Inhibition of alkaline phosphatase by sialic acid.

The interaction of human organ alkaline phosphatases (orthophosphoric-monoester phosphohydrolases (alkaline optimum), EC 3.1.3.1) with sugars was studied. Hexosamines, N-acetylneuraminic acid (NANA or sialic acid), N-acetylmuramic acid and N-acetylglycolylneuraminic acid inhibited human organ alkaline phosphatase activities. Of these, sialic acid was the most effective inhibitor. The pH profiles for the enzymes in the absence and presence of sialic acid were similar. The sialic acid - enzyme complex was more heat stable than the free enzyme between 20 and 45 degrees C. Lineweaver-Burk plots of 1/v versus 1/S at various concentrations of sialic acid showed intersecting straight lines indicating that the mechanism of inhibition was a mixed type. The Ki value obtained from the plots of 1/v versus the square of sialic acid concentration was 0.07 mM for the hepatic, sialidase-treated hepatic, and intestinal alkaline phosphatases. The respective Hill coefficients varied somewhat with the alkaline phosphatase isoenzyme. Hyperbolic curves were obtained when the percentage of remaining activity was plotted against the substrate concentration at different concentrations of sialic acid. The Hill coefficient was lowered in the presence of sialic acid. The sialidase-treated hepatic enzymes used gave the most effective conversion. Partial denaturation of the enzyme with urea, or pronase digestion had a little if any effect on the sialic acid inhibition with constant time.     

The cytosolic and glycosomal glyceraldehyde-3-phosphate dehydrogenase from Trypanosoma brucei. Kinetic properties and comparison with homologous enzymes

The protozoan haemoflagellate Trypanosoma brucei has two NAD-dependent glyceraldehyde-3-phosphate dehydrogenase isoenzymes, each with a different localization within the cell. One isoenzyme is found in the cytosol, as in other eukaryotes, while the other is found in the glycosome, a microbody-like organelle that fulfils an essential role in glycolysis. The kinetic properties of the purified glycosomal and cytosolic isoenzymes were compared with homologous enzymes from other organisms. Both trypanosome enzymes had pH/activity profiles similar to that of other glyceraldehyde-3-phosphate dehydrogenases, with optimal activity around pH 8.5-9. Only the yeast enzyme showed its maximal activity at a lower pH. The glycosomal enzyme was more sensitive to changes in ionic strength below 0.1 M, while the cytosolic enzyme resembled more the enzymes from rabbit muscle, human erythrocytes and yeast. The affinity for NAD of the glycosomal enzyme was 5-10-fold lower than that of the cytosolic, as well as the other enzymes. A similar, but less pronounced, difference was found for its affinity for NADH. These differences are explained by a number of amino acid substitutions in the NAD-binding domain of the glycosomal isoenzyme. In addition, the effects of suramin, gossypol, agaricic acid and pentalenolactone on the trypanosome enzymes were studied. The trypanocidal drug suramin inhibited both enzymes, but in a different manner. Inhibition of the cytosolic enzyme was competitive with NAD, while in the case of the glycosomal isoenzyme, with NAD as substrate, the drug had an effect both on Km and Vmax. The most potent inhibitor was pentalenolactone, which at micromolar concentrations inhibited the glycosomal enzyme and the enzymes from yeast and Bacillus stearothermophilus in a reversible manner, while the rabbit muscle enzyme was irreversibly inhibited.     

The mechanism of peroxidase-mediated cytotoxicity. I. Comparison of horseradish peroxidase and lactoperoxidase

The kinetics of the cytolytic activity expressed by lactoperoxidase and horseradish peroxidase toward erythrocytes in the presence of H2O2 and iodide have been investigated at physiological pH. The action of both enzymes was found to be very similar with respect to their kinetic mechanisms. Both enzymes showed saturation kinetics at higher enzyme concentrations under conditions where substrate concentrations were not limiting. Optimal concentrations of H2O2 and iodide were found to be 40 and 25 microM, respectively, for both enzymes. Higher concentrations of H2O2 inhibited the cytolytic activity. The pH dependence of the cytolytic reaction is also very similar for both enzymes, showing maximal activity at about pH 6.3. Moreover, the cytolytic activities of both enzymes were inhibited by tyrosine, tryptophan, cysteine, and to a lesser extent by histidine. We conclude from these data that the mechanisms of horseradish peroxidase and lactoperoxidase in promoting the lysis of erythrocytes are closely related if not identical.     

Biochemical and Immunological Properties of a Membrane-Bound Brain Metalloendopeptidase: Comparison with Thermolysin-Like Kidney Neutral Metalloendopeptidase

Abstract: Membrane-bound neutral metalloendopeptidase ("enkephalinase") was purified from rabbit brain and compared with a homogeneous preparation of a similar enzyme (EC 3.4.24.11) isolated from rabbit kidney. The two enzymes had the same pH optimum and the same apparent molecular weight. They showed identical specificity toward several synthetic substrates and cleaved both Met- and Leu-enkephalin at the Gly-Phe bond. Minor, but significant, differences were found between the two enzymes in the inhibitory constants determined for phosphoramidon and the N -[1( R,S )-carboxy-2-phenylethyl] derivatives of phenylalanyl and alanyl- p -aminobenzoate. A guinea pig antiserum obtained against the rabbit kidney enzyme showed strong crossreactivity with the rabbit brain enzyme when tested in an anticatalytic immunoinhibition assay. Ouchterlony immunodiffusion experiments gave a pattern of precipitation consistent with partial identity of the two enzymes. The kidney enzyme, however, seemed to contain antigenic determinants not present on the brain enzyme. The data indicate that the two enzymes are identical with respect to specificity, pH optimum, and molecular weight, but show minor, although significant, differences in interaction with active-site-directed inhibitors and specific antisera.     

Biochemical and immunological identity of the hepatic peroxisomal and microsomal trans-2-enoyl CoA hydratase bifunctional protein

In the present study, the hepatic microsomal and peroxisomal bifunctional trans-2-enoyl CoA hydratases were isolated and purified from rats treated with 2% di-(2-ethylhexyl)phthalate for 8 days. These two enzymes (microsomal and peroxisomal) were purified with the identical purification procedures and had identical molecular masses of 76 kDa. A single band was observed on an electrophoretic gel of an equimixture of the two proteins. Both preparations had identical pI's of 8.6 and pH optima of 6.0 for the dehydrogenase (reductase) and 7.5 for the hydratase activity. Two-dimensional gel analysis of an equimixture of the two preparations showed only one band. Ouchterlony double-diffusion analysis showed that an antibody raised against the purified microsomal enzyme interacted at a point with the peroxisomal enzyme, indicating immunologic identity. Western blot analysis demonstrated that the antibody formed a single band with total microsomal and peroxisomal fractions. The antibody inhibited the enzymatic activities of both preparations in a similar manner. Interestingly, the antibody had a markedly greater inhibitory effect on the reductase activity of the two enzyme preparations, and a much less inhibitory effect on the hydratase activity, suggesting that the antigenic determinants reside at or near the catalytic site of the reductase portion of the protein. These results suggest that the microsomal and peroxisomal bifunctional proteins are identical.     

Different molecular forms of D-ribulose-1,5-bisphosphate carboxylase from Rhodopseudomonas sphaeroides.

Ribulose-1,5-bisphosphate (Rbu-P2) carboxylase isolated from Rhodopseudomonas sphaeroides 2.4.1.Ga was separated into two different forms by DEAE-cellulose column chromatography. Both forms, designated Peak I and Peak II have been purified to homogeneity by the criterion of polyacrylamide disc-gel electrophoresis. The Peak I carboxylase has a molecular weight of 550,000, while the Peak II carboxylase is a smaller protein having a molecular weight of approximately 360,000. Sodium dodecyl sulfate electrophoresis revealed a large subunit for both enzymes which migrates similarly to the large subunit of spinach Rbu-P2 carboxylase. The Peak I enzyme also exhibited a small subunit having a molecular weight of 11,000. No evidence for a smaller polypeptide was found associated with the Peak II enzyme. Antisera prepared against the Peak I enzyme inhibited Peak I enzymatic activity, but had no effect on the activity of the Peak II enzyme. The two enzymes exhibited marked differences in catalytic properties. The Peak I enzyme exhibits optimal activity at pH 8.0 and is inhibited by low concentrations of 6-phosphogluconate, while the Peak II enzyme has a pH optimum of 7.2 and is relatively insensitive to 6-phosphogluconate.     

Comparative studies of mouse liver cathepsin B and an analogous tumor thiol proteinase

Cathepsin B (EC 3.4.22.1) and an analogous thiol proteinase were isolated from mouse liver and from a transplantable tumor induced by methylcholanthrene, respectively, by a sequence of steps involving salt fractionation and ion exchange and gel permeation chromatography. Both enzymes are capable of hydrolyzing N-benzyloxycarbonyl-L-Ala-L-Arg-L-Arg-4-methoxy-2-naphthylamide but are weakly active towards N-benzoyl-DL-arginine-2-naphthylamide. The specific activity of the liver enzyme towards these substrates is approximately 14 times greater than that of the tumor enzyme. Both enzymes show a single band with slight difference in mobility when subjected to gel electrophoresis at pH 4.5, but both exhibit a multiple banding pattern when examined by isoelectric focusing. The tumor enzyme has a somewhat higher molecular weight than the liver enzyme (33,000 versus 30,000) and possesses a slightly higher helical content (48% versus 40%) based on CD spectra. Both enzymes display maximum activity in the pH range of 5.5 to 7.0 and are irreversibly denatured above pH 7 and below pH 4. Both enzymes cross-react with antiserum towards the tumor enzyme. The liver enzyme displays a higher catalytic efficiency towards a series of oligopeptide substrates than the tumor enzyme, but is only one-third as active towards N-benzoyl-L-arginine-2-naphthylamide. Both proteinases exhibit similar patterns of inhibition by iodoacetate, chloroquine, leupeptin, antipain, and several peptide chloromethylketones. Despite what appear to be subtle differences in physical properties, amino acid composition data and peptide mapping revealed significant differences between these two enzymes reflective of extensive regions of non-identity. These results suggest that the tumor thiol protease and liver cathepsin B are products of separate genes and that the tumor enzyme is not likely an immediate precursor of the liver enzyme produced by post-translational modification.     

Hepatic cytochrome P-448 and epoxide hydrase: enzymes of nuclear origin are immunochemically identical with those of microsomal origin.

Nuclear and microsomal sources of hepatic cytochrome P-448 and epoxide hydrase were compared using antibodies made against the pure antigens isolated from rat liver microsomes. Both antigens were easily detected in detergent-solubilized nuclei and microsomes from rats using the Ouchterlony double-diffusion technique. Epoxide hydrase from either whole nuclei or nuclear envelope was immunochemically identical with the enzyme isolated from microsomes. Similarly, in rats pretreated with 3-methylcholanthrene, the cytochrome P-448 of nuclear origin was immunochemically indistinguishable from the enzyme derived from microsomes. These results establish the immunochemical identity of these hepatic nuclear and microsomal enzymes and provide a firm basis for applying the knowledge gained with the microsomal system of metabolism to the nuclear system.     

Isolation and characterization of two chondroitin lyases from Bacteroides thetaiotaomicron.

Two chondroitin lyases were isolated from the colon anaerobe Bacteroides thetaiotaomicron. Both enzymes had similar molecular weights (104,000 and 108,000) and similar isoelectric points (8.0 and 7.9, respectively). Both enzymes were active against chondroitin sulfates A, B, and C and unsulfated polysaccharides, such as chondroitin and hyaluronic acid, although one of the enzymes was twice as active against chondroitin as the other enzyme. Both had similar Km values for chondroitin sulfates A and C (40 to 70 micrograms/ml) and for chondroitin (300 to 400 micrograms/ml). Neither enzyme could degrade the highly sulfated mucopolysaccharide heparin, but heparin was a potent inhibitor of the activity of both enzymes. Although enzymes I and II were similar in many respects, a comparison of peptides resulting from partial digestion with N-chlorosuccinimide or papain demonstrated that the two proteins are not related.     

Non-specific alkaline phosphomonoesterases of eight species of digenetic trematodes.

Alkaline phosphatases from different trematodes occupying the same habitat have identical pH otima but different levels of enzyme activities. Isoparorchis hypselobagri, from the fish Wallago attu, shows four to six times more enzyme activity than Fasciolopsis buski, Gastrodiscoides hominis and Echinostoma malayanum, from the pig Sus scrofa, and Fasciola gigantica, Gigantocotyle explanatum, Cotylophoron cotylophorum and Gastrothylax crumenifer, from the buffalo Bubalus bubalis. At least two peaks of activity at different levels of pH were obtained for each trematode examined. Both Gastrodiscoides hominis and Isoparorchis hypselobagri enzymes had three peaks of alkaline phosphatase activity. The optimum temperature for maximum enzyme activity was 40 degrees C, above which rapid inactivation occurred. At temperatures below 40 degrees C, the enzymes of fish and mammalian trematodes did not behave similarly; I. hypselobagri enzyme being active over a wider range of temperature (20 degrees-40 degrees C. Various concentrations of KCN and arsenate proportionately inhibited enzyme activity. NaF Did not significantly influence enzyme activity, while Mg++ and Co++ acted as activators. The extent of inhibition or activation of enzyme activity of different trematodes varied, probably due to species differences. Both inhibition and activation of I. hypselobagri enzyme was higher than in the case of other trematodes.     

Allosteric and non-allosteric phosphofructokinases from Lactobacilli. Purification and properties of phosphofructokinases from L. plantarum and L. acidophilus.

Phosphofructokinase (ATP : D-fructose-6-phosphate 1 phosphotransferase, EC 2.7.1.11) from two different lactobacilli, Lactobacillus plantarum and Lactobacillus acidophilus were isolated and purified. Both enzymes have a molecular weight of 154 000 and consist of four subunits of identical size. Antisera from sheep immunized against the purified phosphofructokinase from L. plantarum showed immunologic cross reaction with the enzyme from L. acidophilus. In spite of the close molecular relationship indicated by the immunologic cross reaction, the kinetic behaviour of the two enzymes was strikingly different. Phosphofructokinase from L. plantarum showed pure Michaelis-Menten behaviour. Phosphofructokinase from L. acidophilus, however, showed sigmoidal substrate saturation curves for fructose 6-phosphate in the presence of slightly alkaline pH and high ATP concentrations; it was activated by fructose 1,6-biphosphate and inhibited by ADP. The results indicate that even enzymes which are structurally very similar may differ greatly with respect to their kinetic and regulatory properties and suggest that allosteric and non-allosteric phosphofructokinases have the same origin in evolution.     

Characteristics of yeast invertase immobilized on porous cellulose beads.

Invertase from Candida utilis was immobilized on porous cellulose beads by an ionic-quanidino bond. The immobilized invertase showed optimum activity between pH 4.0 and 5.4, while the free enzyme had a sharp optimum at pH 4.1. Both temperature profiles were fairly similar up to 55 degrees C. However, above this temperature the immobilized enzyme was more stable than the free enzyme. From the temperature data, the activation energies were found to be 7,322 and 4,052 cal/g mol for the free and the immobilized enzyme, respectively. Candida invertase shows characteristics of substrate inhibition. Both the Km and Ki for the free and the immobilized enzymes were determined. The apparent Ki for the immobilized invertase was much higher than the Ki of the free enzyme, suggesting a diffusion effect. Immobilized invertase molecules deep in the pores only see sucrose concentrations much less than the bulk concentrations. Immobilization, thus, offers certain processing advantages in this regard.     

Recombinant expression and purification of an enzymatically active cysteine proteinase of the protozoan parasite Entamoeba histolytica.

Cysteine proteinases and in particular cysteine proteinase 5 (EhCP5) of Entamoeba histolytica are considered important for ameba pathogenicity. To study EhCP5 in more detail a protocol was elaborated to produce considerable amounts of the enzyme in its active form. The protein was expressed in Escherichia coli as a histidine-tagged pro-enzyme and purified to homogeneity under denaturing conditions in the presence of guanidine-HCl using nickel affinity chromatography. Renaturation was performed by 100-fold dilution in a buffer containing reduced and oxidized thiols, which led to soluble but enzymatically inactive pro-enzyme. Further processing and activation was achieved in the presence of 10 mM DTT and 0.04% SDS at 37 degrees C. Recombinant enzyme (rEhCP5) was indistinguishable from native EhCP5 purified from E. histolytica lysates. Both runs in SDS-PAGE under reducing and nonreducing conditions at positions corresponding to 27 and 29 kDa, respectively, had the same pH optima and displayed similar specific activity against azocasein. Moreover, both enzymes were active against a broad spectrum of biological and synthetic substrates such as mucin, fibrinogen, collagen, human hemoglobin, bovine serum albumin, gelatin, human IgG, Z-Arg-Arg-pNA, and Z-Ala-Arg-Arg-pNA, but not against Z-Phe-Arg-pNA. The identity of rEhCP5 as a cysteine proteinase was confirmed by inhibition with specific cysteine proteinase inhibitors. In contrast, various compounds known to specifically inhibit aspartic, metallo, or serine proteinases had no effect on rEhCP5 activity.     

Characterization of lipases from Staphylococcus aureus and Staphylococcus epidermidis isolated from human facial sebaceous skin

Two staphylococcal lipases were obtained from Staphylococcus epidermidis S2 and Staphylococcus aureus S11 isolated from sebaceous areas on the skin of the human face. The molecular mass of both enzymes was estimated to be 45 kDa by SDS-PAGE. S2 lipase displayed its highest activity in the hydrolysis of olive oil at 32 degrees C and pH 8, whereas S11 lipase showed optimal activity at 31 degrees C and pH 8.5. The S2 lipase showed the property of cold-adaptation, with activation energy of 6.52 kcal/mol. In contrast, S11 lipase's activation energy, at 21 kcal/mol, was more characteristic of mesophilic lipases. S2 lipase was stable up to 45° C and within the pH range from 5 to 9, whereas S11 lipase was stable up to 50 degrees C and from pH 6 to 10. Both enzymes had high activity against tributyrin, waste soybean oil, and fish oil. Sequence analysis of the S2 lipase gene showed an open reading frame of 2,067 bp encoding a signal peptide (35 aa), a pro-peptide (267 aa), and a mature enzyme (386 aa); the S11 lipase gene, at 2,076 bp, also encoded a signal peptide (37 aa), pro-peptide (255 aa), and mature enzyme (399 aa). The two enzymes maintained amino acid sequence identity of 98-99% with other similar staphylococcal lipases. Their microbial origins and biochemical properties may make these staphylococcal lipases isolated from facial sebaceous skin suitable for use as catalysts in the cosmetic, medicinal, food, or detergent industries.     

The purification, properties and characterization of three forms of alpha-L-fucosidase from monkey brain.

alpha-L-Fucosidase (alpha-L-fucoside fucohydrolase, EC 3.2.1.51) has been purified to apparent homogeneity (about 22 000-fold over the crude homogenate) from monkey brain. Values of kinetic constants for the purified enzyme were as follows: pH optimum, 5.0; Km, 0.22 mM; V, 913 mumol/mg per h. alpha-L-Fucose was a competitive inhibitor (Ki, 0.275 mM) of the enzyme. Evidence for the involvement of sulphydryl group(s) and carboxyl group containing amino acid(s) in the catalytic process is presented. The purified enzyme was a tetramer of molecular weight of 285 000 of identical subunits of 73 500 held together by non-covalent forces. Gel filtration studies revealed the presence of three molecular forms of the activity in the purified preparation which appeared to be the tetramer, dimer and monomer. The existence of three types of activities was also aupported by a triphasic heat inactivation profile of the enzyme at 50 or 55 degrees C and the distinctly different pH activity profiles of the differentially heat-inactivated enzymes. Immunodiffusion studies using antibody developed against purified monkey brain alpha-L-fucosidase showed that the monkey brain enzyme had only partial immunological identity with the enzymes from the non-neural tissues of monkey as well as the human and rat liver and the rat brain. However, the monkey brain and liver enzymes appeared to be similar to the human brain and liver enzymes, respectively.     

A comparison of the properties of the pyruvate kinases of the fat body and flight muscle of the adult male desert locust

1. The pyruvate kinases of the desert locust fat body and flight muscle were partially purified by ammonium sulphate fractionation. 2. The fat-body enzyme is allosterically activated by very low (1mum) concentrations of fructose 1,6-diphosphate, whereas the flight-muscle enzyme is unaffected by this metabolite at physiological pH. 3. Flight-muscle pyruvate kinase is activated by preincubation at 25 degrees for 5min., whereas the fat-body enzyme is unaffected by such treatment. 4. Both enzymes require 1-2mm-ADP for maximal activity and are inhibited at higher concentrations. With the fat-body enzyme inhibition by ADP is prevented by the presence of fructose 1,6-diphosphate. 5. Both enzymes are inhibited by ATP, half-maximal inhibition occurring at about 5mm-ATP. With the fat-body enzyme ATP inhibition can be reversed by fructose 1,6-diphosphate. 6. The fat-body enzyme exhibits maximal activity at about pH7.2 and the activity decreases rapidly above this pH. This inactivation at high pH is not observed in the presence of fructose 1,6-diphosphate, i.e. maximum stimulating effects of fructose 1,6-diphosphate are observed at high pH. The flight-muscle enzyme exhibits two optima, one at about pH7.2 as with the fat-body enzyme and the other at about pH8.5. Stimulation of the enzyme activity by fructose 1,6-diphosphate was observed at pH8.5 and above.     

Evidence that isoniazid and ethanol induce the same microsomal cytochrome P-450 in rat liver, an isozyme homologous to rabbit liver cytochrome P-450 isozyme 3a

Cytochrome P-450j has been purified to electrophoretic homogeneity from hepatic microsomes of adult male rats administered ethanol and compared to the corresponding enzyme from isoniazid-treated rats. The enzymes isolated from ethanol- and isoniazid-treated rats have identical chromatographic properties, minimum molecular weights, spectral properties, peptide maps, NH2-terminal sequences, immunochemical reactivities, and substrate selectivities. Both preparations of cytochrome P-450j have high catalytic activity in aniline hydroxylation, butanol oxidation, and N-nitrosodimethylamine demethylation with turnover numbers of 17-18, 37-46, and 15 nmol product/min/nmol of P-450, respectively. A single immunoprecipitin band exhibiting complete identity was observed when the two preparations were tested by double diffusion analysis with antibody to isoniazid-inducible cytochrome P-450j. Ethanol- and isoniazid-inducible rat liver cytochrome P-450j preparations have also been compared and contrasted with cytochrome P-450 isozyme 3a, the major ethanol-inducible isozyme from rabbit liver. The rat and rabbit liver enzymes have slightly different minimum molecular weights and somewhat different peptide maps but similar spectral, catalytic, and immunological properties, as well as significant homology in their NH2-terminal sequences. Antibody to either the rat or rabbit isozyme cross-reacts with the heterologous enzyme, showing a strong reaction of partial identity. Antibody against isozyme 3a specifically recognizes cytochrome P-450j in immunoblots of induced rat liver microsomes. Aniline hydroxylation catalyzed by the reconstituted system containing cytochrome P-450j is markedly inhibited (greater than 90%) by antibody to the rabbit protein. Furthermore, greater than 85% of butanol or aniline metabolism catalyzed by hepatic microsomes from ethanol- or isoniazid-treated rats is inhibited by antibody against isozyme 3a. Results of antibody inhibition studies suggest that cytochrome P-450j is induced four- to sixfold by ethanol or isoniazid treatment of rats. All of the evidence presented in this study indicates that the identical cytochrome P-450, P-450j, is induced in rat liver by either isoniazid or ethanol, and that this isozyme is closely related to rabbit cytochrome P-450 isozyme 3a.     

Purification and properties of mitochondrial and peroxisomal 3-hydroxyacyl-CoA dehydrogenase from rat liver

There are two 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in rat liver, one in mitochondria (type I enzyme), and another in peroxisomes (type II enzyme). In a series of the studies on the properties and the physiological roles of fatty acid oxidation systems in both organelles, the two enzymes were purified and compared for their properties. The final preparations obtained were judged to be homogeneous based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Type I enzyme was composed of two identical subunits of molecular weight of 32,000, whereas type II enzyme was a monomeric enzyme having a molecular weight of 70,000–77,000. These subunit structures were confirmed by the results of fluorescence studies. Both enzymes were different in amino acid compositions, especially in the contents of tryptophan and half-cystine. Antibodies against them formed single precipitin lines for the corresponding enzymes, but not for the others when subjected to an Ouchterlony double-diffusion test. The K_m values of type II enzyme for various substrates were lower than those of type I enzyme except those for acetoacetyl-CoA. As for 3-hydroxyacyl-CoA substrates, both enzymes had lower K_m's for longer-chain substrates. The V for the substrates of C₄C₁₀ were similar for each enzyme, though the value of type II enzyme for C₁₀ substrate was rather lower. The results of fluorescence studies suggested that their dissociation constants for NADH were lower and those for NAD⁺ were higher at lower pH. Both enzymes were specific to l-form of 3-hydroxyacyl-CoA substrate. The optimal pH of the forward reaction of type I and type II enzymes was 9.6 and 9.8, and of the reverse reaction, 4.5 and 6.2, respectively. From these results they were concluded to be completely different enzymes.     

Catalytic activity and substrate specificity of the flavin-containing monooxygenase in microsomal systems: characterization of the hepatic, pulmonary and renal enzymes of the mouse, rabbit, and rat

Inhibitory antibodies against NADPH-cytochrome P-450 reductase, detergent solubilization to dissociate functional interaction between the reductase and cytochrome P-450, and selective trypsin degradation have been used to characterize flavin-containing monooxygenase activity in microsomes from different tissues and species. A comparison of assay methods is reported. The native microsome-bound flavin-containing monooxygenase of mouse, rabbit, and rat liver, lung, and kidney can metabolize compounds containing thiol, sulfide, thioamide, secondary and tertiary amine, hydrazine, and phosphine substituents. Therefore, this enzyme from these common experimental animals has catalytic capabilities similar to those of the well-characterized porcine liver enzyme. True allosteric activation by n-octylamine does not appear to be a property of either the mouse, rabbit, or rat liver enzymes, but is a property of the pig liver and mouse lung enzymes. The microsomal pulmonary flavin-containing monooxygenase of the rabbit has some unique substrate preferences which differ from the mouse lung enzyme. Both the rabbit and mouse pulmonary enzymes have recently been shown to be distinct enzyme forms. However, the rat pulmonary flavin-containing monooxygenase appears to be catalytically identical to the rat liver enzyme, and does not have any of the unusual catalytic properties of either the rabbit or mouse lung enzymes. Enzyme activity of mouse, rabbit, and rat kidney microsomes is qualitatively similar to the hepatic activities. Substrates which saturate the microsome-bound flavin-containing monooxygenase at 1.0 mM, including thiourea, thioacetamide, methimazole, cysteamine, and thiobenzamide, are metabolized at common maximal velocities. This suggests that the kinetic mechanism of the native enzyme is similar to that established for the isolated porcine liver enzyme in that the rate-limiting step of catalysis occurs after substrate binding, and that all substrates capable of saturating the microsomal enzyme should be metabolized at a common maximal velocity.     

Separation and Some Properties of Two Intracellular beta-Glucosidases of Sporotrichum (Chrysosporium) thermophile

Intracellular, inducible beta-glucosidase from the cellulolytic fungus Sporotrichum (Chrysosporium) thermophile (ATCC 42464) was fractionated by gel chromatography or isoelectric focusing into components A and B. Enzyme A (molecular weight 440,000) had only aryl-beta-glucosidase activity, whereas enzyme B (molecular weight 40,000) hydrolyzed several beta-glucosides but had only low activity against o-nitrophenyl-beta-d-glucopyranoside (ONPG). Both enzymes had temperature optima of about 50 degrees C. The pH optimum was 5.6 for enzyme A and 6.3 for enzyme B, respectively. The K(m) (ONPG) value for enzyme A was 0.5 mM, and the corresponding values for enzyme B were 0.18 mM (ONPG) and 0.28 mM (cellobiose). Enzyme B, when tested with ONPG, showed substrate inhibition at a substrate concentration above 0.4 mM which could be released by cellobiitol and other alditols. Enzyme A was isoelectric at pH 4.48, and enzyme B was isoelectric at pH 4.64. Several inhibitors were tested for their action on the activity of enzymes A and B. Both enzymes were found to be concomitantly induced in cultures with either cellobiose or cellulose as carbon source.