Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical Mr of 24,000 whereas a predominant subunit with Mr of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.     

Alkaline phosphatase in the integument of Ceratitis capitata: Developmental profile and functional properties

Electrophoretic analysis of alkaline phosphatase from the integument during development, reveals two bands of enzyme activity. One corresponding to phosphatase activity during pupation and just prior to eclosion and the other during the middle of the pupal stages. On the contrary in the haemolymph there is one band on enzyme activity through all the developmental stages. The haemolymph alkaline phosphatase band does not comigrate with any integumental enzyme band. The developmental profile of the integumental alkaline phosphatase activity has also been compared to that of the haemolymph. It was found that the pattern of activity is completely different. In the integument, two peaks of enzyme activity were found: one just prior to pupation and the other during eclosion. These two peaks do not coincide to that of haemolymph alkaline phosphatase activity. The pH optimum for both enzyme forms of third instar larvae, although broad especially for haemolymph form, was clearly in the alkaline range, with a peak at pH 8.5–9.0. The two isozymes have different affinities for the substrate tyrosine-O-phosphate. Tyrosine-O-phosphate is the preferred substrate for the integumental enzyme form with a K_m of 0.4 mM. We suggest that alkaline phosphates from the integument is specific for the hydrolysis of tyrosine-O-phosphate.     

Two different intrachain cAMP sites in the cAMP-dependent protein kinase of the dimorphic fungus Mucor rouxii

cAMP sites of the cAMP-dependent protein kinase from the fungus Mucor rouxii have been characterized through the study of the effects of cAMP and of cAMP analogs on the phosphotransferase activity and through binding kinetics. The tetrameric holoenzyme, which contains two regulatory (R) and two catalytic (C) subunits, exhibited positive cooperativity in activation by cAMP, suggesting multiple cAMP-binding sites. Several other results indicated that the Mucor kinase contained two different cooperative cAMP-binding sites on each R subunit, with properties similar to those of the mammalian cAMP-dependent protein kinase. Under optimum binding conditions, the [3H]cAMP dissociation behavior indicated equal amounts of two components which had dissociation rate constants of 0.09 min-1 (site 1) and 0.90 min-1 (site 2) at 30 degrees C. Two cAMP-binding sites could also be distinguished by C-8 cAMP analogs (site-1-selective) and C-6 cAMP analogs (site-2-selective); combinations of site-1- and site-2-selective analogs were synergistic in protein kinase activation. The two different cooperative binding sites were probably located on the same R subunit, since the proteolytically derived dimeric form of the enzyme, which contained one R and one C component, retained the salient properties of the untreated tetrameric enzyme. Unlike any of the mammalian cyclic-nucleotide-dependent isozymes described thus far, the Mucor kinase was much more potently activated by C-6 cAMP analogs than by C-8 cAMP analogs. In the ternary complex formed by the native Mucor tetramer and cAMP, only the two sites 1 contained bound cAMP, a feature which has also not yet been demonstrated for the mammalian cAMP-dependent protein kinase.     

Phosphorylation of rat heart glycogen synthase: studies in cardiomyocytes and in vitro phosphorylations with cAMP-dependent kinase and protein phosphatase-1

The phosphorylation of glycogen synthase has been studied in freshly isolated adult rat cardiomyocytes. Six peaks of 32P-labeled tryptic peptides are recovered via C-18 high performance liquid chromatography (HPLC) when synthase is immunoprecipitated from 32P-labeled cardiomyocytes and digested with trypsin. When epinephrine treated cells are used as a source of enzyme, the same HPLC profile is obtained with a dramatic enhancement of 32P recovered in two of the HPLC peaks. In vitro phosphorylation of rat heart synthase by cAMP-dependent protein kinase stimulates the conversion of synthase from the I to the D form and results in the recovery of the same tryptic peptides from the C-18 as is the case for synthase derived from cardiomyocytes. Treatment of cAMP-dependent kinase phosphorylated synthase with protein phosphatase-1 leads to a reactivation of the enzyme and a dephosphorylation of the same tryptic peptides that are selectively phosphorylated in epinephrine treated cardiomyocytes. These results are discussed in relation to hormonal control of glycogen metabolism in cardiac tissue.     

Phytohormonal regulation of S-adenosylmethionine synthetase by gibberellic acid in wheat aleurones.

Gibberellic acid (GA3) brought about a 3-fold stimulation of AdoMet synthetase activity in wheat aleurones. At the qualitative level, three isozymes of AdoMet synthetase were observed by DE-52 chromatography in GA3-treated wheat aleurones. In contrast, the control wheat aleurones showed a single isozyme. Thus the phytohormone (GA3, 1 microM) induced two additional isozymes of AdoMet synthetase in wheat aleurones. The activity of all the three isozymes in GA3-treated aleurones was considerably decreased by the simultaneous presence of abscisic acid (ABA, 10 microM). Cycloheximide (20 micrograms/ml) also significantly lowered the levels of the three isozymes of AdoMet synthetase in Ga3-treated aleurones, thereby suggesting the requirement of de-novo protein synthesis for the complete induction of isozymes. However, wheat aleurones excised from embryonated wheat seeds, did not require the application of GA3 for the induction of two additional isozymes of AdoMet synthetase. Apparently, the transport of GA3 from the embryo to aleurones induced two new isozymes of AdoMet synthetase. Three isozymes of AdoMet synthetase were also observed in wheat embryos excised from germinated wheat grains, without exogenous application of GA3. The molecular weight of all the three isozymes of AdoMet synthetase in wheat system is 181,000. The molecular weight of the subunit of the enzyme is 84,000. The dimeric nature of AdoMet synthetase was established by SDS-PAGE analysis of the purified enzyme. In-vitro hybridization of two flanking isozymic peaks I and III by NaCl-freeze-thaw method resulted in the appearance of an additional middle activity peak (isozyme II). However, no additional isozymic peaks were generated when isozymic peaks I and III were individually given a freeze-thaw treatment. Thus the flanking isozymic peaks I and III represent homodimers that differed in their net charge. In contrast, the middle isozymic activity peak II, when subjected to NaCl-freeze-thaw treatments yielded two additional isozymic peaks, I and III, thereby suggesting its heterodimeric nature. We envisage that the three isozymes in GA3-treated wheat aleurone layers are formed by the random dimerization of two classes of enzyme subunits. The two enzyme subunits which differ in their net charge could be the product of two genes of AdoMet synthetase (SAM1 and SAM2). Based on this assumption, we propose that a single isozyme I in water imbibed control wheat aleurones is the product of SAM1 gene of AdoMet synthetase. The occurrence of three isozymes in GA3-treated aleurones could be ascribed to the expression of an alternate gene of AdoMet synthetase (SAM2 gene).(ABSTRACT TRUNCATED AT 400 WORDS)     

人肝谷胱甘肽转移酶的纯化和性质

谷胱甘肽转移酶(EC 2,5,1,18 Glutathione S-transferases简称GSTs)是一组具有多种生理功能的蛋白质。我们通过105,000×g超速离心,s—已基—谷胱甘肽—Sepharose-6B亲和层析柱和DEAE52纤维柱或CM52纤维柱将人肝粗匀浆纯化为电泳纯的GSTs同工酶。经系和层析柱后GSTs比活比粗匀浆上清液提高54倍,回收率近60％。通过DE52柱将人肝GSTs分离为7个同工酶组分,分别称为c_(DE),A_1,A_2,A_3,A_4,A_5和A_6,经等电聚焦电泳和SDS-pAGE电泳鉴定,其等电点依次为8.60,7.05,6.70,6:60,6.55,6.45和6.4。经CM52柱后得到5个不同的同工酶组分,分别定名为A_(CM),c_1,c_2,c_3和c_4等电点各自为 6.30,7.00,8.50,8.55和8.60。阳离子同工酶(即c_(DE),C_1,C_2,C_3和C_4)的分子量在23,500—24,000道尔顿,阴离子同工酶(A_(CM),A_1-A_6)约为25,000道尔顿。并将亲和层析柱后样品,阳离子同工酶C_(DE)和阴离子同工酶A_(CM)作为抗原,得到兔抗人肝GSTs相应同工酶的抗血清,其抗血清效价经免疫双扩散法测定分别为1:96,1:64,1:16。并对人肝GSTs进行氨基酸组份的测定。     

Isozymes of fructose 6-phosphate,2-kinase:fructose-2,6-bisphosphatase in rat and bovine heart, liver, and skeletal muscle

The distribution of Fructose 6-P,2-kinase:Fructose 2,6-bisphosphatase in rat and bovine heart, liver, and skeletal muscle tissues was examined. With DEAE-cellulose chromatography, two peaks (I and II) of Fru 6-P,2-kinase activity were detected in all tissue extracts. Peak I was the predominant form both in rat and bovine heart tissue, while peak II was the major form in liver and skeletal muscle. Antibodies to heart enzyme reacted specifically with peak I, and antibodies to liver enzyme reacted with peak II from both liver and skeletal muscle. All the isozymes were bifunctional. All the tissues examined contained other isozymes in minor amounts.     

An isozyme of acid alpha-glucosidase with reduced catalytic activity for glycogen

Both the common and a variant isozyme of acid alpha-glucosidase have been purified from a heterozygous placenta with CM-Sephadex, ammonium sulfate precipitation, dialysis, Amicon filtration, affinity chromatography by Sephadex G-100, and DEAE-cellulose chromatography. Three and two activity peaks, from the common and variant isozymes, respectively, were obtained by DEAE-cellulose chromatography using a linear NaCl gradient. The three peaks of activity of the common isozyme were eluted with 0.08, 0.12, and 0.17 M NaCl, whereas the two peaks of the variant, with 0.01 and 0.06 M NaCl. The pH optimum and thermal denaturation at 57 degrees C were the same in all enzyme peaks of both isozymes. Rabbit antiacid alpha-glucosidase antibodies produced against the common isozyme were found to cross-react with both peaks of the variant isozyme. The two isozymes shared antigenic identity and had similar Km's with maltose as substrate. Normal substrate saturation kinetics were observed with the common isozyme when glycogen was the substrate, but the variant produced an S-shaped saturation curve indicating a phase of negative and positive cooperativity at low and high glycogen concentrations, respectively. The activity of the variant was only 8.6% and 19.2% of the common isozyme when assayed with nonsaturating and saturating concentrations of glycogen, respectively. A similar rate of hydrolysis of isomaltose by both isozymes was found indicating that the reduced catalytic activity of the variant isozyme toward glycogen is not the result of a reduced ability of this enzyme to cleave the alpha-1,6 linkages of glycogen.     

Purification and characterization of human liver beta-galactosidase from a patient with the adult form of GM1 gangliosidosis and a normal control

beta-Galactosidases were purified to homogeneity from livers of a normal control and a patient with the adult form of GM1 gangliosidosis. The purification was achieved by chromatography on DEAE-Sepharose fast flow, Con A-Sepharose, p-aminophenyl-1-thio-beta-D-galactopyranoside-Sepharose, and QAE-Mono Q. The normal and mutant enzymes were purified about 5000-fold with a yield of 10% and 1800-fold with a yield of 34%, respectively, and could hydrolyze 4-methylumbelliferyl-beta-D-galactoside, GM1 ganglioside, and asialofetuin. The purified normal enzyme was eluted from a TSK gel G-4000SW column as three symmetrical peaks of protein which were coincident with the three peaks of enzyme activity. The enzyme in these three peaks had apparent molecular weights of 800,000 (polymer), 140,000 (dimer), and 65,000 (monomer), whereas the mutant enzyme was eluted as two symmetrical peaks of protein and enzyme activity. The apparent molecular weight of a major monomeric form of the enzyme (beta-galactosidase A) was 60,000, and no dimeric form of the enzyme existed. Normal and mutant purified enzyme preparations migrated as a single major protein band with apparent molecular weights of 65,000 or 60,000, respectively, by SDS-polyacrylamide gel electrophoresis after treatment with mercaptoethanol. On isoelectric focussing, the mutant enzyme migrated more anodally than the normal enzyme. The mutant enzyme also had altered enzyme properties, such as pH optimum, Km values, substrate specificity and heat-stability. These data on the characteristics of the purified enzyme preparations provide the first direct evidence that patients with the adult form of GM1 gangliosidosis have a structurally altered beta-galactosidase.     

Proton magnetic resonance studies of carbonic anhydrase. I. Identification of histidine resonances.

Nuclear magnetic resonance (nmr) spectra of human carbonic anhydrase B recorded in deuterium oxide reveal seven discrete single proton resonances between 7 and 9 ppm downfield from sodium 2,2-dimethyl-i-silapentane-5-sulfonate. Simplification of spectra by use of Fremy's salt, comparison of peak widths at intersections, and evaluation of the results of inhibition and modification experiments permit determination of the pH dependencies of these resonances. Five of these peaks change position with increasing pH; three move upfield by approximately 95 Hz and two move downfield by 10 and 23 Hz. The first three reflect residues with pK values of 7.23, 6.98, and 6 and can be assigned to the C-2 protons of histidines. The two remaining pH dependent resonances reflect groups with pK values of 8.2 and 8.24. Their line widths and T1 values are comparable to those of the first group, and they also appear to reflect C-H protons of histidines. Despite the structural and functional similarities of the B and C isozymes of human carbonic anhydrase, few of the low field resonances appear to be common to both. Six histidine C-2 protons are observed in the C enzyme and reflect groups with pK values of approximately 7.3, 6.5, 5.7, 6.6, 6.6, and 6.4. A seventh peak contains two protons and moves upfield with increasing pH without titrating. A final resonance to low field moves downfield with increasing pH and reflects a group with a pK between 6 and 7. Its behavior resembles that of peak 1 of the human B enzyme, and it also appears to be a histidine C-H proton. This peak may reflect a conserved residue in the two isozymes that plays an important role in enzymatic function, as discussed in the following paper.     

Partial Characterization of Glutathione S-Transferase Isozymes Induced by the Herbicide Safener Benoxacor in Maize

The effects of the dichloroacetamide safener benoxacor on maize (Zea mays L. var Pioneer 3906) growth and glutathione S-transferase (GST) activity were evaluated, and GST isozymes induced by benoxacor were partially separated, characterized, and identified. Protection from metolachlor injury was closely correlated with GST activity, which was assayed with metolachlor as a substrate, as benoxacor concentration increased from 0.01 to 1 [mu]M. GST activity continued to increase at higher benoxacor concentrations (10 and 100 [mu]M), but no further protection was observed. Total GST activity with metolachlor as a substrate increased 2.6- to 3.8-fold in response to 1 [mu]M benoxacor treatment. Total GST activity from maize treated with or without 1 [mu]M benoxacor was resolved by fast protein liquid chromatography anion-exchange chromatography into four major activities, designated activity peaks A, B, C, and D in their order of elution. These GST activity peaks were enhanced to varying degrees by benoxacor. Activity peak B showed the least induction, whereas activity peak A was absent constitutively and thus highly induced by benoxacor. In contrast to earlier reports, there appear to be not one, but at least two, major constitutive isozymes (activity peaks A and D) having activity with metolachlor as substrate; there were at least three such isozymes in benoxacor-treated maize (activity peaks A, C, and D). The elution volumes of activity peaks A, B, C, and D were compared with those of partially purified maize GST I and GST II; also, the reactivity of polypeptides in these activity peaks with antisera to GST I or GST I/III (mixture) was evaluated. Evidence from these experiments indicated that activity peak B contained GST I, and activity peak C contained GST II and GST III. Activity peaks A and D contained unique GSTs that may play a major role in metolachlor metabolism and in the safening activity of benoxacor in maize. Isozymes present in activity peaks A and D were not detected in earlier reports because of the very low activity with the artificial substrate 1-chloro-2,4-dinitrobenzene. Immunoblotting experiments also indicated the presence of numerous unidentified GST subunits, including multiple subunits in chromatography fractions containing single peaks of GST activity; this is indicative of the likely complexity and diversity of the maize GST enzyme family.     

Variations in elastaselike esterase activities in human leucocytes during cell maturation.

Granules of human peripheral blood leucocytes contain four well-characterized elastase isozymes and one or two slow-moving elastaselike esterases (SE) which have not been as well characterized. SE are capable of hydrolyzing typical elastase synthetic sybstrates such as N-acetyl-dl-alanine-alpha-naphthyl ester (Ac-DL-Ala-1-ONap) and N-t-butyloxycarbonyl-L-alanine-p-nitrophenyl ester (Boc-Ala-ONp), but unlike the highly basic elastase isozymes, SE barely migrate into 13% acrylamide gels during cationic electrophoresis at pH 4.3. Hydrolysis of Ac-DL-Ala-1-ONap by SE requires the presence of Triton in the gel, and hydrolysis of Boc-Ala-ONp by the same enzyme(s) is also enhanced in the presence of the detergent. Triton is not required for these activities, in the case of the elastase isozymes. Diisopropylfluorophosphate (Dip-F) inactivates both SE and the elastase isozymes, whereas Ac-(Ala)2-Pro-AlaCH2Cl (a powerful inactivator of the leucocyte elastase isozymes at 10-4 M concentration) does not inactivate SE at the same concentration. Immunochemical studies revealed antigenic cross-reaction between the rapidly migrating leucocyte elastase isozymes and SE. Two preparations of leucocyte granules from nonleukemic bone marrow cells showed no activity of the rapidly migrating elastase isozymes, but did contain SE activity. SE may be a precursor or zymogen form of the elastase isozymes, present in immature cells and partly retained through later stages of development.     

Purification, partial characterization, and reactivity with aromatic compounds of two laccases from Marasmius quercophilus strain 17

Two isozymes of laccase were obtained from an induced liquid culture of Marasmius quercophilus with p-hydroxybenzoic acid as the inducer. Both the constitutive and the induced isozyme have a molecular mass of 60 kDa as determined by polyacrylamide gel electrophoresis. Using isoelectric focusing, we found three isozymes with the constitutive enzyme (pI 4, 4.2, 4.4) and four of the induced form (pI 4.75, 4.85, 4.95, 5.1). We observed certain differences between these two isozymes; the specific activity of the induced isozyme was twice as high, and two optimum pH levels (5 and 6) were observed with the induced isozyme (only one, pH 5, for the constitutive isozyme). However, both of these enzymes have the same thermal stability and the same temperature for their highest activity (80 degrees C). Furthermore, the reactivity of both these enzymes with aromatic compounds was similar. The use of mediators extended the oxidized substrate range of the laccases studied. Various products of degradation were observed, depending on the mediator used. When laccase was used alone, the decrease of the signal corresponding to the aromatic cycle, without any formations of other peaks at different wavelengths, suggested polymerisation of aromatic compounds.     

Alkaline phosphatase isozymes of Xenopus laevis embryos and tissues.

Alkaline phosphatase was obtained by treating embryos of Xenopus laevis with n-butanol at different developmental stages from gastrula to tadpole; the enzyme was also obtained from adult kidney, liver, and intestinal mucosa. Purification was carried out by gel filtration and polyacrylamide gel electrophoresis. The enzyme activity is chromatographically spearated into two peaks, with molecular weights of approximately 200,000 and 400,000. Alternatively, two groups may be characterized on the basis of their electrophoretic mobilities, which correspond to the different molecular weight classes. Effects of pH, temperature, inhibitors, and substrate concentration were studied. The kinetic and physical properties of the two alkaline phosphatase isozymes are similar, and are comparable to the properties reported for this enzyme from other vertebrates. Alkaline phosphatase activity increased sharply at the gastrula stage and reached a plateau at the late tailbud stage. During this period there was an 18-fold increase in activity.     

Novel dehydroepiandrosterone benzimidazolyl derivatives as 5α-reductase isozymes inhibitors

5α-R isozymes (types 1 and 2) play an important role in prostate gland development because they are responsible for intraprostatic dihydrotestosterone (DHT) levels when the physiological serum testosterone (T) concentration is low. In this study, we synthesized seven novel dehydroepiandrosterone derivatives with benzimidazol moiety at C-17, and determined their effect on the activity of 5α-reductase types 1 and 2. The derivatives with an aliphatic ester at C-3 of the dehydroepiandrosterone scaffold induced specific inhibition of 5α-R1 activity, whereas those with a cycloaliphatic ester (cyclopropyl, cyclobutyl, or cyclopentyl ring) or an alcohol group at C-3 inhibited the activity of both isozymes. Derivatives with a cyclohexyl or cycloheptyl ester at C-3 showed no inhibitory activity. In pharmacological experiments, derivatives with esters having an alcohol or the aliphatic group or one of the three smaller cycloaliphatic rings at C-3 decreased the diameter of male hamster flank organs, with the cyclobutyl and cyclopentyl esters exhibiting higher effect. With exception of the cyclobutyl and cyclopentyl esters, these compounds reduced the weight of the prostate and seminal vesicles.     

Characterization of vitamin K-dependent carboxylase from the livers from the adult ox and dicoumarol-treated calf.

The properties of the microsomal vitamin K-dependent carboxylase from the livers of the adult ox and dicoumarol-treated calf were investigated. The enzymes from both sources utilized glutamic residues of synthetic peptides as substrates and could be solubilized with Triton X-100 similarly to the enzyme from vitamin K-deficient rat liver. Under the optimal assay conditions, the microsomes from calf liver had peptide carboxylase activity comparable with that of the rat liver microsomes and 6.5-fold that of adult ox liver microsomes. The apparent Km for reduced vitamin K and the ionic strength optima of the calf and adult ox enzyme clearly differ from those of the rat enzyme. Pyridoxal phosphate activated the adult ox carboxylase only slightly, whereas the calf enzyme was activated by pyridoxal phosphate as effectively as was the enzyme from the vitamin K-deficient rat. Mn2+ activated the adult ox enzyme 9-fold and calf enzyme 22-fold under optimal conditions (no KCl). Three other divalent metal cations (Ca2+, Ba2+, and Mg2+) activated the adult ox and calf enzymes to about half the extent caused by Mn2+, KCl inhibited this activation. The vitamin K-dependent carboxylase from the dicoumarol-treated calf is apparently more tightly bound to the microsomal membrane than is the adult ox enzyme. In many other respects (pH optimum), temperature optimum, Km values for peptide substrate, substrate specificity, inhibitor effects), the properties of the adult ox and calf enzymes resemble closely those of the rat enzyme.     

Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis

The release of two mitochondrial proteins, cytochrome c and apoptosis-inducing factor (AIF), into the soluble cytoplasm of cells undergoing apoptosis is well established. Using spectrophotometric determination of enzyme activity, the accumulation of adenylate kinase (AK) activity in the cytosolic fraction of apoptotic cells has also been observed recently. However, three isozymes, AK1, AK2 and AK3, have been characterized in mammalian cells and shown to be localized in the cytosol, mitochondrial intermembrane space and mitochondrial matrix, respectively, and it is unknown which one of these isozymes accumulates in the cytosol during apoptosis. We now demonstrate that in apoptotic cells only AK2 was translocated into the cytosol concomitantly with cytochrome c. The amount of AK1 in cytosol, as well as the amount of matrix-associated AK3, remained unchanged during the apoptotic process. Thus, our data suggest that only intermembrane proteins are released from mitochondria during the early phase of the apoptotic process.     

Developmental changes of chicken liver AMP deaminase.

The AMP deaminase activity measured in crude chicken liver extract did not change significantly during development. The livers of 10- and 14-day chick embryos, 1-day, 5-, 10- and 16-week-old chickens and adult hens were examined for the existence of multiple forms of AMP deaminase. Phosphocellulose column chromatography revealed the existence of two peaks of enzyme activity in the liver of 10- and 16-week-old chickens and adult hens. Kinetic studies with the preparations of AMP deaminase revealed sigmoid-shaped substrate-saturation curves at all developmental stages and hyperbolic-shaped saturation curves for the enzyme form appearing in 10-week-old chickens. All AMP deaminases investigated were susceptible to activation by ATP and inhibition by Pi. Kinetic and regulatory properties as well as pH optima of all the enzyme preparations tested indicate that AMP deaminase isolated from the embryos and from 1-day-old chicks was similar to the form I isolated from adult hens and differed significantly from the form II of this enzyme.     

Two immunologically distinct human acidic beta-galactosidase A isozymes

Two acidic beta-galactosidase isozymes (designated A1 and A2) were separated by isoelectric focusing from beta-galactosidase A of human liver. Kinetic studies with 4-methylumbelliferyl-beta-D-galactopyranoside substrate revealed similar parameters for both. The Km value was 0.32 mmol/1 for A1 and 0.30 mmol/1 for A2 and Vmax values of 59.3 and mumol min-1 mg-1, respectively. The pH optimum was 4.2 for beta-galactosidase A1 and 4.5 for the A2 form. The A1 enzyme form was shown to be more heat labile than the A2. Significant differences were observed with antibody preparations against the two enzyme forms. Using the anti-A1 antibodies two precipitin arcs with residual enzymatic activity were obtained by immunoelectrophoresis of beta-galactosidase A whereas only one with anti-A2 antibodies. Anti-A1 precipated 85% of the original activity present in beta-galactosidase A and only 56% could be precipated by anti-A2. The possibility of common structural components is suggested.     

Three isozymes of catechol 1,2-dioxygenase (pyrocatechase), alpha alpha, alpha beta, and beta beta, from Pseudomonas arvilla C-1

Three isozymes of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas arvilla C-1 were separated using DEAE-Toyopearl chromatography. The specific activities of each isozyme were similar to one another. The molecular weights of isozymes 1, 2, and 3 were estimated to be approximately 67,000, 64,000, and 59,000, respectively, from gel filtration. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, isozymes 1 and 3 gave a single protein band, corresponding to Mr = 32,000 and 30,000, respectively, and isozyme 2 gave two bands corresponding to Mr = 32,000 and 30,000. These results indicated that isozymes 1 and 3 were homodimers, while isozyme 2 was a heterodimer. The NH2-terminal sequences up to 20 residues of these three isozymes confirmed that isozymes 1, 2, and 3 consisted of beta beta, alpha beta, and alpha alpha, respectively, based on our previous data (Nakai, C., Kagamiyama, H., Saeki, Y., and Nozaki, M. (1979) Arch. Biochem. Biophys. 195, 12-22). Properties of these isozymes such as absorption spectrum, iron content, substrate specificity, and kinetic constants were similar to one another. Subunit exchange between the different isozymes and dissociation of the isozymes into subunits was not observed under nondenaturing conditions. Available evidence indicates that these isozymes exist naturally in the bacterium and were not due to artifacts caused by purification.