Analysis of androgen-5 alpha-reductase by an enzyme kinetic method

Analysis of androgen-5 alpha-reductase (A-5 alpha-R) is commonly done by measuring distinct testosterone (T) metabolites after tissue incubation. In the present study, A-5 alpha-R analysis was performed according to the principles of Michaelis-Menten, i.e. by evaluation of KM and Vmax data of the enzyme. 48 tissue samples (foreskin) of healthy boys in the age group 6 months to 8 years were examined. Incubation was carried out using increasing concentrations of 3H-T (8-208 nM) as substrate, followed by extraction and thin layer chromatography (TLC) of the reaction products. Zone detection and quantitation of radioactivity was done by a computing TLC scanner. The specific radioactivity of the metabolites was evaluated by high resolution radio gas chromatography. KM values ranged from 81.8 to 118.1 nM and Vmax from 8.9 to 30.1 pmol/mg . h. Coefficient of variation was smaller for KM (0.1) than for Vmax (0.38). It is recommended to do A-5 alpha-R analysis by evaluation of the enzyme kinetics as this is a reproducible method giving accurate biochemical data of the qualitative and quantitative characteristics of the enzyme.     

Enhancement of polyphenol bio-activities by enzyme reaction

Using β-glucosidase to hydrolyze glycosides into aglycones, the present study attempted to improve the bio-activity of the extract from mulberry leaves. When varying the ethanol fraction, pH, and temperature of the extract, the optimum conditions for the enzyme reaction were identified as a 10% ethanol fraction in the extract, pH 5.0, and 40 °C temperature. Under these optimum conditions, the enzyme reaction produced a remarkable increase in the anti-oxidation and tyrosinase inhibition activities of the extract by as much as 219.5% and 230.9%, respectively. This improved bio-activity of the extract was due to the hydrolysis of the glycoside polyphenols rutin, isoquercitrin, and astragalin into the aglycone polyphenols quercetin and kaempferol. Furthermore, the enzymatic hydrolysis of the extract by β-glucosidase also produced some additional benefits that are critical factors for the skin absorption of bio-active ingredients, including an improved hydrophobicity (239.41%) and reduced mean molecular weight (from 387.3 to 291.4), resulting in a significantly enhanced skin permeability (513%).     

Analysis of the kinetics of cyclic AMP hydrolysis by the cyclic GMP-stimulated cyclic nucleotide phosphodiesterase

The kinetics of cAMP hydrolysis by the purified calf liver cGMP-stimulated cyclic nucleotide phosphodiesterase were analyzed in the absence or presence of a number of competitive inhibitors of the methylxanthine type according to a two-site competitive model for allosteric enzymes. Methylxanthines were also classified by graphical analysis of classical competition kinetics at saturating cAMP. This treatment yielded Km/KI ratios which estimated the relative effectiveness of the binding of substrate and inhibitors to the "high affinity" (ES complex) state without establishing individual equilibrium-binding constants of cAMP and inhibitors for specific enzyme states. Individual binding constants for substrate and inhibitors were estimated directly by fitting primary data to the rate equation for the two-site competitive model. The equilibrium dissociation constants for cAMP to the "high" (KS) and "low affinity" (AKS) states were 2.4 +/- 0.8 and 410 +/- 140 microM, respectively. Dissociation constants for various inhibitors to the high (BKI) and low affinity (KI) states were also estimated. The ratio KS/BKI, which directly compared the equilibrium-binding constants of substrate and inhibitors to the high affinity state (ES complex), was in excellent agreement with Km/KI ratios derived from graphical analysis. Whereas a number of the methylxanthine analogues were more effective or as effective as cAMP in binding to the low affinity or "ligand-free" state, only isobutylmethylxanthine was effective as cAMP in binding to the high affinity state (1-methyl-3-isopropylxanthine, and 1,3-dipropylxanthine were somewhat less effective). These findings suggested that allosteric transitions might alter the topography of specific hydrophobic domains at cyclic nucleotide-binding sites and that structural determinants were more stringent for binding to the high affinity state than to the low affinity state.     

Cyclization reaction catalyzed by branching enzyme.

The action of branching enzyme (EC 2.4.l.l8) from Bacillus stearothermophilus on amylose was analyzed. The enzyme reduced the molecular size of amylose without increasing the reducing power. This result could not be explained by the normal branching reaction model. When the product was treated with glucoamylase (an exo++-type amylase), a resistant component remained. The glucoamylase-resistant component was easily digested by an endo-type alpha-amylase or by isoamylase plus glucoamylase. These results suggested that the glucoamylase-resistant component was a cyclic glucan composed of alpha-1,4- and alpha-l,6-glucosidic linkages. In other words, it was suggested that branching enzyme catalyzed cyclization of the alpha-l,4-glucan chain of the amylose molecule to form an alpha-l,6-glucosidic linkage, thereby forming two smaller molecules. Mass spectrometry also supported the cyclic nature of the product.     

DFT study on gas-phase hydrodeoxygenation of guaiacol by various reaction schemes

Guaiacol is an important phenolic component present in pyrolytic bio-oils; and in this work its hydrodeoxygenation (HDO) by various reaction schemes has been considered within the framework of density functional theory. In this computational study, primarily three reaction schemes for the HDO of guaiacol are considered. In the first reaction scheme (RS 1), guaiacol undergoes hydrogenolysis at O–CH₃ bond site of methoxy group to produce catechol and methane followed by HDO of catechol forming phenol and water, followed by HDO of phenol producing benzene and water and finally benzene leading to cyclohexane formation. In the second reaction scheme (RS 2), guaiacol undergoes hydrogenolysis at C_aromatic–O bond of methoxy group producing phenol and methanol followed by hydrotreatment of phenol to form cyclohexane along with same intermediates as in the first reaction scheme. In the third reaction scheme (RS 3), HDO of guaiacol compound at C_aromatic–OH sigma bond produces anisole and water; and then anisole follows two secondary pathways to produce cyclohexane. In this computational study, the transition state optimisations, vibrational frequency and IRC calculations are carried out by B3LYP functional with 6-311+g(d,p) basis set using Gaussian 09 and Gauss View 5 software package.     

Analysis of the amino terminus of maize branching enzyme II by polymerase chain reaction random mutagenesis

Maize endosperm branching enzyme II (mBEII) plays a pivotal role in determining the quality of starch by catalyzing the synthesis of the alpha-1,6-branch points. While the central (alpha/beta)8-barrel and the C-terminal domains of mBEII have been analyzed previously, the possible role of its amino terminus in catalysis is still poorly understood. Because the amino terminus of mBEII shares very little sequence homology with other amylolytic enzymes, the Met1-Gly276 region of mBEII was randomly mutagenized under error-prone PCR conditions. Subsequent screening by a heterologous complementation system, utilizing an Escherichia coli strain devoid of the endogenous glycogen branching enzyme (glgB-), led to the recovery of mBEII mutants with altered iodine-staining patterns and reduced branching enzyme activities. The NR-625 mutant enzyme, which lacks the N-terminal 39 residues of mBEII due to a frameshift mutation introduced during the random mutagenesis, retained more than 70% of the wild-type activity. The chain transfer pattern and substrate preference of the truncated enzyme were almost identical to those of the wild-type mBEII. It appears that the N-terminal 39 residues of mBEII are neither required for catalysis nor involved in chain transfer. On the other hand, the Gln-to-Arg substitution at position 270 of mBEII resulted in the loss of more than 90% of branching activity. The Gln270 of mBEII, located at the beginning of the (alpha/beta)8-barrel domain, may be required for maximum enzyme activity.     

A study of the reaction catalysed by the liver branching enzyme

The mechanism of action of liver branching enzyme has been studied by using as substrate two polysaccharides in which the non-reducing ends had been labelled by incubation with phosphorylase and a trace amount of [¹⁴C]glucose 1-phosphate. After these polysaccharides had been treated with branching enzyme, their structure was analysed by periodate oxidation, by degradation with phosphorylase and amylo-(1→6)-glucosidase and by degradation with pullulanase. All the results indicate that the branching enzyme catalyses the transfer from (1→4)- to (1→6)-linkage of a chain of glucose units, the minimum length of which is six glucose units. A maltodextrin containing 16 glucose units was not acted on by the branching enzyme.     

A graph-theoretic method for the basic reproduction number in continuous time epidemiological models

In epidemiological models of infectious diseases the basic reproduction number is used as a threshold parameter to determine the threshold between disease extinction and outbreak. A graph-theoretic form of Gaussian elimination using digraph reduction is derived and an algorithm given for calculating the basic reproduction number in continuous time epidemiological models. Examples illustrate how this method can be applied to compartmental models of infectious diseases modelled by a system of ordinary differential equations. We also show with these examples how lower bounds for can be obtained from the digraphs in the reduction process.     

Optimization of fermentation reaction conditions for preparation of PATE enzyme by Erwinia carotovora IFO3830 by TFCCRD method.

The fermentation conditions for preparation of polygalacturonic acid transeliminase (PATE) enzyme by Erwinia carotovora IFO3830 were optimized for seed ratio, vibration rate, and temperature by the TFCCRD method. The results indicated that the optimum fermentation conditions for E. carotovora IFO 3830 were that seed ratio, vibration rate, and temperature were 5% v/v, 113 min(-1), and 29 degrees C, respectively.     

Eicosanoid forming enzyme mRNA in human tissues. Analysis by quantitative polymerase chain reaction

The key enzymes in the formation of eicosanoids, including leukocyte 5-lipoxygenase (5LX), platelet 12-lipoxygenase (12LX), reticulocyte 15-lipoxygenase (15LX), prostaglandin G/H synthase cyclooxygenase, and leukotriene A4 (LTA) hydrolase have been studied extensively in recent years. Little is known, however, about the regulation of these enzymes at the gene level. We have developed a quantitative polymerase chain reaction (PCR) assay to quantify the mRNAs for these five enzymes, as well as for cytoplasmic beta-actin (bACT) mRNA. Human erythroleukemia (HEL) cells, which display megakaryocytic/erythroid characteristics, were selected as a source of RNA to characterize the assay. These cells expressed mRNA for bACT, LTA, cyclooxygenase, and 12LX (in decreasing order). mRNA for 5LX and 15LX was undetectable. Bronchoalveolar lavage fluid cells obtained from asthmatic patients, primarily alveolar macrophages, contained mRNA for bACT, LTA, 5LX, cyclooxygenase, and 15LX (in decreasing order). Treatment of HEL cells with phorbol 12-myristate 13-acetate or steroid administration to asthmatic patients apparently selectively regulated certain of these target genes. The utility of this assay in quantifying mRNA for the various target genes in blood cells, including platelets from patients with chronic myelogenous leukemia, has also been demonstrated. Studies on the regulation of genes for enzymes involved in the leukotriene and prostaglandin biosynthetic pathways, especially when only small tissue samples are available, will be facilitated with this approach.     

Enzymatic synthesis of alkylglucosides by amphiphilic phase enzyme reaction

Alkyl--glucosides were synthesized with -glucosidase, from almond, in an amphiphilic phase reaction system using different water-miscible cosolvents. By means of stepwise addition of glucose, we obtained about 1.5 times higher concentration of hexylglucoside than that of normal reaction. Using the same way, octyl-, decyl- and dodecylglucoside were synthesized and their concentrations were 6.64, 5.3 and 4.48 g l^–1, respectively.     

Mechanism of the adenylate cyclase reaction. Stereochemistry of the reaction catalyzed by the enzyme from Brevibacterium liquefaciens

Adenylate cyclase from Brevibacterium liquefaciens (ATCC 14929) catalyzes the formation of the RP-diastereomer of adenosine 3':5'-cyclic monophosphorothioate from the SP-diastereomer of adenosine-5'-(1-thiotriphosphate). The reaction catalyzed by this adenylate cyclase proceeds with inversion of configuration at phosphorus, indicating that the cyclization reaction is direct and does not involve formation of an adenylated enzyme intermediate.     

Stimulation of bovine endothelial cell angiotensin-I-converting enzyme activity by cyclic AMP-related agents

Bovine pulmonary artery endothelial cells in culture were used to assess the influence of cyclic nucleotides, isoproterenol (beta adrenergic agonist), and theophylline (phosphodiesterase inhibitor) on angiotensin-I-converting enzyme (ACE) activity of the cells and culture medium. Dibutyryl cAMP (10(-3) M) but not cAMP or dibutyryl cGMP stimulated angiotensin-I-converting enzyme (ACE) activity of cells in culture approximately 50-100% but had little influence on ACE activity of the medium. Theophylline at 10(-3) M concentration produced a three- to fourfold stimulation of both cellular and medium ACE activity. Isoproterenol by itself had no effect on cellular ACE activity but produced a stimulatory effect at 10(-7)-10(-5) M concentration after pretreatment of cells for 24 hr with 10(-4) M theophylline. The results support the concept that ACE activity of endothelial cells is influenced by the cyclic AMP system. ACE activity in cells and that released into medium may be under different regulatory controls.     

Activation of a new plasma enzyme by antigen-antibody reaction.

The change of enzyme activity in immunized rabbit plasma after addition of the homologous antigen was examined. The activities of N alpha-tosyl-L-arginine methyl ester (TAMe) and N alpha-tosyl-L-lysine methyl ester (TLMe) hydrolysis increased about 15 to 18 days after immunization. This increase was especially marked before the maximal rise of antibody content, and is thought to be related to the IgM antibody not to the IgG antibody. Enzyme activation was strongly inhibited by chelation of Ca2+ with 5 mM disodium ethylenediamine tetraacetate (EDTA), but not by other protease inhibitors, such as epsilon amino-caproic acid (epsilon-ACA), bovine lung kallikrein inhibitor (Trasylol) or soybean trypsin inhibitor (SBTI).