Purification and characterization of enkephalinase B from rat brain membrane

Enkephalinase B from rat brain membrane which hydrolyzes enkephalin at the Gly-Gly bond was purified about 9400-fold to apparent electrophoretic homogeneity. The enzyme, which has a molecular weight of 82,000, consists of a single polypeptide chain. The enzyme has a pH optimum of 6.0-6.5 and is stable in the neutral pH region. The Km values of Met-enkephalin and Leu-enkephalin for this enzyme were 5.3 X 10(-5) M and 5.0 X 10(-5) M, respectively. The enzyme was inactivated by metal chelators, EDTA and o-phenanthroline and restored by the addition of divalent metal ions, Zn2+, Mn2+ or Fe2+, but was not inhibited by bestatin, amastatin, phosphoramidon or captopril. The enzyme hydrolyzed Met-enkephalin and Leu-enkephalin effectively. Although the enzyme belongs to the dipeptidyl aminopeptidase class, enkephalin-related peptides such as Leu-enkephalin-Arg, dynorphin (1-13) or alpha-endorphin and other biologically active peptides examined were hardly, or not at all, hydrolyzed. It was assumed that enkephalinase B functions mainly in enkephalin degradation in vivo.     

Purification and characterization of two distinct dipeptidyl aminopeptidases in soluble fraction from monkey brain and their action on enkephalins

Two distinct dipeptidyl aminopeptidases, which were designated DPP-A and DPP-B, were purified from soluble fraction of monkey brain using Leu-enkephalin as the substrate. The enzymes were purified 187 and 136 fold, respectively. Both enzymes showed the optimum pH in neutral range. Their molecular weights were almost equal and were estimated to be about 100,000. Their Km values with Leu-enkephalin as the substrate were 5.6 X 10(-5) and 1.1 X 10(-5) M, respectively. Among synthesized substrates, the highest affinity of the enzymes was toward arginyl-arginine beta-naphthylamide with the Km values of 6.25 X 10(-5) and 6.41 X 10(-5) M, respectively. Both enzyme activities were inhibited by the metal-chelators DFP and PCMB. Two hundred fifty microM arphamenine A inhibited DPP-A and -B with inhibition of 36.6% and 44.1%, respectively. Beta-endorphin, ACTH, and glucagon inhibited only DPP-B, while beta-lipotropin and angiotensin II inhibited both DPP-A and -B when Leu-enkephalin was used as the substrate.     

Effect of temperature and pH on phosphate transport through brush border membrane vesicles in rats

The kinetics of sodium gradient dependent phosphate uptake by the renal brush border membrane vesicles of the rat have ben studied under various conditions of temperature and pH. From 7 to 30 degrees C the Lineweaver-Burk plots are linear, and the apparent Km progressively increases from 54 to 91 microM. Above 30 degrees C, the apparent Km continues to increase to reach 135 microM at 40 degrees C, but a break is observed in the Lineweaver-Burk plots at the substrate concentration of 300 microM. The existence of this break, confirmed by the Eadie-Hofstee plot supports the hypothesis of a dual mechanism of phosphate transport, one for low concentrations of substrate with a Km of 100 microM and the other for high concentrations with a Km of approximately 240 microM. When the two components of the Eadie-Hofstee plot are analyzed according to a nonlinear regression program, these two values of Km become 70 microM and 1.18 mM, respectively. The Vmax continuously increases with temperature. However, the Arrhenius plot (In Vmax vs. 1/TK) shows an abrupt discontinuity at 23 degrees C. pH experiments were performed at 35 degrees C. In the absence of a proton gradient, increasing the pH from 6.5 to 7.5 and 8.5 decreases the apparent Km from 341 to 167 and 94 microM, respectively. When only the divalent form of phosphate is considered as the substrate, the apparent Km does not vary anymore with the pH and remains around the mean value of 105 microM. The uniformity of the apparent Km for the total phosphate uptake, when only the divalent phosphate is considered as being the substrate, suggests that this divalent form is the only one which is transported. Whatever the substrate considered, total phosphate or divalent phosphate, the highest Vmax is obtained at pH 7.5 which probably approximates the optimum pH inside the vesicles for the phosphate uptake.     

Properties of a soluble polyprenyl phosphate: UDP-D-glucose glucosyltransferase.

A soluble enzyme that catalyzes the transfer of D-glucose from UDP-D-glucose to dolichyl phosphate has been prepared by sonic oscillation of Acanthamoeba castellani cysts. The product of catalysis is dolichyl beta-D-glucosyl phosphate. The enzyme requires a divalent cation, either magnesium or manganese, and the presence of a reducing agent for maximum activity. Solanesyl phosphate and ficaprenyl phosphate are alternative substrates, apparently at lower rates, but GDP-D-glucose, UDP-D-glucuronic acid, UDP-N-acetyl-D-glucosamine, and UDP-D-xylose are not substrates. The temperature optimum is 30 degrees C, the pH optimum is pH 7.0, the Km for UDP-Glc is 9.1 microM and for dolichyl phosphate it is 4.5 microM. Uridine monophosphate and UDP are inhibitors of the reaction, UDP causing reversal and UMP being a competitive inhibitor of UDP-Glc with a Ki of 62 microM. The enzyme can be stored indefinitely below -20 degrees C, is stable for several days at 4 degrees C, but is half-inactivated within 2 h at 30 degrees C and completely inactivated within 10 min at 52 degrees C.     

Human placental dipeptidyl aminopeptidase III: hydrolysis of enkephalins and its stimulation by cobaltous ion

The degradation of enkephalin and related peptides by highly purified dipeptidyl aminopeptidase III (EC 3.4.14.4) was studied. The enzyme releases the N-terminal dipeptide units from substrates greater in length than the tetrapeptide. The enzyme exhibits an optimum of pH 7.5, Km of 81 microM and Vmax of 0.043 mumole/min for Leu-enkephalin. Its activity was markedly stimulated by Co2+, with both the Km and Vmax being increased. Among the enkephalin-related peptides examined, des-Tyr1-Leu-enkephalin was the most rapidly hydrolyzed with Co2+, but only slight stimulation was observed with Co2+.     

Sulphur metabolism in Paracoccus denitrificans. Purification, properties and regulation of serine transacetylase, O-acetylserine sulphydrylase and beta-cystathionase.

1. Serine transacetylase, O-acetylserine sulphydrylase and beta-cystathionase were purified from Paracoccus denitrificans strain 8944. 2. Serin transacetylase was purified 150-fold. The enzyme has a pH optimum between 7.5 and 8.0, is specific for L-serine and is inhibited by sulphydryl-group reagents. The apparent Km values for serine and acetyl-CoA are 4.0 - 10(-4) and 1.0 - 10(-4) M, respectively. Serine transacetylase is strongly inhibited by cysteine. 3. O-Acetylserine sulphydrylase was purified 450-fold. The enzymes has a sharp pH optimum at pH 7.5. In addition to catalysing the synthesis of cysteine, O-acetylserine sulphydrylase catalyses the synthesis of selenocysteine from O-acetylserine and selenide. The Km values for sulphide and O-acetylserine are 2.7 - 10(-3) and 1.25 - 10(-3) M, respectively. The enzyme was stimulated by pyridoxal phosphate and was inhibited by cystathionine, homocysteine and methionine. 4. beta-Cystathionase was purified approx. 50-fold. beta-Cystathionase has a pH optimum between pH 9.0 and 9.5, is sensitive to sulphydryl-group reagents, required pyridoxal phosphate for maximum activity and has an apparent Km for cystathionine of 4.2 - 10 (-3) M. beta-Cystathionase also catalyses the release of keto acid from lanthionine, djenkolic acid and cystine. Cysteine, O-acetylserine, homocysteine and glutathione strongly inhibit beta-cystathionase activity and homocysteine and methionine represses enzyme activity. 5. O-Acetylserine lyase was identified in crude extracts of Paracoccus denitrificans. The enzyme is specific for O-acetyl-L-serine, requires pyridoxal phosphate and is inhibied by KCN and hydroxylamine. The enzyme has a high Km value for O-acetylserine (50--100 mM).     

Pyruvate dehydrogenase complex of ascites tumour. Activation by AMP and other properties of potential significance in metabolic regulation.

1. AMP is an activator of the pyruvate dehydrogenase complex of the Ehrlich--Lettré ascites tumour, increasing its V up to 2-fold, with Ka of 40 microM at pH 7.4. This activation appears to be an allosteric effect on the decarboxylase subunit of the complex. 2. The pyruvate dehydrogenase complex has a Km for pyruvate within the range 17--36 microM depending on the pH, the optimum pH being approx. 7.4, with a V of approx. 0.1 unit/g of cells. The rate-limiting step is dependent on the transformation of the enzyme--substrate complex. The Km for CoA is 15 microM. The Km for NAD+ is 0.7 mM for both the complex and the lipoamide dehydrogenase. The complex is inhibited by acetyl-CoA competitively with CoA; the Ki is 60 microM. The lipoamide dehydrogenase is inhibited by NADH and NADPH competitively with NAD+, with Ki values of 80 and 90 microM respectively. In the reverse reaction the Km values for NADH and NADPH are essentially equal to their Ki values for the forward reaction, the V for the latter being 0.09 of that of the former. Hence the reaction rate of the complex in vivo is likely to be markedly affected by feedback isosteric inhibition by reduced nicotinamide nucleotides and possibly acetyl-CoA.     

Comparison of the substrate specificity of adenosine 3':5'-monophosphate- and guanosine 3':5'-monophosphate-dependent protein kinases. Kinetic studies using synthetic peptides corresponding to phosphorylation sites in histone H2B.

The substrate specificities of cyclic GMP-dependent and cyclic AMP-dependent protein kinases have been compared by kinetic analysis using synthetic peptides as substrates. Both enzymes catalyzed the transfer of phosphate from ATP to calf thymus histone H2B, as well as to two synthetic peptides, Arg-Lys-Arg-Ser32-Arg-Lys-Glu and Arg-Lys-Glu-Ser36-Tyr-Ser-Val, corresponding to the amino acid sequences around serine 32 and serine 36 in histone H2B. Serine 38 in the latter peptide was not phosphorylated by either enzyme. Cyclic GMP-dependent kinase and cyclic AMP-dependent kinase catalyzed the incorporation of 1.1 and 2.0 mol of phosphate/mol of histone H2B, respectively. The phosphorylation of histone H2B, respectively. The phosphorylation of histone H2B by cyclic GMP-dependent kinase showed two distinct optima as the magnesium concentration was increased. However, the phosphorylation of either synthetic peptide by this enzyme was depressed at high magnesium concentrations. As the pH of reaction mixtures was elevated from pH 6 to pH 9, the rate of phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase continually increased. Acetylation of the NH2 terminus of the peptide did not qualitatively affect this pH profile, but did increase the Vmax value of the enzyme 3-fold. The apparent Km and Vmax values for the phosphorylation of Arg-Lys-Arg-Ser32-Arg-Lys-Glu by cyclic GMP-dependent kinase were 21 microM and 4.4 mumol/min/mg, respectively. The synthetic peptide Arg-Lys-Glu-Ser36-Tyr-Ser-Val was a relatively poor substrate for cyclic GMP-dependent kinase, exhibiting a Km value of 732 microM, although the Vmax was 12 micromol/min/mg. With histone H2B as substrate for the cyclic GMP-dependent kinase, two different Km values were apparent. The Km values for cyclic AMP-dependent kinase for either synthetic peptide were approximately 100 microM, but the Vmax for Arg-Lys-Arg-Ser32-Arg-Lys-Glu was 1.1 mumol/min/mg, while the Vmax for Arg-Lys-Glu-Ser36-Tyr-Ser-Val was 16.5 mumol/min/mg. These data suggest that although the two cyclic nucleotide-dependent protein kinases have similar substrate specificities, the determinants dictated by the primary sequence around the two phosphorylation sites in histone H2B are different for the two enzymes.     

Musa paradisiaca stem juice as a source of peroxidase and ligninperoxidase

Musa paradisiaca stem juice has been shown to contain peroxidase activity of the order of 0.1 enzyme unit/ml. The Km values of this peroxidase for the substrates guaiacol and hydrogen peroxide are 2.4 and 0.28 mM respectively. The pH and temperature optima are 4.5 and 62.5 degrees C respectively. Like other peroxidases, it follows double displacement type mechanism. At low pH, Musa paradisiaca stem juice exhibits ligninperoxidase type activity. The pH optimum for ligninperoxidase type activity is 2.0 and the temperature optimum is 24 degrees C. The Km values for veratryl alcohol and n-propanol are 66 and 78 microM respectively.     

Inactivation of an aminoaldehyde dehydrogenase is responsible for fragrance in rice

Rice (Oryza sativa) has two betaine aldehyde dehydrogenase homologs, BAD1 and BAD2, encoded on chromosome four and chromosome eight respectively. BAD2 is responsible for the characteristic aroma of fragrant rice. Complementary DNA clones of both BAD1 and BAD2 were isolated and expressed in E. coli. BAD2 had optimum activity at pH 10, little to no affinity towards N-acetyl-gamma-aminobutyraldehyde (NAGABald) with a Km of approximately 10 mM and moderate affinity towards gamma-guanidinobutyraldehyde (GGBald) and betaine aldehyde (bet-ald) with Km values of approximately 260 microM and 63 microM respectively. A lower Km of approximately 9 microM was observed with gamma-aminobutyraldehyde (GABald), suggesting BAD2 has a higher affinity towards this substate in vivo. The enzyme encoded on chromosome four, BAD1, had optimum activity at pH 9.5, showed little to no affinity towards bet-ald with a Km of 3 mM and had moderate affinity towards GGBald, NAGABald and GABald with Km values of approximately 545, 420 and 497 microM respectively. BAD1 had a half life roughly double that of BAD2. We discuss the implications of these findings on the pathway of fragrance generation in Basmati and Jasmine rice and the potential of rice to accumulate the osmoprotectant glycine betaine.     

Purification and properties of mitochondrial malate dehydrogenase of Toxocara canis muscle

Mitochondrial malate dehydrogenase was purified from muscle extracts of Toxocara canis by means of Sephadex G-100 gel filtration, DEAE-Sephadex ion-exchange chromatography and 5'AMP-Sepharose 4B affinity chromatography. The purified enzyme showed an optimum pH for the reduction of oxaloacetate of 7.3 in Tris-HCl buffer and of pH 7.5-7.8 in phosphate buffer. The m-MDH showed values of 3.2 kcal/mol and 10.5 kcal/mol for the energy of activation, calculated from the Arrhenius equation. The mitochondrial enzyme was found to be more susceptible to thermal inactivation as compared with the cytosolic isoenzyme. Kinetic experiments showed that the m-MDH of Toxocara canis is inhibited by excess oxaloacetate but not by excess NADH. The apparent Km for oxaloacetate reduction was 53 microM and 0.54 mM for L-malate oxidation.     

Purification and characterization of two extracellular beta-glucosidases from Trichoderma reesei.

A major beta-glucosidase I and a minor beta-glucosidase II were purified from culture filtrates of the fungus Trichoderma reesei grown on wheat straw. The enzymes were purified using CM-Sepharose CL-6B cation-exchange and DEAE Bio-Gel A anion-exchange chromatography steps, followed by Sephadex G-75 gel filtration. The isolated enzymes were homogeneous in SDS-polyacrylamide gel electrophoresis and isoelectric focusing. beta-Glucosidase I (71 kDa) was isoelectric at pH 8.7 and contained 0.12% carbohydrate; beta-glucosidase II (114 kDa) was isoelectric at pH 4.8 and contained 9.0% carbohydrate. Both enzymes catalyzed the hydrolysis of cellobiose and p-nitrophenyl-beta-D-glucoside (pNPG). The Km and kcat/Km values for cellobiose were 2.10 mM, 2.45.10(4) s-1 M-1 (beta-glucosidase I) and 11.1 mM, 1.68.10(3) s-1 M-1 (beta-glucosidase II). With pNPG as substrate the Km and kcat/Km values were 182 microM, 7.93.10(5) s-1 M-1 (beta-glucosidase I) and 135 microM, 1.02.10(6) s-1 M-1 (beta-glucosidase II). The temperature optimum was 65-70 degrees C for beta-glucosidase I and 60 degrees C for beta-glucosidase II, the pH optimum was 4.6 and 4.0, respectively. Several inhibitors were tested for their action on both enzymes. beta-Glucosidase I and II were competitively inhibited by desoxynojirimycin, gluconolactone and glucose.     

Properties of a highly purified mitochondrial deoxyguanosine kinase

Deoxyguanosine kinase, purified over 6000-fold from beef liver mitochondria by means of deoxyguanosine-3'-(4-aminophenyl phosphate)-Sepharose affinity chromatography, was nearly homogeneous. It phosphorylates only deoxyguanosine and deoxyinosine among the natural nucleosides, with apparent Km values of 4.7 and 21 microM, respectively. Among nucleoside analogs tested, only arabinosylguanine (Ki = 125 microM) and 8-aza-deoxyguanosine (Ki = 450 microM) competed with deoxyguanosine. The relative molecular mass of the enzyme is 56,000, as determined by equilibrium sedimentation, and sodium dodecyl sulfate-gel electrophoresis suggests two subunits of Mr 28,000. The pH optimum for enzyme activity is 5.5, but optimum enzyme stability is seen at pH 7.0. Triton X-100 increased the stability of the enzyme markedly. ATP is the best phosphate donor at pH 5.5, but pyrimidine triphosphates such as dTTP and UTP are more efficient donors at pH 7.4. The activation energy, at pH 5.5, was estimated to be 10.9 kcal/mol. Amino acid modification experiments suggest the involvement of arginine, cysteine, and probably histidine. The inactivation of the enzyme by modification of these amino acid residues was time and pH dependent. Both substrates protected the enzyme from inactivation in every case but that of photooxidation by Rose Bengal, where only deoxyguanosine prevented inactivation.     

Human kidney cathepsins B and L. Characterization and potential role in degradation of glomerular basement membrane.

Cathepsins B and L were purified from human kidney. SDS/polyacrylamide-gel electrophoresis demonstrated that cathepsins B and L, Mr 27000-30000, consist of disulphide-linked dimers, subunit Mr values 22000-25000 and 5000-7000. The pH optimum for the hydrolysis of methylcoumarylamide (-NHMec) substrates (see below) is approx. 6.0 for each enzyme. Km and kcat. are 252 microM and 364s-1 and 2.2 microM and 25.8 s-1 for the hydrolysis of Z-Phe-Arg-NHMec (where Z- represents benzyloxycarbonyl-) by cathepsins B and L respectively, and 184 microM and 158 s-1 for the hydrolysis of Z-Arg-Arg-NHMec by cathepsin B. A 10 min preincubation of cathepsin B (40 degrees C) or cathepsin L (30 degrees C) with E-64 (2.5 microM) results in complete inhibition. Under identical conditions Z-Phe-Phe-CHN2 (0.56 microM) completely inhibits cathepsin L but has little effect on cathepsin B. Incubation of glomerular basement membrane (GBM) with purified human kidney cathepsin L resulted in dose-dependent (10-40 nM) GBM degradation. In contrast, little degradation of GBM (less than 4.0%) was observed with cathepsin B. The pH optimum for GBM degradation by cathepsin L was 3.5. Cathepsin L was significantly more active in degrading GBM than was pancreatic elastase, trypsin or bacterial collagenase. These data suggest that cathepsin L may participate in the lysosomal degradation of GBM associated with normal GBM turnover in vivo.     

Enzymes of vitamin B6 degradation. Purification and properties of 4- and 5-pyridoxolactonases

4-Pyridoxolactone and 5-pyridoxolactone, formed by dehydrogenation of pyridoxal or isopyridoxal during the bacterial degradation of vitamin B6 by Pseudomonas MA-1 and Arthrobacter Cr-7, respectively, are hydrolyzed to the corresponding acids by distinct inducible lactonases which were purified to homogeneity. 4-Pyridoxolactonase from Pseudomonas MA-1 has an Mr of 54,000 and contains two probably identical subunits of Mr = 28,600. It has a pH optimum of 7.0, a Km of 5.9 microM, and a Vmax at 25 degrees C of 35.2 mumol X min-1 X mg-1. 5-Pyridoxolactonase from Arthrobacter Cr-7 has an Mr of 65,200 and also contains two probably identical subunits of Mr = 32,800. It has a pH optimum of 7.1-7.7, a Km of 300 microM, and a Vmax at 25 degrees C of 21.5 mumol-1 X min-1 X mg-1. The two lactonases require no added cofactors or metal ions; their activities are inhibited by sulfhydryl reagents but are not affected by metal-chelating reagents. Although the two lactonases are entirely specific for their respective substrates, 4-pyridoxolactone is a competitive inhibitor (KI = 52 microM) for 5-pyridoxolactonase, and 5-pyridoxolactone is a competitive inhibitor (KI = 48 microM) for 4-pyridoxolactonase.     

Purification and characterization of cathepsin L from skeletal muscle of the lizard Agama stellio stellio

Cathepsin L from skeletal muscle of the lizard Agama stellio stellio was purified to homogeneity by ion-exchange and gel-permeation chromatography. The molecular weight of the cathepsin L is estimated to be 34 kD, and its isoelectric point is 5.5. The cathepsin L has a pH optimum of 6.1, requires a thiol-reducing reagent for activation, and is inhibited by cysteine protease inhibitors. The Km and kcat values for Z-Phe-Arg-MCA as substrate are 1.4 microM and 6.2 sec-1, respectively. This enzyme readily hydrolyzes proteins such as insulin B chain, hemoglobin, and serum albumin.     

GDPmannose dolicholphosphate mannosyltransferase of chicken liver mitochondria

Chicken liver mitochondria contain enzymes for the dolichol cycle. GDPmannose dolicholphosphate mannosyltransferase has been solubilized with Emulgen 909 and purified. The purified enzyme was not homogeneous, but highly specific for GDPmannose and dolichyl phosphate. The enzyme activity was stimulated by MgCl2 (3 mM optimum) and exhibited a pH optimum at around 7.2. Bisubstrate kinetic analysis indicated that the enzyme follows a sequential mechanism. The Km values for GDPmannose and dolichyl phosphate were 0.43 and 14.3 microM, respectively. The purified enzyme was labile and lost its activity on storage at 0 degree C overnight or incubation at 30 degrees C or higher temperature. Inactivation could be prevented by the addition of heat-denatured mitochondrial extract. Further investigation revealed that phospholipids and dolichyl phosphate are responsible for the stabilization. Single addition of either phospholipid or dolichyl phosphate showed little activity, but the combination of these lipids enhanced the stabilizing activity greatly. Eight naturally occurring phospholipids were tested and found to be effective in combination with dolichyl phosphate. Among these, sphingomyelin was the most effective. Dolichol could partially substitute dolichyl phosphate but worked at higher concentrations.     

The role of aldehyde dehydrogenases in the malonic dialdehyde metabolism in the rat liver

The enzymes catalyzing the NAD-dependent oxidation of malonic dialdehyde (MDA) were isolated from rat liver extracts. Upon 5'-AMP-Sepharose chromatography MDA dehydrogenase was separated into two isoforms, I and II. Isoform I was eluted from the affinity carrier with a 0.1 M phosphate buffer pH 8.0. This isoform had a broad substrate specificity towards aliphatic and aromatic aldehydes. Kinetic studies showed that short- and medium-chain aliphatic aldehydes (C2-C6) were characterized by the lowest Km values and the highest Vmax values. The Km' values for MDA and acetaldehyde were 2.8 microM and 0.69 microM, respectively. Isoform II was eluted with a 0.1 M phosphate buffer pH 8.0 containing 0.5 mM NAD, was the most active with medium- and long-chain aliphatic aldehydes (C6-C11) and had Km values for MDA and acetaldehyde equal to 37 microM and 52 microM, respectively. Isoform I was much more sensitive towards disulfiram inhibition than isoform II. Both isoforms had an identical molecular mass (93 kD) upon gel filtration. It is concluded that MDA dehydrogenase isoform I is identical to mitochondrial aldehyde dehydrogenase having a low Km for acetaldehyde, whereas isoform II may be localized in liver cytosol. The role of aldehyde dehydrogenases in the metabolism of aldehydes derived from lipid peroxidation is discussed.     

Purification and characterization of glutamate decarboxylase from Neurospora crassa conidia.

L-Glutamate decarboxylase, an enzyme under the control of the asexual developmental cycle of Neurospora crassa, was purified to homogeneity from conidia. The purification procedure included ammonium sulfate fractionation and DEAE-Sephadex and cellulose phosphate column chromatography. The final preparation gave a single band on sodium dodecyl sulfate-polyacrylamide gels with a molecular weight of 33,200 +/- 200. A single band coincident with enzyme activity was found on native 7.5% polyacrylamide gels. The molecular weight of glutamate decarboxylase was 30,500 as determined by gel permeation column chromatography at pH 6.0. The enzyme had an acidic pH optimum and showed hyperbolic kinetics at pH 5.5 with a Km for glutamic acid of 2.2 mM and a Km for pyridoxal-5'-phosphate of 0.04 microM.     

Chick embryo liver beta-glucuronidase. Comparison of activity on natural and artificial substrates.

The beta-glucuronidase in homogenates of 12-day chick embryo livers catalyzed the release of glucuronic acid from 4-methylumbelliferyl-beta-D-glucuronide and from the nonreducing terminals of the hexasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4 at rates of 143, 114, and 108 nmol of glucuronic acid/h/mg of protein, respectively, when assayed at pH 3.5 in 0.05 M sodium acetate buffer. During a 60-fold purification of the enzyme, the ratios of the activities on these substrates did not change. When 4-methylumbelliferyl-beta-D-glucuronide was used as substrate the enzyme was active at pH values from 3.0 to 5.5, with maximal activity between pH values 4.0 and 4.5. Concentrations of NaCl from 0.15 to 0.3 M inhibited the activity at low pH values but activated the enzyme between pH 4.0 and 5.5. The enzyme was active on the chondroitin-6-SO4 hexasaccharide from pH 3.0 to 5.5, with a broad optimum between 3.0 and 4.5. NaCl inhibited the activity on the oligosaccharide substrate at all pH values. Eadie-Scatchard plots of rates of 4-methylumbelliferyl-beta-D-glucuronide hydrolysis at substrate concentrations ranging from 2 to 1000 microM showed multiple kinetic forms of the enzyme, a form with a Km of approximately 11 microM, and a second form with a Km of approximately 225 microM. The pH optimum of the low Km form was 3.5 to 4.0; that of the high Km form was pH 4.5. NaCl inhibited the activity of the low Km form, but activated the high Km form of the enzyme. Chondroitin SO4 oligosaccharides competed with 4-methylumbelliferyl-beta-D-glucuronide for the low Km form of the enzyme but had little effect on the hydrolysis of 4-methylumbelliferyl-beta-D-glucuronide by the high Km form of the enzyme. The activities of the beta-glucuronidase on tetra-, hexa-, octa-, and decasaccharides of chondroitin-6-SO4 and chondroitin-4-SO4, measured using a new assay procedure which can detect the formation of 1 nmol of product, were similar, although rates were somewhat lower for the higher oligosaccharides. With the exception of the chondroitin-4-SO4 tetrasaccharide, all of the oligosaccharide substrates saturated the enzyme at concentrations of 20 to 30 microM, indicating Km values of less than 10 to 15 microM for the oligosaccharides. Highly purified beta-glcuronidases from human placenta and from rat preputial gland also showed multiple kinetic forms when assayed using 4-methylumbelliferyl-beta-D-glucuronide as substrate.