Comparative studies on dihydrofolate reductases from Plasmodium falciparum and Aotus trivirgatus.

Dihydrofolate reductase (E.C. 1.5.1.3) from Plasmodium falciparum and from its host, the owl monkey (Aotus trivirgatus), were partially purified and characterized. The molecular weight of the parasite enzyme was estimated to be over 10 times as high as that of the host enzyme. The host enzyme had 2 pH optima whereas the parasite enzyme only one. The activity of the host enzyme was greatly stimulated by KCl and urea, while that of the parasite enzyme was inhibited at high concentrations of such chaotropic agents. Km of the parasite enzyme was significantly higher than that of the host enzyme. The parasite enzyme had much lower Ki for pyrimethamine than the host enzyme. Dihydrofolate reductases isolated from pyrimethamine-resistant and pyrimethamine sensitive strains of P. falciparum were found to be similar.     

Enzymatic modification of vegetable protein: Immobilization of Penicillium duponti enzyme on reconstituted collagen and the use of the immobilized-enzyme complex for solubilizing vegetable protein in a recycle reactor

Penicillium duponti enzyme was immobilized on reconstituted collagen by macromolecular complication, impregnation, and covalent crosslinking techniques. The immobilization of the enzyme on collagen has a twofold purpose: (1) providing a protein microenvironment for the proteolytic enzyme; and (2) extending the useful life the enzyme once immobilized on the collagen matrix. Two types of collagen were used, one produced by the United States Department of Agriculture and the other produced by FMC. The USDA collagen contained unhydrolyzed telepeptide linkages and required pretreatment to reduce collagenaselike activity of the enzyme. Activity analysis of the immobilized enzyme complex showed that membranes with enzyme loading less than 10 mg enzyme/g of wet membrane in the reactor were dimensionally stable. The degree of crosslinking was an important parameter. Membranes with structural opening up to three times the initial dry thickness were found to be the maximum limit for controlled release of enzyme from the collagen membrane during enzymatic reaction. Higher activities and better stability of the enzyme in collagen membrane were found for covalent crosslinking of the enzyme to treated collagen films. The hydrolysis of soybean vegetable protein with the immobilized enzyme in a recycle reactor at enzyme loading of mg/g of wet membrane at 40°C, pH 3.4, produced 56.5% of soluble protein in 10h. The production is equivalent to 1.84 h total contact time between the substrate and the immobilized enzyme. The average productivity based on a stable enzyme activity and 20g of dry membrane was 329 mg of protein/g/mg of active enzyme immobilized. The productivity of the free enzyme in a batch reactor was 62.5 mg protein/h/mg enzyme.     

Immobilization–stabilization of a new recombinant glutamate dehydrogenase from <Emphasis Type="Italic">Thermus thermophilus</Emphasis>

The genome of Thermus thermophilus contains two genes encoding putative glutamate dehydrogenases. One of these genes (TTC1211) was cloned and overexpressed in Escherichia coli. The purified enzyme was a trimer that catalyzed the oxidation of glutamate to alpha-ketoglutarate and ammonia with either NAD+ or NADP+ as cofactors. The enzyme was also able to catalyze the inverse reductive reaction. The thermostability of the enzyme at neutral pH was very high even at 70 degrees C, but at acidic pH values, the dissociation of enzyme subunits produced the rapid enzyme inactivation even at 25 degrees C. The immobilization of the enzyme on glyoxyl agarose permitted to greatly increase the enzyme stability under all conditions studied. It was found that the multimeric structure of the enzyme was stabilized by the immobilization (enzyme subunits could be not desorbed from the support by boiling it in the presence of sodium dodecyl sulfate). This makes the enzyme very stable at pH 4 (e.g., the enzyme activity did not decrease after 12 h at 45 degrees C) and even improved the enzyme stability at neutral pH values. This immobilized enzyme can be of great interest as a biosensor or as a biocatalyst to regenerate both reduced and oxidized cofactors.     

Purification and biochemical characterization of recombinant rat liver phenylalanine hydroxylase produced in Escherichia coli.

Phenylalanine hydroxylase, important in phenylalanine metabolism in mammals, is regulated through short-term (activation) and long-term (induction) mechanisms. To help elucidate the structure-function relationships involved in the activation of this enzyme, we have isolated and characterized full-length cDNA clones to rat phenylalanine hydroxylase. Recombinant rat phenylalanine hydroxylase was placed into an expression vector in Escherichia coli. The enzyme has been purified to homogeneity and its physical and catalytic properties have been characterized. The molecular weight and the fluorescence emission spectrum of the recombinant enzyme were identical to those of the native enzyme. The recombinant enzyme could be activated by incubation with phenylalanine or lysolecithin or by phosphorylation, as is the rat liver enzyme. The extent of activation is the same as that for the native enzyme in each case except for phenylalanine, which activates the recombinant enzyme only 5- to 10-fold rather than the 15- to 30-fold activation observed with the native enzyme. The kinetic constants determined for the recombinant enzyme are also essentially the same as those reported for the native enzyme. We conclude that this enzyme is essentially identical to the native enzyme and should be very useful in the future study of this important hydroxylase.     

Enzyme synthesis in the regulation of hepatic `malic'' enzyme activity

A homogeneous preparation of ;malic' enzyme (EC 1.1.1.40) from livers of thyroxine-treated rats was used to prepare in rabbits an antiserum to the enzyme that reacts monospecifically with the ;malic' enzyme in livers of rats in several physiological states. Changes in enzyme activity resulting from modification of the state of the animal are hence due to an altered amount of enzyme protein. The antiserum has been used to precipitate out ;malic' enzyme from heat-treated supernatant preparations of livers from both adult and neonatal rats, in a number of physiological conditions, that had been injected 30min earlier with l-[4,5-(3)H]leucine. The low incorporations of radioactivity into the immunoprecipitable enzyme have permitted the qualitative conclusion that changed enzyme activity in adult rats arises mainly from alterations in the rate of enzyme synthesis. The marked increase in ;malic' enzyme activity that occurs naturally or as a result of thyroxine treatment of the weanling rat is likewise due to a marked increase in the rate of enzyme synthesis possibly associated with a concurrent diminished rate of enzyme degradation.     

Limited proteolysis of maize NADP-malic enzyme

The incubation of maize malic enzyme at 37 degrees C with trypsin at a ratio of 150:1 of malic enzyme to trypsin caused rapid and complete inactivation of enzyme activity. The inactivation was caused by fairly specific cleavage of the enzyme monomer (62 kDa) into 40 kDa and 20 kDa fragments. The intensity of 40 kDa band increased with the time of treatment of enzyme with trypsin from 2 to 30 min. Substrates, especially NADP (25 microM) provided almost total protection against trypsin inactivation of the enzyme activity. The studies carried out with various other endoproteases indicated that endoprotease Lys-C was most effective in inactivating malic enzyme activity. The kinetic properties of the truncated enzyme have been studied. The Km value for malate in case of native and modified enzyme was found to be identical. Km NADP for the modified enzyme was slightly higher indicating that after proteolysis the enzyme affinity for NADP had decreased. Limited proteolysis with trypsin did not show any appreciable change in fluorescence properties of the modified enzyme. Binding of NADPH to the enzyme was not affected after modification.     

DISTRIBUTION AND PROPERTIES OF ANGIOTENSIN CONVERTING ENZYME OF RAT BRAIN

Abstract— Angiotensin converting enzyme of rat brain was studied using Hip-His-Leu as substrate. The highest specific activity of the enzyme was associated with the microsomal fraction. The specific activity of the microsomal enzyme in several regions of the rat brain varied significantly. For example, the specific activities of the striatal and pituitary enzymes were about 10-fold greater than that of the cerebral cortical enzyme. The enzyme required chloride ion; moreover, activity was inhibited in the presence of disodium EDTA or O-phenanthroline, effects suggesting that the converting enzyme of brain is a metalloprotein. SQ-20881, a nonapeptide that inhibits converting enzyme in peripheral tissue, was a potent inhibitor of the enzyme of brain. In addition to Hip-His-Leu, the microsomal fraction was capable of liberating C terminal dipeptides from angiotensin I, Hip-Gly-Gly and Z-Gly- Gly-Val. The broad substrate specificity of the enzyme suggests that, in addition to the possible contribution of the enzyme to the brain renin-angiotensin system, other naturally occurring peptides might also be substrates for the enzyme.     

Synthesis and degradation of phenylalanine ammonia-lyase of Rhodosporidium toruloides.

The regulation of the enzyme phenylalanine ammonia-lyase (PAL), which is of potential use in oral treatment of phenylketonuria, was investigated. Antiserum against PAL was prepared and was shown to be monospecific for the enzyme by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The native enzyme and two inactive mutant forms of the enzyme were purified to homogeneity by immunoaffinity chromatography, using anti-PAL immunoglobulin G-Sepharose 4B. Both mutant enzymes contained intact prosthetic groups. The formation of PAL catalytic activity after phenylalanine was added to yeast cultures was paralleled by the appearance of enzyme antigen. During induction, uptake of [3H]leucine into the enzyme was higher than uptake into total protein. Our results are consistent with de novo synthesis of an enzyme induced by phenylalanine, rather than activation of a proenzyme. The half-lives of PAL and total protein were similar in both exponential and stationary phase cultures. No metabolite tested affected the rate of enzyme degradation. Glucose repressed enzyme synthesis, whereas ammonia reduced phenylalanine uptake and pool size and so may repress enzyme synthesis through inducer exclusion. The synthesis of enzyme antigen by a mutant unable to metabolize phenylalanine indicated that this amino acid is the physiological inducer of the enzyme.     

The purification and properties of the trimethoprim-resistant dihydrofolate reductase mediated by the R-factor, R388.

The R-factor R388 mediates the production of a trimethoprim-resistant dihydrofolate reductase. This enzyme has a different molecular weight and pH profile to the trimethoprim-sensitive enzyme of the Escherichia coli host. The R-factor mediated enzyme was separated completely from the host E. coli enzyme by DEAE-cellulose ion-exchange chromatography. The purified R-factor enzyme was about 20 000 times less susceptible to trimethoprim than the E. coli enzyme and although it was inhibited competitively by trimethoprim, its inhibitor constant (Ki) was 20 000 times greater than that of the host enzyme. The R388 and E. coli enzymes also differed in their substrate specificity requirements. In addition, the R388 enzyme suprisingly conferred high level resistance to the broad spectrum dihydrofolate reductase inhibitor, amethopterin. The possible origins of the R388 enzyme are discussed.     

Fabrication of butyrylcholinesterase sensor using polyurethane-based ion-selective membranes.

A simple method of enzyme immobilization was investigated which is useful for fabrication of enzyme sensors based on polymeric ion-selective membranes. The enzyme membrane was built by coating a thin hydrophilic polyurethane (HPU) film directly mixed with an enzyme over an underlying polyurethane (PU)-based ion-selective membrane. This highly simple method of enzyme immobilization was applied to the fabrication of a potentiometric butyrylcholinesterase-based biosensor for the determination of organophosphorus pesticides. The enzyme was well entrapped within the HPU film and the intrinsic potentiometric response of the underlying ion-selective PU membrane was not influenced significantly by the outer HPU/enzyme membrane. The enzyme electrode was optimized by changing systematically the composition of the enzyme membrane to evaluate the effect of the changes on sensor response. The sensor was successfully applied to the analysis of paraoxon, an organophosphorus pesticide.     

Purification and characterization of 2,6-beta-D-fructan 6-levanbiohydrolase from Streptomyces exfoliatus F3-2

Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a beta-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60 degrees C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50 degrees C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only beta-2,6-linkage of levan, but also beta-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-beta-D-fructan 6-levanbiohydrolase (EC 3.2.1.64).     

Potent fibrinolytic enzyme from a thermophilic Streptomyces megasporus strain SD5

As a therapeutic agent in thrombosis the fibrinolytic enzymes are of interest and the search for a new enzyme continues. A strong fibrin-specific fibrinolytic enzyme was purified from the cell-free spent broth of thermophilic Streptomyces megasporus strain SD5. The crude enzyme was concentrated using ammonium sulphate, dialysis and lyophilization. Approximately 0.11 mg ml(-1) crude enzyme with a specific activity of 4.2 U microg(-1) was obtained. Post-electrophoretic reactivity revealed a monomeric form of the enzyme with a molecular weight of 35 kDa. The optimum pH and temperature for production of the enzyme were 8 and 55 degrees C, respectively. The enzyme was resistant to a broad range of pH ranging from 6 to 9 and temperature ranging from 37 to 60 degrees C. The enzyme was a chymotrypsin-like serine peptidase and the activity of the enzyme was N-terminal-dependent. The in vitro clot lysis by the enzyme at 37 degrees C was encouraging.     

Aminoacetone oxidase from goat liver. Formation of methylglyoxal from aminoacetone

An enzyme which oxidizes aminoacetone to methylglyoxal has been purified from the particulate fraction of goat liver. Polyamines, such as spermidine and spermine, are also good substrates for this enzyme. The pH optimum for aminoacetone oxidation was found to be 8.2. The apparent Km values of the enzyme for aminoacetone and spermidine were 0.009 and 0.095 mM, respectively. The subunit molecular weight of the enzyme was 93,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The apparent molecular weight of the native enzyme was 186,000 by gel filtration. The enzyme is highly sensitive to carbonyl group reagents. The enzyme is not inhibited by monoamine and diamine oxidase inhibitors.     

Immobilization of the exo-maltohexaohydrolase by the irradiation method

Exomaltohexaohydrolase (E.C.3.2.1.98) was immobilized by radiocopolymerization of some synthetic monomers which were mixed in various combinations. Irradiation was carried out while the mixture of monomers and enzymes was frozen in petroleum ether-dry-ice bath. Recovery of the immobilized enzyme was 44-75%.The optimum pH of the enzyme slightly shifted to the acidic side. The pH stability was improved remarkably by immobilization. The enzyme was stable retaining more than 90% of its original activity in the range pH 4-11. The optimum reaction temperature of the enzyme increased about 2 degrees C. Heat stability was also improved by immobilization, and that the enzyme retained about 40% of its original activity after treatment at 75 degrees C for 15 min. The immobilized enzyme was stable to the repeated use of 20 cycles. The K(m) value of the enzyme for short-chain amylose was almost the same as that of native enzyme. When soluble starch was used as the substrate, the K(m), value of the enzyme was three times as large as that of native enzyme. Effects of various metal ions and inhibitors on the immobilized enzyme were also studied compared to the native enzyme.     

Effects of phospholipids on L-lactate dehydrogenase from membranes of Escherichia coli. Activation and stabilization of the enzyme with phospholipids.

Membrane-bound L-lactate dehydrogenase was freed from the detergent used during purification. The detergent-free enzyme had about one-half the specific activity of the enzyme in 1.0% Tween 80, and was only partially sensitive to the specific antibody. This enzyme was activated about 3-fold with phosphatidylglycerol, cardiolipin, or a mixture of phospholipids. The phospholipid-activated enzyme had a similar Km value for L-lactate to that of the membrane enzyme and was completely inhibited by the specific antibody. On heat treatment, the phospholipid-activated enzyme was more stable than detergent-free enzyme and was as stable as membrane-bound enzyme. The alpha helical content of the enzyme increased 1.7-fold during preincubation with these lipids and the alpha helix became more stable during heat treatment than that of the detergent-free enzyme. These results suggest that the enzyme showed monomolecular dispersion in the lipid bilayer and that its conformation, including its active site and secondary structure, was different from that of the detergent-free enzyme. Phosphatidylethanolamine, dilauroyl lecithin and lecithin from egg yolk had none of the above effects on the activity or the secondary structure of the enzyme. On the other hand, mixtures of each of these lipids and cholate had essentially similar effects to phosphatidylglycerol.     

Immobilized phospholipase A2 from cobra venom. Prevention of substrate interfacial and activator effects

The activation of cobra venom phospholipase A2 by activators (containing phosphorylcholine moieties) appears to depend upon the aggregation state of the enzyme, and the presence of a lipid-water interface. The characteristics of this activation were studied by comparing the behavior of the enzyme immobilized on an agarose gel to that of the soluble enzyme. The immobilized enzyme displays only a few per cent of the soluble enzyme activity toward micellar dipalmitoyl-phosphatidylcholine (PC). However, the relative loss of activity is much less with micellar dipalmitoylphosphatidylethanolamine or soluble diheptanoyl-PC. The affinity for Ca2+ is increased about 10-fold by immobilization while the apparent pKa of the enzyme is decreased by 0.5-0.8 pH units. Activation energies are similar for the two enzyme forms and are independent of the physical state of the substrate used. Catalytic constants of the enzyme toward monomeric PC are not changed by immobilization. Yet, activators of the soluble enzyme have negligible effect on the immobilized enzyme, either in the presence or absence of an interface. Monomeric activators promote the binding of the soluble enzyme to the immobilized form. Apparently, immobilization mainly produces monomerically constrained enzyme which cannot be activated under any condition, whereas normally, activators in the presence of lipid-water interfaces induce the formation of enzyme dimers or possibly higher order aggregates.     

Substrate-dependent dissociation of malate thiokinase.

Malate thiokinase has been purified to apparent homogeneity by employing conventional purification techniques along with affinity chromatography. The enzyme is composed of two nonidentical subunits (alpha subunit Mr=34,000, beta subunit Mr=42,500) to yield an alpha 4 beta 4 structure for the native enzyme. Phosphorylation of the enzyme by ATP occurs exclusively on the alpha subunit. The phosphorylated enzyme is acid labile and base stable consistent with phosphorylation of a histidine residue. Dephosphorylation of the enzyme is promoted by ADP, succinate, malate, and coenzyme A plus inorganic phosphate. Phosphorylation of the enzyme leads to a reversible change in the sedimentation properties of the enzyme; the native enzyme exhibits an S20,w of approximately 10, whereas the phosphoenzyme exhibits an S20,w of approximately 7. Formation of the 7 S form of the enzyme is also observed when coenzyme A and succinyl-CoA interact with the enzyme. The ratio of alpha to beta subunits in both the 10 S and 7 S forms of the enzyme is approximately 1.0, suggesting that the 7 S form of the enzyme has an alpha 2 beta 2 structure.     

Differences in redox and kinetic properties between NAD-dependent and O2-dependent types of rat liver xanthine dehydrogenase

Reductive titrations of a NAD-dependent type (type-D) and an O2-dependent type (type-O) of rat liver xanthine dehydrogenase showed that only the type-D enzyme formed a pronounced stable FAD semiquinone (FADH*). The FAD semiquinone was less stabilized in the presence of NAD. The Vmax value for xanthine-NAD activity of type-D enzyme was close to that for xanthine-O2 activity of type-O enzyme, while the Vmax value for xanthine-O2 activity of type-D enzyme was about one-fourth of that of type-O enzyme. The Km value for O2 of type-D enzyme was about five times as large as that of type-O enzyme. The absorbance spectrum of type-D enzyme during turnover with xanthine and O2 as substrates showed a considerable amount of FADH* formation, but that with xanthine and NAD as substrates showed only a negligible one. Low xanthine-O2 activity of type-D enzyme, as compared with that of type-O enzyme, seems to be explained by the conformational change occurring in conversion from type-O to type-D enzyme, which results in different reactivity of FAD to molecular oxygen and a higher fraction of FADH* during turnover. The binding of NAD may possibly increase the fraction of FADH2, resulting in a Vmax value of xanthine-NAD activity almost as high as that of xanthine-O2 activity of type-O enzyme.     

Immunological characterization of maize starch branching enzymes

Highly purified fractions of three starch branching enzymes from developing maize (Zea mays L.) endosperm were used to prepare antisera in rabbits. In double diffusion experiments, no immunoprecipitate was observed when branching enzyme IIa or IIb was tested against branching enzyme I antiserum. No immunoprecipitate was formed when branching enzyme I was tested against branching enzyme IIa or IIb antiserum. Increasing amounts of antisera in the above combinations also failed to inhibit enzyme activity. Branching enzyme IIa antiserum cross-reacted and formed spurs with branching enzyme IIb when compared with branching enzyme IIa antigen. Comparison of branching enzyme IIb antiserum with branching enzyme IIa also resulted in an immunoprecipitate. Increasing levels of branching enzyme IIa antiserum inhibited branching enzyme IIb as did the reciprocal combination. The data indicated that branching enzymes IIa and IIb are immunologically similar while branching enzyme I is distinct. The data supports the classification of starch branching enzymes based on genetic, kinetic, and chromatographic properties.     

Purification and properties of ferrochelatase from Chironomidae larvae

Ferrochelatase with an M_r of 42,700 Da and a pI of 7.35 has been purified to homogeneity from chironomidae larvae. The activity of the enzyme reached maximum at pH 7.8 and decreased with the increase of pH. The enzyme activity varied with temperature and showed maximum activity around 37°C. The purified enzyme was active towards protoporphyrin but inactive towards other porphyrins. The specific enzyme activity of ferrochelatase from chironomidae is about 10-fold higher than that of the rat. Electrophoresis of the purified fractions shows that the enzyme contains only one single polypeptide. The soluble ferrochelatase contained one mole of iron in each mole of the enzyme. The N-terminal sequence analysis of the enzyme shows a high percentage of conserved regions of the enzyme among other species. The enzyme properties are similar to those of the mammalian ferrochelatases except with slightly higher specific activity. Chironomidae ferrochelatase appeared to be more heat resistant and less susceptible than its mammalian equivalent to inhibition by lead.