the holoenzyme with cyclic AMP resulted in shifting to a position of a smaller molecular size.
The results indicate the occurrence of multiple forms of protein kinases in membrane fractions of brain with respect to substrate specificity and physical property.     

Displacement of a DNA binding protein by Dda helicase

Bacteriophage T4 Dda helicase has recently been shown to be active as a monomer for unwinding of short duplex oligonucleotides and for displacing streptavidin from 3′-biotinylated oligonucleotides. However, its activity for streptavidin displacement and DNA unwinding has been shown to increase as the number of Dda molecules bound to the substrate molecule increases. A substrate was designed to address the ability of Dda to displace DNA binding proteins. A DNA binding site for the Escherichia coli trp repressor was introduced into an oligonucleotide substrate for Dda helicase containing single-stranded overhang. Here we show that a Dda monomer is insufficient to displace the E.coli trp repressor from dsDNA under single turnover conditions, although the substrate is unwound and the repressor displaced when the single-stranded overhang is long enough to accommodate two Dda molecules. The quantity of product formed increases when the substrate is able to accommodate more than two Dda molecules. These results indicate that multiple Dda molecules act to displace DNA binding proteins in a manner that correlates with the DNA unwinding activity and streptavidin displacement activity. We suggest a cooperative inchworm model to describe the activities of Dda helicase.     

The secondary substrate binding site of the <Emphasis Type="Italic">Pseudoalteromonas haloplanktis</Emphasis> GH8 xylanase is relevant for activity on insoluble but not soluble substrates

Previously, it has been demonstrated that the glycoside hydrolase family 8 xylanase from the psychrophylic bacterium Pseudoalteromonas haloplanktis (XPH) can bind substrate non-catalytically on the surface of its catalytic module. In the present study, the functional relevance of this secondary binding site (SBS) for the enzyme is investigated by site-directed mutagenesis and evaluation of activity and binding properties of mutant variants on a range of structurally different homoxylan and heteroxylan substrates. The SBS had an impact on the activity on insoluble substrates, whereas the activity on soluble substrates remained unaffected. Unexpectedly, the activity on a soluble oligomeric substrate was also affected for some mutants and results on a chromophoric polymeric model substrate were in contrast with the trends observed on the corresponding natural substrate. All in all, results show that the impact of the SBS on the activity of XPH is in part analogous to the functioning of some carbohydrate-binding modules in modular enzymes.     

Modulation of calcineurin phosphotyrosyl protein phosphatase activity by calmodulin and protease treatment

Calcineurin (CN) dephosphorylated [32P] phosphotyrosyl glutamine synthetase, a model phosphoprotein substrate containing approximately 1 mol of phosphotyrosine per mol subunit. Phosphatase activity with and without calmodulin (CaM) was greatly stimulated by Mn2+; with Ca2+, even in the presence of CaM, activity was very low. CaM-stimulated phosphatase activity exhibited deactivation with time; initial rates declined markedly after 2-3 min. The Michaelis constant for substrate (3 microM) was identical whether 2 or 12 min assays (with CaM) were used suggesting that the decreased rate of hydrolysis did not result from a decrease in affinity for the phosphoprotein substrate. Limited proteolysis of CN by chymotrypsin increased phosphatase activity 2-3 times that of CaM-supported activity; however, addition of CaM to assays with protease-activated CN reduced activity to that observed for non-proteolyzed enzyme. These data suggest that, in addition to stimulation, CaM can inhibit certain activated conformations of the phosphatase.     

L-gamma-(Threo-beta-methyl)glutamyl-L-alpha-aminobutyrate, a selective substrate of alpha-glutamyl cyclotransferase

L-gamma(Threo-beta-methyl)glutamyl-L-alpha-aminobutyrate was was prepared and found to be an excellent substrate of gamma-glutamyl cyclotransferase; in contrast to gamma-glutamyl-glutamine and other good substrates of cyclotransferase, the new substrate is not acted upon by gamma-glutamyl transpeptidase. gamma-Glutamyl cyclotransferase converts the new substrate to alpha-aminobutyrate and 3-methyl-5-oxoproline; the latter compound is not a substrate of 5-oxoprolinase. These properties of L-gamma-(threo-beta-methyl)glutamyl-L-alpha-aminobutyrate facilitate its use in selectively determining cyclotransferase activity in biological materials that have transpeptidase activity. Thus, the new substrate was used here for the determination of the cyclotransferase activity of homogenates of various mouse tissues. The new substrate was also used to examine gamma-glutamyl cyclotransferase activity in vivo; thus, the rate of respiratory 14CO2 formation after administration of L-gamma-(threo-beta-methyl)glutamyl-L-alpha-amino[14C]butyrate to mice provides a valid measure of cyclotransferase activity. beta-Aminoglutaryl-L-alpha-aminobutyrate is a competitive inhibitor of cyclotransferase (apparent Ki, 0.6 mM). Administration of beta-amino-glutaryl-L-alpha-aminobutyrate to mice out only decreased the level of 5-oxoproline in the kidney of control mice, but also of mice in which kidney 5-oxoproline levels were increased by administration of methionine. Administration of beta-aminoglutaryl-L-alpha-aminobutyrate to mice decreased the in vivo metabolism of L-(threo-beta-methyl)glutamyl-L-alpha-amino[14C]butyrate as indicated by a marked decrease in the rate of respiratory 14CO2 formation. The findings indicate that gamma-glutamyl cyclo-transferase is a major in vivo catalyst for the formation of 5-oxoproline.