Isolation and characterization of the 1,4-beta-D-glucan glucanohydrolases of Talaromyces emersonii.

Culture filtrates of Talaromyces emersonii were found to contain four endocellulases termed I, II, III and IV, the last having the greatest electrophoretic mobility towards the anode in homogeneous 5%-(w/v)-polyacrylamide gels at pH 4.5. All four are glycoproteins, the carbohydrate contents being: I, 27.7%; II, 29.0%; III, 44.7%; IV, 50.8. Each form is eluted as a single peak corresponding to an Mr value of 68000 on gel filtration at pH 3.5 and as a single band corresponding to an Mr value of 35000 on reductive sodium dodecyl sulphate/polyacrylamide-gradient-gel electrophoresis. However, we believe that the latter represents the native Mr value. The pI values for each lie between pH 2.8 and 3.2. Activity in each case is optimal at pH 5.5-5.8 and at 75-80 degrees C. Half-life values at pH5 and 75 degrees C were from 2 to 4h. The specific activity with any individual substrate was much the same for each enzyme, as was the ratio of activity from one substrate to the next. Possible reasons for the observation that plots of velocity versus substrate concentration are sigmoidal are discussed. We believe that the finding of four endocellulases reflects differential glycosylation of a single enzyme form rather than genetically determined differences in primary structure.     

Multiple forms of myeloperoxidase from human neutrophilic granulocytes: evidence for differences in compartmentalization, enzymatic activity, and subunit structure

Multiple forms of myeloperoxidase from normal human neutrophilic granulocytes obtained from a single donor can be resolved by carboxymethyl (CM)-cellulose ion-exchange column chromatography into three forms (I, II, and III) designated in order of elution of adsorbed enzyme using a linear salt gradient. Selective solubilization of individual forms of the enzyme by detergent (form I) or high-ionic-strength procedures (forms II and III) suggested that these forms of the enzyme were compartmentalized differently. All three forms were purified by a combination of preferential extraction, manipulation of ionic strength, and ion-exchange and molecular sieve chromatography. Purified forms II and III had similar specific activities for a variety of substrates. Form I was less active toward several of these same substrates, most notably iodide, with a specific activity about one-half that of forms II and III. All forms had similar spectral properties characteristic of a type alpha heme. The amino acid compositions of the three forms were similar, yet significant differences were found in selected residues such as the charged amino acids. Native polyacrylamide gel electrophoresis resolved small differences in mobility between the forms which were consistent with the charge heterogeneity observed on CM-cellulose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis data were consistent with the generally accepted subunit structure of two heavy chains and two light chains. All three forms contained a small-molecular-weight subunit of Mr 11,500. Form I contained a large subunit of Mr 63,000, while forms II and III contained a corresponding subunit of Mr approximately 57,500. We conclude that heterogeneity of human myeloperoxidase is accompanied by differences in cellular compartmentalization, enzymatic activity, and subunit structure.     

Differences between glycogen synthases from rat and rabbit skeletal muscle as indicated by phosphopeptide maps

Glycogen synthase I was purified from rat skeletal muscle. On sodium dodecyl sulfate polyacrylamide gel electrophoresis, the enzyme migrated as a major band with a subunit Mr of 85,000. The specific activity (24 units/mg protein), activity ratio (the activity in the absence of glucose-6-P divided by the activity in the presence of glucose-6-P X 100) (92 +/- 2) and phosphate content (0.6 mol/mol subunit) were similar to the enzyme from rabbit skeletal muscle. Phosphorylation and inactivation of rat muscle glycogen synthase by casein kinase I, casein kinase II (glycogen synthase kinase 5), glycogen synthase kinase 3 (kinase FA), glycogen synthase kinase 4, phosphorylase b kinase, and the catalytic subunit of cAMP-dependent protein kinase were similar to those reported for rabbit muscle synthase. The greatest decrease in rat muscle glycogen synthase activity was seen after phosphorylation of the synthase by casein kinase I. Phosphopeptide maps of glycogen synthase were obtained by digesting the different 32P-labeled forms of glycogen synthase by CNBr, trypsin, or chymotrypsin. The CNBr peptides were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and the tryptic and chymotryptic peptides were separated by reversed-phase HPLC. Although the rat and rabbit forms of synthase gave similar peptide maps, there were significant differences between the phosphopeptides derived from the N-terminal region of rabbit glycogen synthase and the corresponding peptides presumably derived from the N-terminal region of rat glycogen synthase. For CNBr peptides, the apparent Mr was 12,500 for rat and 12,000 for the rabbit. The tryptic peptides obtained from the two species had different retention times. A single chymotryptic peptide was produced from rat skeletal muscle glycogen synthase after phosphorylation by phosphorylase kinase whereas two peptides were obtained with the rabbit enzyme. These results indicate that the N-terminus of rabbit glycogen synthase, which contains four phosphorylatable residues (Kuret et al. (1985) Eur. J. Biochem. 151, 39-48), is different from the N-terminus of rat glycogen synthase.     

Comparison between the monomeric and binary-complex forms of procarboxypeptidase A from whole pig pancreas.

Two different forms of procarboxypeptidase A (I and II) were obtained from pig pancreas extracts. The Mr values, the pattern found on polyacrylamide-gel electrophoresis in the presence of sodium dodecyl sulphate, and the sedimentation coefficients indicate that form I is a binary complex formed by two different subunits, whereas form II is a monomer. The carboxypeptidase A-precursor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx.sor subunit of form I and the form II monomer are very similar with respect to Mr value, amino acid composition and fragmentation by CNBr and iodosobenzoic acid. The activation process of both forms is unspecific with respect to the activating enzyme, the peptide released during activation is unusually long (Mr approx. 12500) and, in the case of the binary complex, the activation with trypsin follows a rather complex pattern, suggesting that the accompanying subunit of form I might play a modulating role in the activation process. Although the appearance of enzymic activity is rather slow, a protein with an Mr equivalent to that of active carboxypeptidase A is found very early in the activation process. Both zymogens are glycoproteins (so far no carbohydrate has been reported in any procarboxypeptidase A) and both contain two strongly bound Zn2+ ions/molecule. Other chemical and physical properties were also determined.     

Purification and characterization of two porcine liver nuclear histone acetyltransferases

Two forms of porcine histone acetyltransferase (types I and II) have been purified to apparent homogeneity from liver nuclei. Both activities are extracted from nuclei by 0.5 M NaCl and display a native Mr of 110,000 as determined by gel filtration. Saline enzyme extracts were subject to ammonium sulfate precipitation and sequential chromatography on Q-Sepharose, Sephacryl S-200, hydroxylapatite, and Mono Q supports. The histone acetyltransferase type I fraction contains three polypeptide chains with apparent Mr values of 105,000, 62,000, and 45,000, respectively, by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Cyanogen bromide peptide mapping and immunoblotting suggest that the Mr 62,000 and 45,000 polypeptides are derived by cleavage of the Mr 105,000 polypeptide. Histone acetyltransferase type II contains two different subunits with apparent Mr values of 50,000 and 40,000, respectively. The amino acid composition, heat inactivation profiles, and Michaelis constants with respect to both acetyl coenzyme A and histones were indistinguishable for types I and II. However, affinity-purified polyclonal antibodies to both forms of the enzyme do not cross-react; cyanogen bromide-derived in situ cleavage digest patterns show few similarities; and the turnover number for type I is approximately 15-fold lower than that for type II. We estimate that there is one enzyme molecule for every 500 nucleosomes. The existence of two distinct forms of nuclear histone acetyltransferase in pig liver suggests that they may have separate functions in vivo.     

Human placental lysyl oxidase. Purification, partial characterization, and preparation of two specific antisera to the enzyme

Lysyl oxidase from human placentas gave four catalytically active forms on DEAE-cellulose chromatography in 6 M urea. The first tow of these were combined to form pool I and the remaining two to form pool II. Pool I was purified to homogeneity, while the final pool II enzyme usually had one minor contaminant. The molecular weight of both enzyme pools was identical, being about 30,000 by gel filtration in 6 M urea and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. No distinct differences were found between the two pools in amino acid composition, specific activity, or the use of various substrates. Two antisera were prepared, one to the total enzyme protein (pools I and II) and the other to pool I. Both antisera inhibited and precipitated crude placental lysyl oxidase, the two enzyme pools, and crude human skin fibroblast enzyme, there being no differences between the various enzyme forms. Both antisera also stained the two enzyme pools in immunoblotting of denatured proteins. The data suggest that there are no major catalytic, molecular, or immunological differences between the multiple forms of human lysyl oxidase. An antiserum prepared to any of the enzyme forms can, therefore, probably be used to study the total enzyme protein.     

Secretion of both partially unfolded and folded apoproteins of dimethyl sulfoxide reductase by spheroplasts from a molybdenum cofactor-deficient mutant of Rhodobacter sphaeroides f. sp. denitrificans.

Spheroplasts prepared from a molybdenum cofactor-deficient mutant of Rhodobacter sphaeroides f. sp. denitrificans secreted dimethyl sulfoxide (DMSO) reductase which had no molybdenum cofactor and therefore no activity, whereas those from wild-type cells secreted the active reductase. The inactive DMSO reductase proteins were separated by nondenaturing electrophoresis into two forms: form I, with the same mobility as the native enzyme, and form II, with slower mobility. Both forms had the same mobility on denaturing gel. Form I and active DMSO reductase had the same profile on gel filtration chromatography. Form II was eluted a little faster than the native enzyme, suggesting that DMSO reductase form II was not an aggregated form but a compactly folded form very similar to the native enzyme. Form II was digested by trypsin and denatured with urea, whereas form I was unaffected, like native DMSO reductase. These results suggested that form II was a partially unfolded but compactly folded apoprotein of DMSO reductase.     

Human epidermal transglutaminase. Preparation and properties.

A transglutaminase from human hair follicle-free epidermis was purified to homogeneity using gel filtration and ion exchange chromatography. The enzyme had an apparent Mr = 51,000 +/- 2,000 by sodium dodecyl sulfate electrophoresis, 100,000 +/- 5,000 by discontinuous gel electrophoresis, and 50,000 +/- 2,000 by gel filtration in Bio-Gel A-0.5m agarose. The enzyme cross-linked Factor XIII-free fibrinogen forming gamma dimers and alpha polymers. Either calcium or strontium was necessary for enzyme activity. In the presence of calcium, enzyme activity was increased by heating at 56 degrees or by treating with dimethylsulfoxide. Activation required calcium and occurred in the presence of serine protease inhibitors. The activated and native enzyme had apparently identical mobilities in acrylamide disc electrophoresis and sodium dodecyl sulfate electrophoresis. The Km values for two substrates in the reaction, casein and putrescine, were very similar for the native and the activated enzyme. The activated enzyme had a larger elution volume on Bio-Gel A-0.5m in the presence of calcium than did the native enzyme. The detailed mechanism of activation remains to be determined.     

Cloning and expression of the Clostridium thermohydrosulfuricum alpha-amylase-pullulanase gene in Escherichia coli

An alpha-amylase-pullulanase gene from Clostridium thermohydrosulfuricum DSM 3783 was cloned in Escherichia coli on a 7.0 kb EcoRI fragment using a lambda vector. The gene produced, from an indigenous promoter, active thermostable alpha-amylase-pullulanase, seemingly mostly a soluble intracellular enzyme in E. coli. Gel filtration separated the active enzyme produced into three peaks, each having both alpha-amylase and pullulanase activities. Immunoblotting after SDS-PAGE revealed more than ten alpha-amylase-pullulanase specific polypeptides; the biggest of these had an Mr of about 165,000, whereas the smallest enzymically active polypeptide had an Mr of about 100,000. Despite the marked degeneration of its constituent polypeptides, the apparent temperature optimum of the enzyme (80-85 degrees C) was only some 5 degrees C lower and the heat stability the same as that of the extracellular alpha-amylase-pullulanase produced by the native host. Oligonucleotide probes prepared according to the NH2-terminal amino acid sequences of the enzyme and its satellite polypeptide (a polypeptide associated with the extracellular enzyme of the native host) hybridized to different regions of the 7.0 kb DNA insert.     

Purification and physicochemical characterization of a human cytotoxic factor produced by a human haemic cell line.

A cytotoxic factor, produced by a human lymphoblastoid cell line [Karpas (1977) Br. J. Cancer 35, 152--160; Karpas (1977) Br. J. Cancer 36, 437--445], was purified both from the cell extracts and from the culture medium containing the cell lysate, by using ammonium sulphate precipitation, DEAE-cellulose chromatography, gel filtration and affinity chromatography on concanavalin A--Sepharose and on [3H]amino-ethanol--glass beads. Two factors, Factor I and Factor II, were separated by DEAE-cellulose chromatography. Factor I was eluted from this column at 30 mM-aminoethanol/HCl buffer, pH 8.0, whereas Factor II was bound strongly to DEAE-cellulose and was eluted only at 325 mM-aminoethanol/HCl buffer, pH 8.0. The purified Factor I migrated as a single band on polyacrylamide-gel electrophoresis. Its isoelectric point, pI, was 8.0 +/- 0.3. Its sedimentation coefficient, S20,w, was 3.5 +/- 0.1 S and its apparent molecular weight, Mr, was 65 000 +/- 1000 as determined by sedimentation-velocity and sedimentation-equilibrium measurements. A linear relationship between molecular weight and concentration was found in equilibrium runs, suggesting a non-spherical shape of the molecule. Factor I is not a glycoprotein, inasmuch as it does not bind to concanavalin A--Sepharose. It consists of two subunits (Mr 32 000 +/- 4000), migrating on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis as a single band. Factor II had pI 6.0 +/- 0.4 and Mr 75 000 +/- 3000. Factors I and II are thus different proteins.     

Intramolecular crosslinking of gamma-glutamyl transpeptidase

gamma-Glutamyl transpeptidase (rat kidney) is a heterodimeric glycoprotein (subunit molecular weights 52,000 and 25,000). In addition to its single-chain biosynthetic precursor (Mr 78,000), glycosylated high molecular weight forms (Mr 85,000-95,000) have been reported in various rat tissues as well as during in vitro translation of its mRNA. Studies reported here suggest that these might be attributed to the anomalous behavior of intramolecularly crosslinked species. Thus, chemical crosslinking of the purified enzyme (as well as enzyme on the renal brush border membranes) by bifunctional reagents such as dimethyl suberimidate and by an active site-directed reagent, diazotized p-amino-hippurate, produces stable heterodimers which exhibit molecular weights identical to that of the native enzyme when subjected to gel filtration. However, when subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the crosslinked species exhibit apparent Mr values of 85,000 to 110,000, depending upon the crosslinking agent used. Protein glycosylation alone does not account for such anomalous electrophoretic behavior; the extent and the regions of the enzyme involved in formation of crosslinks appear to exert considerable constraints upon their conformation even in denaturing media.     

Purification and characterization of the extracellular cyclic AMP phosphodiesterase of Dictyostelium discoideum

Extracellular phosphodiesterase for adenosine 3':5'-monophosphate [EC 3.1.4.17] was purified from the supernatant of aggregation phase culture of Dictyostelium discoideum, and two types (type I and type II) of the enzyme were found. The type I enzyme was not absorbed on DEAE-Sephacel at pH 8.5 and had an apparent molecular weight of about 67,000 daltons. In contrast, the type II enzyme was adsorbed on DEAE-Sephacel and had an apparent molecular weight of about 120,000 daltons. The Km values of the two types were similar (2-4 microM). Upon SDS polyacrylamide gel electrophoresis analyses, however, both types produced the same bands with molecular weights of 55,000 and 57,000, indicating that they are two different forms composed of common constituents. During the growth phase, the two types of the enzyme were present in culture supernatant in roughly equal amounts, but type II accumulated predominantly in the aggregation phase, suggesting that the ratio of activity of the two forms is under developmental control. Rabbit antiserum prepared against purified type II enzyme cross-reacted with type I as well as membrane-bound enzyme, indicating that the three classes of the enzyme possess some common sequence.     

Purification and properties of GM1 ganglioside beta-galactosidases from bovine brain

Two GM1-beta-galactosidases, beta-galactosidases I, and II, have been highly purified from bovine brain by procedures including acetone and butanol treatments, and chromatographies on Con A-Sepharose, PATG-Sepharose, and Sephadex G-200. beta-Galactosidase I was purified 30,000-fold and beta-galactosidase II 19,000-fold. Both enzymes appeared to be homogeneous, as judged from the results of polyacrylamide disc gel electrophoresis. Enzyme I had a molecular weight of 600,000-700,000 and enzyme II one of 68,000, as determined on gel filtration. On sodium dodecyl sulfate polyacrylamide slab gel electrophoresis under denaturing conditions, enzyme II gave a single band with a molecular weight of 62,000, while enzyme I gave two minor bands with molecular weights of 32,000 and 20,000 in addition to the major band at 62,000. Both enzymes liberated the terminal galactose from GM1 ganglioside and lactosylceramide but not from galactosylceramide. Enzyme I showed a pH optimum of 4.0 and was heat stable, while enzyme II showed a pH optimum of 5.0 and lost 50% of its activity in 15 min at 45 degrees C. Enzyme I showed a pI of 4.2 and enzyme II one of 5.9.     

Isolation and characterization of two 3-phosphatases that hydrolyze both phosphatidylinositol 3-phosphate and inositol 1,3-bisphosphate.

Inositol-polyphosphate 3-phosphatase catalyzes the hydrolysis of the 3-position phosphate bond of inositol 1,3-bisphosphate (Ins(1,3)P2) to form inositol 1-monophosphate and inorganic phosphate (Bansal, V.S., Inhorn, R.C., and Majerus, P.W. (1987) J. Biol. Chem. 262, 9444-9447). Phosphatidylinositol 3-phosphatase catalyzes the analogous reaction utilizing phosphatidylinositol 3-phosphate (PtdIns(3)P) as substrate to form phosphatidylinositol and inorganic phosphate (Lips, D.L., and Majerus, P.W. (1989) J. Biol. Chem. 264, 19911-19915). We now demonstrate that these enzyme activities are identical. Two forms of the enzyme, designated Type I and II 3-phosphatases, were isolated from rat brain. The Type I 3-phosphatase consisted of a protein doublet that migrated at a relative Mr of 65,000 upon sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. The Mr of this isoform upon size-exclusion chromatography was 110,000, suggesting that the native enzyme is a dimer. The Type II enzyme consisted of equal amounts of an Mr = 65,000 doublet and an Mr = 78,000 band upon SDS-polyacrylamide gel electrophoresis. This isoform displayed an Mr upon size-exclusion chromatography of 147,000, indicating that it is a heterodimer. The Type II 3-phosphatase catalyzed the hydrolysis of Ins(1,3)P2 with a catalytic efficiency of one-nineteenth of that measured for the Type I enzyme, whereas PtdIns(3)P was hydrolyzed by the Type II 3-phosphatase at three times the rate measured for the Type I 3-phosphatase. The Mr = 65,000 subunits of the two forms of 3-phosphatase appear to be the same based on co-migration on SDS-polyacrylamide gels and peptide maps generated with Staphylococcus aureus protease V8 and trypsin. The peptide map of the Mr = 78,000 subunit was different from that of the Mr = 65,000 subunits. Thus, we propose that the differing relative specificities of the Type I and II 3-phosphatases for Ins(1,3)P2 and PtdIns(3)P are due to the presence of the Mr = 78,000 subunit of the Type II enzyme.     

Multiple forms of rat-liver dihydropteridine reductase identified by their differing isoelectric points

Purified rat-liver dihydropteridine reductase is homogeneous by gel filtration (Mr approximately 51,000), sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Mr approximately 25,500), and native polyacrylamide gel electrophoresis, suggesting that the enzyme is composed of two identical subunits. However, analysis by isoelectric focusing has revealed three enzyme forms with approximate isoelectric points of 6.5, 5.9, and 5.7 (designated forms, I, II, and III, respectively). The three forms, isolated in 65% yield by preparative chromatofocusing, are stable in 0.05 M phosphate buffer, pH 6.8, containing 1 mM beta-mercaptoethanol and exhibit similar kinetic constants when the catalytic activities of the isolated forms are compared with quinonoid dihydrobiopterin as substrate. All forms generate complexes with the enzymatic cofactor NADH which are also detectable by IEF. When examined further by IEF under denaturing conditions in 6 M urea the enzyme demonstrates a differing subunit composition for its three forms. Two distinct subunits, designated alpha and beta, can be identified, and additional evidence suggests that the native enzyme forms I, II, and III represent the three differing dimeric combinations alpha alpha (form I), alpha beta (form II), and beta beta (form III).     

Microbial metabolism of quinoline and related compounds. VII. Quinoline oxidoreductase from Pseudomonas putida: a molybdenum-containing enzyme

The quinoline oxidoreductase from Pseudomonas putida was purified 50-fold to homogeneity with 21% recovery, using ammonium sulfate precipitation, hydrophobic interaction-, anion exchange-, and gel chromatography. The Mr of the native enzyme was calculated to be 300,000 by gel filtration. SDS-polyacrylamide gel electrophoresis of the enzyme revealed three protein bands corresponding to Mr 85,000, 30,000 and 20,000. The enzyme contained 8 atoms of iron, 8 atoms of acid-labile sulfide, 2 molecules of FAD, and the molybdenum cofactor, molybdopterin. Besides quinoline, the quinoline oxidoreductase also catalysed the conversion of 5-, 6-, 7- and 8-hydroxyquinoline and 8-chloroquinoline to the corresponding 2-oxo compounds. The incorporated oxygen atom was derived from water. Cyanide and methanol were effective inhibitors.     

Affinity purification of human deoxycytidine kinase: avoidance of structural and kinetic artifacts arising from limited proteolysis

Homogeneous deoxycytidine kinase has been isolated from leukemic human T-lymphoblasts by affinity chromatography based on a multisubstrate analog, deoxycytidine 5'-adenosine 5"'-P1,P4-tetraphosphate (dCp4A). Chromatography of extract treated with protease inhibitors yielded a monomeric polypeptide, inasmuch as the Mr of the native protein, 59,300, is comparable to the value of 52,000 from sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isoelectric pH was 6.1. But, enzyme isolated without protease inhibitors exhibited two fragments of Mr = 30,000 and 33,000, suggesting that proteolytic cleavage of the parental polypeptide had occurred during affinity chromatography. Both the parental and proteolyzed enzymes phosphorylated deoxyadenosine and deoxyguanosine, as well as deoxycytidine. However, the proteolyzed enzyme had an increased apparent Km for deoxycytidine. In consequence of this, a mixture of the two forms produced bimodal kinetic plots, whereas linear kinetics were displayed by each form alone.     

Polyclonal and monoclonal antibodies to human lysyl hydroxylase and studies on the molecular heterogeneity of the enzyme.

Human placental lysyl hydroxylase gave two bands in SDS/polyacrylamide-slab-gel electrophoresis: a broad, diffuse, major band corresponding to an apparent Mr of 80,000-85,000, and a sharp minor band with Mr 78,000. Mouse and chick-embryo lysyl hydroxylases gave only the broad, diffuse band, whereas the sharp band could not be detected. Polyclonal antibodies were prepared to the two bands of the human enzyme separately, and monoclonal antibodies were prepared to the whole purified enzyme preparation. Both types of polyclonal antibody inhibited and precipitated the enzyme activity, and both stained the two polypeptide bands in immunoblotting after SDS/polyacrylamide-gel electrophoresis. Only one out of five monoclonal antibodies inhibited the enzyme activity, whereas they all precipitated the activity when studied with antibody coupled to Sepharose. All five monoclonal antibodies stained the whole broad band in immunoblotting, and at least three of them also stained the sharp band. Peptide maps produced from the two polypeptide species by digestion with Staphylococcus aureus V8 protease were highly similar. Experiments with endoglycosidase H demonstrated that the Mr-80,000-85,000 polypeptide contains asparagine-linked carbohydrate units, which are required for maximal lysyl hydroxylase activity. The data suggest that the lysyl hydroxylase dimer consists of only one type of monomer, the heterogeneity of which is due to differences in glycosylation.     

Purification and properties of mitochondrial uracil-DNA glycosylase from rat liver

Uracil-DNA glycosylase from rat liver mitochondria, an inner membrane protein, has been purified approximately 575,000-fold to apparent homogeneity. During purification two distinct activity peaks, designated form I and form II, were resolved by phosphocellulose chromatography. Form I constituted approximately 85% while form II was approximately 15% of the total activity; no interconversion between the forms was observed. The major form was purified as a basic protein with an isoelectric point of 10.3. This enzyme consists of a single polypeptide with an apparent Mr of 24,000 as determined by recovering glycosylase activity from a sodium dodecyl sulfate-polyacrylamide gel. A native Mr of 29,000 was determined by glycerol gradient sedimentation. The purified enzyme had no detectable exonuclease, apurinic/apyrimidinic endonuclease, DNA polymerase, or hydroxymethyluracil-DNA glycosylase activity. A 2-fold preference for single-stranded uracil-DNA over a duplex substrate was observed. The apparent Km for uracil residues in DNA was 1.1 microM, and the turnover number is about 1000 uracil residues released per minute. Both free uracil and apyrimidinic sites inhibited glycosylase activity with Ki values of approximately 600 microM and 1.2 microM, respectively. Other uracil analogues including 5-(hydroxymethyl)uracil, 5-fluorouracil, 5-aminouracil, 6-azauracil, and 2-thiouracil or analogues of apyrimidinic sites such as deoxyribose and deoxyribose 5'-phosphate did not inhibit activity. Both form I and form II had virtually identical kinetic properties, and the catalytic fingerprints (specificity for uracil residues located in a defined nucleotide sequence) obtained on a 152-nucleotide restriction fragment of M13mp2 uracil-DNA were almost identical. These properties differentiated the mitochondrial enzyme from that of the uracil-DNA glycosylase purified from nuclei of the same source.     

Isozymes of alpha-galactosidase from Bacillus stearothermophilus

Two molecular forms of alpha-galactosidase (EC 3.2.1.22) synthesized constitutively by Bacillus stearothermophilus, strain AT-7, have been purified. alpha-Galactosidase I (with the substrate p-nitrophenyl alpha-D-galactopyranoside (PNPG)) has a pH optimum of 6 and half-life at 65 degrees C of > 2 h at low protein concentration. alpha-Galactosidase II has a pH optimum of 7 with PNPG and a half-life at 65 degrees C of about 3 min. The isozymes also differ with respect to their Km with PNPG and melibiose. Both enzymes are inhibited competitively by D-galactose, melibiose, and Tris. With the beta-glycosides cellobiose and lactose either noncompetitive or mixed-type inhibition is observed, with the pattern dependent on both the pH and the isozyme. The two isozymes have similar Arrhenius activation energies (about 20 kcal/mol, 1 kcal = 4.184 kJ). Their molecular weights, estimated by disc gel electrophoresis, are alpha-galactosidase I, 280 000 +/- 30 000 and alpha-galactosidase II, 325 000 +/- 15 000. Dodecyl sulfate gel electrophoresis gave a single band for each enzyme. The respective molecular weights, 81 000 +/- 500 for alpha-galactosidase I and 84 000 +/- 500 for alpha-galactosidase II, suggest that both enzymes consist of four subunits.