Interaction of membranous enzymes with membranous lipid substrates. Hydrolysis of diacylglycerol by lipase in rat brain microsomes.

The phospholipids in rat brain microsomes were labeled with tritium by intracerebral administration of radioactive fatty acids and converted to diacylglycerol with phospholipase C. The latter lipid was hydrolyzed in situ at pH 4.8, to monoacylglycerol and fatty acid by the endogenous microsomal lipase. This paper provides an experimental approach to determine whether the lipid was degraded by enzyme molecules residing in its own membrane (intramembrane interaction) or an adjacent membrane (intermembrane interaction). Direct interaction between separate membranes containing enzyme or substrate showed the existence of the inter-membrane route while dilution experiments provided evidence for the presence of the intramembrane interaction as well. A probable difference in the mechanisms of these two interactions is suggested by different shapes of the curves that describe the reaction rate as a function of the endogenous substrate. The curve resulting from the intermembrane interaction was hyperbolic while that representing the intramembrane route was of a parabola-like shape. Competition experiments suggested that when given a choice between the two, the enzyme utilized preferentially the substrate molecules in its own membrane.     

Enhancement of Ca2+ release from limestone by microbial extracellular carbonic anhydrase

Three experimental systems were set up to investigate whether carbonic anhydrase (CA) from microorganisms actually plays the driving role in limestone dissolution. In one, redistilled water served as negative control. In a second, microbial origin CA enzyme solution supplemented with CA special inhibitor acetazolamide (AZ) served as positive control. A third contained a crude enzyme solution of microbial CA. The results showed that the amount of released Ca2+ from limestone in a CA non-inhibited system increased by 2.4 times compared to a CA inhibited system, and increased by 11.7 times compared to the redistilled water control. These experiments demonstrated that microbial origin CA significantly enhanced Ca2+ release from limestone (P < 0.01), and therefore, proved the significant driving effect of microbial CA on limestone dissolution. The results also suggested that microbially derived CA might exert an important influence on biokarst process.     

Design and analysis of experiments on random drift and inbreeding depression

While the genetic consequences of inbreeding and small population size are of fundamental importance in many areas of biology, empirical research on these phenomena has proceeded in the absence of a well-developed statistical methodology. The usual approach is to compare observed means and variances with the expectations of Wright's neutral, additive genetic model for quantitative characters. If the observations deviate from the expectations more than can be accounted for by sampling variance of the parameter estimates, the null hypothesis is routinely rejected in favor of alternatives invoking evolutionary forces such as selection or nonadditive gene action. This is a biased procedure because it treats sequential samples from the same populations as independent, and because it ignores the fact that the expectations of the neutral additive genetic model will rarely be realized when only a finite number of lines are studied. Even when genes are perfectly additive and neutral, the variation among the properties of founder populations, the random development of linkage disequilibrium within lines, and the variance in inbreeding between lines reduce the likelihood that Wright's expectations will be realized in any particular set of lines. Under most experimental designs, these sources of variation are much too large to be ignored. Formulas are presented for the variance-covariance structure of the realized within- and between-line variance under the neutral additive genetic model. These results are then used to develop statistical tests for detecting the operation of selection and/or inbreeding depression in small populations. A number of recommendations are made for the optimal design of experiments on drift and inbreeding, and a method is suggested for the correction of data for general environmental effects. In general, it appears that we can best understand the response of populations to inbreeding and finite population size by studying a very large number (>100) of self-fertilizing or full-sib mated lines in parallel with one or more stable control populations.     

A Serum Enzyme Increasing Effect of Non-Toxic Herring Meal FED to Sheep

In a series of experiments it was demonstrated that highly increased activities of aspartate aminotransferase (= GOT), total lactate dehydrogenase (LDH), and α-hydroxybutyrate dehydrogenase (HBD) occurred in serum of sheep on herring meal feeding. The alanine aminotransferase (A1AT = GPT) level remained unchanged. The enzyme increase was apparently not related to the liver toxic agent dimethylnitrosamine (DMNA) occasionally occurring in lethal doses in meals produced from raw materials preserved with excesses- of nitrite. Histological changes of the liver or other tissues were never detected in experimental animals killed at a stage with very high serum enzyme values. Diets equivalent in digestible crude protein consisting of vegetable as well as of animal protein sources other than fish meal, did not give rise to elevated serum enzyme values. Electrophoretic separation of the LDH isoenzymes in serum of herring meal-fed sheep showed an increased percentage of the LDH1 fraction, which is predominant in liver, heart, and kidney. Determination of enzyme activities in various tissues resulted in a markedly higher concentration in livers from herring meal-fed animals than in sheep fed casein at an equal protein level. It is suggested that herring meal may have a special promoting effect on the de novo synthesis of the enzymes concerned. As a practical consequence of the experiments it must be emphasized that serum determinations of these enzymes for diagnostic purposes will give a false picture of the clinical condition of sheep fed even moderate amounts of herring meal.     

Role of Energized States of (Na<Superscript>+</Superscript> + K<Superscript>+</Superscript>-ATPase in the Sodium Pump

IN an earlier paper¹ we have presented a model for a sodium pump based on the operation of the adenosine triphosphatase component of membranes which is sensitive to ouabain and is activated by sodium and potassium; that is (Na⁺+K⁺)-ATPase. We attempted to correlate the biochemical properties of this enzyme system as they were then known with the essential properties of Na⁺ transport systems. The model suggested further experiments which could clarify the role of (Na⁺ + K ⁺)-ATPase in ion transport and some experimental evidence is now available for the stoichiometry of ouabain binding to isolated enzyme preparations^2,3 although differences in the experimental techniques which have been used make the data equivocal.     

Bridging experiments, models and simulations: an integrative approach to validation in computational cardiac electrophysiology

Computational models in physiology often integrate functional and structural information from a large range of spatiotemporal scales from the ionic to the whole organ level. Their sophistication raises both expectations and skepticism concerning how computational methods can improve our understanding of living organisms and also how they can reduce, replace, and refine animal experiments. A fundamental requirement to fulfill these expectations and achieve the full potential of computational physiology is a clear understanding of what models represent and how they can be validated. The present study aims at informing strategies for validation by elucidating the complex interrelations among experiments, models, and simulations in cardiac electrophysiology. We describe the processes, data, and knowledge involved in the construction of whole ventricular multiscale models of cardiac electrophysiology. Our analysis reveals that models, simulations, and experiments are intertwined, in an assemblage that is a system itself, namely the model-simulation-experiment (MSE) system. We argue that validation is part of the whole MSE system and is contingent upon 1) understanding and coping with sources of biovariability; 2) testing and developing robust techniques and tools as a prerequisite to conducting physiological investigations; 3) defining and adopting standards to facilitate the interoperability of experiments, models, and simulations; 4) and understanding physiological validation as an iterative process that contributes to defining the specific aspects of cardiac electrophysiology the MSE system targets, rather than being only an external test, and that this is driven by advances in experimental and computational methods and the combination of both.     

O-O bond cleavage and alkane hydroxylation in methane monooxygenase

Several new aspects of the O-O bond cleavage and alkane hydroxylation mechanisms have been studied by hybrid density functional theory in this reinvestigation of methane monooxygenase. As concerning key intermediates in these reactions, a new important low-lying state is found, described either as Fe2(III,V) or as Fe2(III,IV)O. A fully optimized transition state for O-O bond cleavage has been determined. It is suggested that the large difference in optimal size (as determined in gas phase) of the complex, before and after the O-O bond cleavage, leads to an additional driving force for the reaction, not considered previously. The strain of the enzyme is estimated to lead to a driving force in the forward direction of about 5 kcal/mol, which could explain some of the pH dependence found in recent experiments. For the hydroxylation reaction, a clean hydrogen abstraction transition state leading to a substrate radical is again found, in contrast to interpretations of radical clock experiments. An explanation, based on new results, is suggested that could account for both the experimental and theoretical results.     

Isozyme variation in roe deer in relation to their population history in Britain

The level of genetic variation in 245 roe deer (Cupreolus cupreolus L.), taken from 15 populations across Britain, was estimated by investigating electrophoretic polymorphism at 15 enzyme loci. The proportion of polymorphic loci and the average heterozygosity of roe deer in Britain were estimated as 13.3% and 3.2%, respectively. Allele frequencies at one locus suggested the existence of an east-west cline of an allelic variant in southern Britain which may have originated from an introduction of roe from continental Europe in the last century. These results are discussed in relation to the theoretical expectations for this species and the documented historical background of roe deer inBritain.     

Biophysical aspects of neutron scattering from vibrational modes of proteins

This review describes a major portion of the published work on neutron scattering experiments aimed at measuring large scale motions in proteins. The importance of these motions for enzyme function and oxygen transport is indicated. The theory applicable to each type of neutron scattering measurement is given and results are discussed with a view to biological relevance. New experiments are suggested and a comparison of neutron scattering data is made with results from other techniques such as raman scattering, infrared absorption, photolysis and molecular dynamics simulations.     

Glutathione: an endogenous substrate for thiopurine methyltransferase?

An investigation was initiated to determine if glutathione is an endogenous substrate for thiopurine methyltransferase. Glutathione, as well as S-methylglutathione, were each capable of inhibiting the enzyme in a concentration-dependent manner, which suggested competitive and product inhibition, respectively. However, radiochromatography revealed that S-methylation of glutathione is not a catalytic activity of this sulfhydryl transmethylase. Subsequent experiments indicated that the inhibitory effects of both glutathione and S-methylglutathione on thiopurine methyltransferase may be due to their acidic natures, changing the reaction mixture pH away from the optimal range for the enzyme.     

Pore diffusion model for a two-substrate enzymatic reaction: Application to galactose oxidase immobilized on porous glass particles

An analysis of the pore diffusion model involving a two-substrate enzymatic reaction is presented. The resulting equations have been applied to the case of galactose oxidase catalyzed oxidation of galactose when the enzyme is immobilized on porous glass particles. The physical constants of the system were obtained by theoretical predictions and the enzyme concentration in the porous medium was derived from the experimental results. The calculations were performed with the assumption that the kinetic parameters of the enzyme remain unchanged upon immobilization. The theoretically calculated effectiveness factors were compared with the experimental effectiveness factors determined from the batch kinetic experiments and were found to be in agreement. The results are presented as effectiveness factor plots graphed as functions of bulk galactose and oxygen concentrations. The model was extended in order to study the effect of external mass transfer coefficients and pore enzyme concentrations on the effectiveness factors.     

Horseradish peroxidase—catalyzed hydroxylation of phenol: I. Thermodynamic analysis

We analyzed the horseradish peroxidase (HRP)—catalyzed hydroxylation of phenol in the presence of dihydroxy-fumaric acid and oxygen. All of the intermediate forms of the enzyme are reviewed. The last step of hydroxylation, consisting of the production of OH^• radicals that further react on phenol, is emphasized. Possible OH^• radicals production reactions were compiled and analyzed with respect to the available thermodynamic data. Some results of electrochemical experiments were also used to choose the correct set of reactions. At the end of analysis only two reactions for producing OH^• seemed to be consistent with the thermodynamic and experimental data. Neither of these reactions involved compound III or any other intermediate form of HRP. The last step of hydroxylation was thus totally independent of the pure catalytic cycle of the enzyme. As a consequence, HRP cannot be used as an hydroxylation enzyme in place of the P₄₅₀ cytochrome, as is sometimes suggested.     

Origin of Polysaccharide Depolymerase Associated with Bacteriophage Infection

Analyses, by construction of phage growth curves, indicated that the polysaccharide depolymerase was synthesized by Pseudomonas aeruginosa strains B and BI after infection with phage 2. The kinetics of biosynthesis of the depolymerase were found to parallel closely the rate of formation of phage-directed virions, and alterations in the experimental conditions of infection were reflected by alterations in the production of enzyme. Infection with other Pseudomonas phages, 84 and 1197, did not result in the synthesis of depolymerase. The enzyme was not detectable in uninfected cultures, and no evidence was obtained for the existence of inhibitors or activators of enzyme activity in extracts of uninfected or infected cells. The results of experiments employing chloramphenicol or an auxotorphic mutant (BI arg(-)) suggested that protein synthesis de novo was essential for production of the enzyme. Various mutants of phage 2 (pdp(1), pdp(2)), which alter the synthesis of the polysaccharide depolymerase, have been isolated. These experimental results strongly support the role of the phage genome in the synthesis of this enzyme.     

Properties of Crystalline α-Glucosidase from Mucor javanicus

Substrate and inhibitor specificities, and transglucosylation action of crystalline α-glucosidase from the mycelia of Mucor javanicus have been investigated. The enzyme hydrolyzed maltose, methyl-α-maltoside, and soluble starch liberating glucose, but little or not phenyl-α-glucoside, methyl-α-glucoside, sucrose, isomaltose, panose and dextran. The enzyme hydrolyzed phenyl-α-maltoside to glucose and phenyl-α-glucoside. The enzyme acted also as a glucosyltransferase when it was incubated with glucosyl donor such as maltose. Maltotriose was the principal transglucosylation product formed from maltose. The enzyme also catalyzed transglucosylation from maltose to riboflavin, pyridoxine, esculin and rutin. Tris and turanose inhibited the enzyme activity, but PCMB and EDTA did not. It is suggested that the enzyme activity is closely related to the histidine residue in the active center, from the inhibition experiments using diazonium-1-H-tetrazole and rose bengal.     

Validation and characterization of uninhibited enzyme kinetics performed in multiwell plates

Several systematic errors may occur during the analysis of uninhibited enzyme kinetic data using commercially available multiwell plate reader software. A MATLAB program is developed to remove these systematic errors from the data analysis process for a single substrate-enzyme system conforming to Michaelis-Menten kinetics. Three experimental designs that may be used to validate a new enzyme preparation or assay methodology and to characterize an enzyme-substrate system, while capitalizing on the ability of multiwell plate readers to perform multiple reactions simultaneously, are also proposed. These experimental designs are used to (i) test for enzyme inactivation and the quality of data obtained from an enzyme assay using Selwyn's test, (ii) calculate the limit of detection of the enzyme assay, and (iii) calculate Km and Vm values. If replicates that reflect the overall error in performing a measurement are used, the latter two experiments may be performed with internal estimation of the error structure. The need to correct for the systematic errors discussed and the utility of the proposed experimental designs were confirmed by numerical simulation. The proposed experiments were conducted using recombinant inducible nitric oxide synthase preparations and the oxyhemoglobin assay.     

Insights on a new PDI-like family: structural and functional analysis of a protein disulfide oxidoreductase from the bacterium Aquifex aeolicus

A potential role in disulfide bond formation in the intracellular proteins of thermophilic organisms has recently been attributed to a new family of protein disulfide isomerase (PDI)-like proteins. Members of this family are characterized by a molecular mass of about 26kDa and by two Trx folds, each comprising a CXXC active site motif. We report on the functional and structural characterization of a new member of this family, which was isolated from the thermophilic bacterium Aquifex aeolicus (AaPDO). Functional studies have revealed the high catalytic efficiency of this enzyme in reducing, oxidizing and isomerizing disulfide bridges. Site-directed mutagenesis experiments have suggested that its two active sites have similar functional properties, i.e. that each of them imparts partial activity to the enzyme. This similarity was confirmed by the analysis of the enzyme crystal structure, which points to similar geometrical parameters and solvent accessibilities for the two active sites. The results demonstrated that AaPDO is the most PDI-like of all prokaryotic proteins so far known. Thus, further experimental studies on this enzyme are likely to provide important information on the eukaryotic homologue.     

Kinetic analysis of a general model of activation of aspartic proteinase zymogens

Starting from a simple general reaction mechanism of activation of aspartic proteinase zymogens involving an uni- and a bimolecular simultaneous route, the time course equation of the concentration of the zymogen and of the activated enzyme have been derived. From these equations, an analysis quantifying the relative contribution to the global process of the two routes has been carried out for the first time. This analysis suggests a way to predict the time course of the relative contribution as well as the effect of the initial zymogen and activating enzyme concentrations, on the relative weight. An experimental design and kinetic data analysis is suggested to estimate the kinetic parameters involved in the reaction mechanism proposed. Finally, we apply some of our results to experimental data obtained by other authors in experimental studies of the activation of some aspartic proteinase zymogens.     

Functional characteristics of nuclear 5 alpha-reductase from rat ventral prostate

The subcellular distribution and functional characteristics of 5 alpha-reductase (3-oxo-5 alpha-steroid: NADP+ 4-ene-oxidoreductase, EC 1.3.1.22) from rat ventral prostate were studied and compared to the 5 alpha-reductase from female rat liver. Tissue fractionation retained main enzymic activity in the microsomal fraction of rat liver, while 5 alpha-reductase from rat prostate was localized in the nuclear membrane with a specific activity 160 times that of the initial homogenate. The purity of nuclear envelopes was checked by electron microscopy. Solubilization experiments indicated that the hepatic 5 alpha-reductase is attached to the endoplasmic reticulum as a peripheral protein, while the nuclear prostatic enzyme is an integral membrane protein. Incubation experiments with phospholipases suggested a decisive role of the surrounding phospholipids for the prostatic enzyme activity. To elucidate the characteristics of hydrogen transfer of the enzyme, the effect of flavins and different other cofactors on 5 alpha-reductase activity in isolated prostatic nuclei were studied. Our findings indicate that in rat ventral prostate the conversion of testosterone to 5 alpha-dihydrotestosterone proceeds by a direct hydrogen transfer from NADPH to testosterone. Concerning these parameters the behaviour of hepatic 5 alpha-reductase is absolutely different from the prostatic enzyme. The localization of 5 alpha-reductase within the nuclear envelope of rat ventral prostate as an integral membrane protein seems to be of physiological significance with regard to the action of androgens.