The separation and characterization of the methylumbelliferyl beta-galactosidases of human liver.

1. A previously uncharacterized form of human liver acid beta-galactosidase (EC 3.2.1.23), possibly a dimer of molecular weight 160 000, was resolved by gel filtration. It has the same ability to hydrolyse GM1 ganglioside as the two other acid beta-galactosidase forms. 2. The low-molecular-weight forms of acid beta-galactosidase undergo salt-dependent aggregation. 3. The high-molecular-weight component may consist of the low-molecular-weight forms bound to membrane fragments. It can be converted completely into a mixture of these forms. 4. The neutral beta-galactosidase activity can be resolved into two forms by DEAE-cellulose chromatography. They differ in their response to Cl-ions. 5. A new nomenclature is suggested for the six beta-galactosidases so far found in human liver. 6. The enzymic constituents of the beta-galactosidase bands resolved by electrophoresis were re-examined. The A band contains three components. A two-dimensional electrophoretic procedure for resolving the A band is described. 7. The effect of neuraminidase treatment on the behaviour of beta-galactosidases in various separation systems is examined.     

Dog GM1 gangliosidosis: characterization of the residual liver acid beta-galactosidase.

The residual liver acid beta-galactosidase activity from the first documented case of GM1 gangliosidosis in dogs was partially purified and characterized with respect to kinetic properties, thermostability, isoelectric point, molecular weight, and antigenicity. The GM1 dog liver beta-galactosidase appears to be identical with the normal dog liver enzyme in all properties examined. The canine disease is strikingly different from the human disease in the amount of enzyme that is present in the tissue. Unlike the human disease, in which normal amounts of catalytically defective beta-galactosidase are present, in dog GM1 gangliosidosis, only 1% of normal beta-galactosidase protein is detectable.     

Intra- and extracellular beta-galactosidases from Bifidobacterium bifidum and B. infantis: molecular cloning, heterologous expression, and comparative characterization

Three beta-galactosidase genes from Bifidobacterium bifidum DSM20215 and one beta-galactosidase gene from Bifidobacterium infantis DSM20088 were isolated and characterized. The three B. bifidum beta-galactosidases exhibited a low degree of amino acid sequence similarity to each other and to previously published beta-galactosidases classified as family 2 glycosyl hydrolases. Likewise, the B. infantis beta-galactosidase was distantly related to enzymes classified as family 42 glycosyl hydrolases. One of the enzymes from B. bifidum, termed BIF3, is most probably an extracellular enzyme, since it contained a signal sequence which was cleaved off during heterologous expression of the enzyme in Escherichia coli. Other exceptional features of the BIF3 beta-galactosidase were (i) the monomeric structure of the active enzyme, comprising 1,752 amino acid residues (188 kDa) and (ii) the molecular organization into an N-terminal beta-galactosidase domain and a C-terminal galactose binding domain. The other two B. bifidum beta-galactosidases and the enzyme from B. infantis were multimeric, intracellular enzymes with molecular masses similar to typical family 2 and family 42 glycosyl hydrolases, respectively. Despite the differences in size, molecular composition, and amino acid sequence, all four beta-galactosidases were highly specific for hydrolysis of beta-D-galactosidic linkages, and all four enzymes were able to transgalactosylate with lactose as a substrate.     

Characteristics of β-galactosidase in the mucosa of the small intestine of infant rats. Physicochemical properties

1. The characteristics of acid and neutral beta-galactosidases isolated chromatographically from homogenates of the mucosa of the jejunum and ileum of suckling rats were studied. 2. The minimal molecular weight of the acid beta-galactosidase, as estimated by gel filtration on Sephadex G-200, was in the range 83000-105000, whereas for the neutral beta-galactosidase the estimated molecular weight was in the range 360000-510000. 3. The acid and neutral beta-galactosidases were inhibited competitively by galactono-(1-->4)-lactone, with respective K(i) values of 0.15mm and 1.1mm. Only the acid beta-galactosidase was inhibited competitively by sodium galactonate (K(i) 0.17mm). 4. Heat inactivation of both beta-galactosidases occurred according to first-order kinetics. The neutral enzyme was more labile, but bovine serum albumin protected acid enzyme only. 5. Urea treatment inactivated both beta-galactosidases, the neutral beta-galactosidase being more sensitive than the acid beta-galactosidase. 6. No differences were found between preparations from the jejunum and ileum.     

Hydrolysis of galactosylceramide is catalyzed by two genetically distinct acid beta-galactosidases

Two genetically distinct acid beta-galactosidases are apparently involved in the hydrolysis of galactosylceramide in fibroblasts. These beta-galactosidases were activated by different bile salts. The classical galactosylceramidase (galactosylceramidase I, EC 3.2.1.46) was activated by sodium taurocholate, while the other galactosylceramidase (galactosylceramidase II) was activated by sodium cholate. The former was genetically lacking in globoid cell leukodystrophy (GLD) and the latter in GM1 gangliosidosis. Galactosylceramidase II cross-reacted with antibody raised against purified GM1 ganglioside beta-galactosidase (EC 3.2.1.23) from the human placenta. The purified beta-galactosidase had galactosylceramidase II activity, which was competitively inhibited by GM1 ganglioside. Thus, galactosylceramidase II seems to be identical to GM1 ganglioside beta-galactosidase and lactosylceramidase II. Galactosylceramidase II had a very low affinity for galactosylsphingosine. In the galactosylceramide-loading tests using fibroblasts from patients with GLD and GM1 gangliosidosis, both cell lines hydrolyzed the incorporated galactosylceramide, with lower rates than control fibroblasts but higher than the fibroblasts from patients with I-cell disease, in which both galactosylceramidase I and II were deficient. These results indicate that galactosylceramide is hydrolyzed by two genetically distinct beta-galactosidases and explain well that galactosylsphingosine but not galactosylceramide accumulates in the brain of patients with GLD.     

Molecular characteristics of beta-galactosidase secreted by Penicillium canescens

Extracellular beta-galactosidase from P. canescens culture medium was purified by ion-exchange chromatography on DEAE and CM-Sepharose CL-6B and gel filtration. The enzyme active form was shown to be a monomer with a molecular weight of about 120 kDa; the isoelectric point is 6.7 and the sedimentation coefficient is 6.5. In terms of physico-chemical and catalytic properties, the purified enzyme is similar to beta-galactosidases of other fungi of genus Penicillium. The amino acid composition and the NH2-terminal sequence of 24 residues non-homologous to the corresponding sequences of bacterial and yeast beta-galactosidases were determined.     

Expression and nucleotide sequence of the Lactobacillus bulgaricus beta-galactosidase gene cloned in Escherichia coli.

The Lactobacillus bulgaricus beta-galactosidase gene was cloned on a ca. 7-kilobase-pair HindIII fragment in the vector pKK223-3 and expressed in Escherichia coli by using its own promoter. The nucleotide sequence of the gene and approximately 400 bases of 3'- and 5'-flanking sequences was determined. The amino acid sequence of the beta-galactosidase, deduced from the nucleotide sequence of the gene, yielded a monomeric molecular mass of ca. 114 kilodaltons, slightly smaller than the E. coli lacZ and Klebsiella pneumoniae lacZ enzymes but larger than the E. coli evolved (ebgA) beta-galactosidase. The cloned beta-galactosidase was found to be indistinguishable from the native enzyme by several criteria. From amino acid sequence alignments, the L. bulgaricus beta-galactosidase has a 30 to 34% similarity to the E. coli lacZ, E. coli ebgA, and K. pneumoniae lacZ enzymes. There are seven regions of high similarity common to all four of these beta-galactosidases. Also, the putative active-site residues (Glu-461 and Tyr-503 in the E. coli lacZ beta-galactosidase) are conserved in the L. bulgaricus enzyme as well as in the other two beta-galactosidases mentioned above. The conservation of active-site amino acids and the large regions of similarity suggest that all four of these beta-galactosidases evolved from a common ancestral gene. However, these enzymes are quite different from the thermophilic beta-galactosidase encoded by the Bacillus stearothermophilus bgaB gene.     

Cloning and expression of the beta-galactosidase genes from Lactobacillus reuteri in Escherichia coli

Heterodimeric beta-galactosidase of Lactobacillus reuteri L103 is encoded by two overlapping genes, lacL and lacM. The lacL (1887bp) and lacM (960bp) genes encode polypeptides with calculated molecular masses of 73,620 and 35,682Da, respectively. The deduced amino acid sequences of lacL and lacM show significant identity with the sequences of beta-galactosidases from other lactobacilli and Escherichia coli. The coding regions of the lacLM genes were cloned and successfully overexpressed in E. coli using an expression system based on the T7 RNA polymerase promoter. Expression of lacL alone and coexpression of lacL and lacM as well as activity staining of both native and recombinant beta-galactosidases suggested a translational coupling between lacL and lacM, indicating that the formation of a functional beta-galactosidase requires both genes. Recombinant beta-galactosidase was purified to apparent homogeneity, characterized and compared with the native beta-galactosidase from L. reuteri L103.     

Thermostable beta-galactosidase from the archaebacterium Sulfolobus solfataricus. Purification and properties

A thermophilic and thermostable beta-galactosidase activity was purified to homogeneity from crude extracts of the archaebacterium Sulfolobus solfataricus, by a procedure including ion-exchange and affinity chromatography. The homogeneous enzyme had a specific activity of 116.4 units/mg at 75 degrees C with o-nitrophenyl beta-galactopyranoside as substrate. Molecular mass studies demonstrated that the S. solfataricus beta-galactosidase was a tetramer of 240 +/- 8 kDa composed of similar or identical subunits. Comparison of the amino acid composition of beta-galactosidase from S. solfataricus with that from Escherichia coli revealed a lower cysteine content and a lower Arg/Lys ratio in the thermophilic enzyme. A rabbit serum, raised against the homogeneous enzyme did not cross-react with beta-galactosidase from E. coli. The enzyme, characterized for its reaction requirements and kinetic properties, showed a thermostability and thermophilicity notably greater than those reported for beta-galactosidases from other mesophilic and thermophilic sources.     

Purification and characterization of isoforms of beta-galactosidases in mung bean seedlings

Five isoforms of beta-galactosidase (EC 3.2.1.23), designated as beta-galactosidases I-V, were isolated from five-day-old mung bean (Vigna radiata) seedlings. Beta-galactosidases II and III were purified to electrophoretic homogeneity by a procedure involving acid precipitation, ammonium sulfate fractionation, chromatography on diethylaminoethyl-cellulose (DEAE-Cellulose) and con A-Sepharose. and chromatofocusing. Beta-galactosidases I, II and III have the same molecular mass of 87 kDa. comprising two nonidentical subunits with molecular masses of 38 and 48 kDa, while beta-galactosidases IV and V have molecular masses of 45 and 73 kDa, respectively. All the enzymes were active against p-nitrophenyl-beta-D-galactoside, and to a lesser extent, p-nitrophenyl-alpha-L-arabinoside and p-nitrophenyl-beta-D-fucoside. The enzymes were inhibited by D-galactono-1,4-lactone, D-galactose, Hg2+, Ag+ and sodium dodecyl sulfate (SDS). Beta-galactosidases I, II and III were shown to be competitively inhibited by either D-galactono-1, 4-lactone or D-galactose. Isoforms I, II and III have a common optimal pH of 3.6, while isoforms IV and V have pH optima at 3.8 and 4.0, respectively. Isoelectric points of isoforms I, II and III were 7.7, 7.5 and 7.3, respectively. Double immunodiffusion analysis indicated that beta-galactosidases I, II, III and V are immunologically similar to each other, while beta-galactosidase IV shares partially identical antigenic determinants with the other four isoforms. The purified beta-galactosidases II and III were capable of releasing D-galactose residue from the hemicellulose fraction isolated from mung bean seeds.     

β-galactosidase activity in fibroblasts and tissues from sheep with a lysosomal storage disease

Tissues and fibroblasts of sheep affected with an inherited, neuronal lysosomal storage disease expressed a deficiency of beta-galactosidase activity. Cerebrum, kidney, lung, spinal cord, and spleen from affected sheep had less than 8% of the beta-galactosidase activity present in the respective tissues of normal sheep. No evidence for the presence of an endogenous inhibitor in affected sheep was detected by mixing studies. Liver of affected sheep expressed a deficiency of beta-galactosidase activity only in the presence of the beta-D-glycosidase inhibitors, glucono-delta-lactone and 2,5-dihydroxymethyl-3,4-dihydroxypyrrolidine. In these studies, we demonstrated the existence of tissue-specific beta-galactosidases in sheep and showed that the affected sheep have a deficiency of the lysosomal beta-galactosidase. Our results suggest that the high residual beta-galactosidase activity in liver of affected sheep can be attributed to a nonlysosomal beta-galactosidase that has a neutral pH optimum and may be under temporal regulation.     

Determining the evolutionary potential of a gene

In addition to information for current functions, the sequence of a gene includes potential information for the evolution of new functions. The wild-type ebgA (evolved beta-galactosidase) gene of Escherichia coli encodes a virtually inactive beta-galactosidase, but that gene has the potential to evolve sufficient activity to replace the lacZ gene for growth on the beta-galactoside sugars lactose and lactulose. Experimental evidence, which has suggested that the evolutionary potential of Ebg enzyme is limited o two specific amino acid replacements, is limited to examining the consequences of single base- substitutions. Thirteen beta-galactosidases homologous with the Ebg beta-galactosidase are widely dispersed, being found in gram-negative and gram-positive eubacteria and in a eukaryote. A comparison of Ebg beta-galactosidase with those 13 beta-galactosidases shows that Ebg is part of an ancient clade that diverged from the paralogous lacZ beta- galactosidase over 2 billion years ago. Ebg differs from other members of its clade at only 2 of the 15 active-site residues, and the two mutations required for full Ebg beta-galactosidase activity bring Ebg into conformity with the other members of its clade. We conclude that either these are the only acceptable amino acids at those positions, or all of the single-base-substitution replacements that must arise as intermediates on the way to other acceptable amino acids are so deleterious that they constitute a deep selective valley that has not been traversed in over 2 billion years. The evolutionary potential of Ebg is thus limited to those two replacements.      

Galactosylation at side chains of branched cyclodextrins by various beta-galactosidases.

The galactosyl transfer reaction to branched cyclodextrins (CDs) was investigated using lactose as a donor substrate and branched CDs as acceptors by various beta-galactosidases. Bacillus circulans beta-galactosidase synthesized galactosyl transfer products to branched CDs, of which the galactose residues were linked at side chains of branched CDs, not directly at CD rings. Aspergillus oryzae and Penicillium multicolor beta-galactosidases also produced derivatives galactosylated at side chains of branched CDs. The structures of main transgalactosylation products of branched CDs by these beta-galactosidases seem to be different from those by B. circulans beta-galactosidase, judging from the retention times on high performance liquid chromatography.     

Structure and expression of dog apolipoprotein A-I, E, and C-I mRNAs: implications for the evolution and functional constraints of apolipoprotein structure

Dog apolipoprotein (apo) C-I, A-I, and E cDNA clones were identified in a dog liver cDNA library in lambda gt10 by hybridization to synthetic oligonucleotide probes with the corresponding human DNA sequences. The longest clone for each apolipoprotein was completely sequenced. The apoC-I cDNA sequence predicts a protein of 62 residue mature peptide preceded by a 26 amino acid signal peptide. The apoA-I cDNA sequence predicts a 242 residue mature peptide, a 6 residue pro-segment, and an 18 residue signal peptide. The apoE cDNA, which lacks the signal peptide region, predicts a mature peptide of 291 amino acid residues. Slot blot hybridization of total RNA isolated from various dog tissues to dog apoC-I, A-I, and E cDNA probes indicates that apoC-I mRNA is detectable in liver only, apoA-I mRNA is present in liver and small intestine, though the concentration in the latter tissue is only approximately 15% of that in the liver, and apoE mRNA is present in multiple tissues including liver, jejunum, urinary bladder, ileum, colon, brain, kidney, spleen, pancreas, and testis with relative concentrations (%) of 100, 17.5, 7.5, 6.9, 5.9, 5.5, 5.0, 3.3, 1.0, and 1.0, respectively. These tissue distributions indicate that nascent lipoprotein particles produced in the dog small intestine would contain apoA-I and apoE but not apoC-I. The widespread tissue distribution of apoE mRNA indicates that like other mammals, peripheral synthesis of apoE contributes significantly to the total apoE pool in dog. We next compared the cDNA sequences among different vertebrate species for apoC-I (human and dog), A-I (human, rat, dog, rabbit and chicken), and E (human, rat, dog and rabbit) and calculated the rate of nucleotide substitution for each gene. Our results indicate that apoC-I has evolved rather rapidly and that on the whole, apoA-I is more conservative than apoE, contradictory to an earlier suggestion. ApoA-I is also more conservative than a region (residues 4204-4536) at the carboxyl-terminal portion, but less conservative than a region (residues 595-979) at the amino-terminal portion of apoB-100. Some regions in each of the apolipoproteins studied are better conserved than others and the rate of evolution of individual regions seems to be related to the stringency of functional requirements. Finally, we estimate that the human apoC-I pseudogene arose more than 35 million years ago, becoming nonfunctional soon after its formation.     

Purification and characterization of the beta-galactosidase of Aeromonas formicans

Rohlfing, S. R. (Western Reserve University, Cleveland, Ohio), and I. P. Crawford. Purification and characterization of the beta-galactosidase of Aeromonas formicans. J. Bacteriol. 91:1085-1097. 1966.-The beta-galactosidase of Aeromonas formicans was purified by diethylaminoethyl cellulose chromatography and gel filtration on Sephadex G-200. The properties of the enzyme molecule were compared with purified beta-galactosidase from Escherichia coli. The sedimentation coefficients and electrophoretic mobilities of the two enzymes were not significantly different; the electrophoretic mobility of urea-produced subunits of the two enzymes was also similar. The stabilities of the two enzymes to denaturing agents provided measurable differences; E. coli beta-galactosidase is relatively more heat-stable and more resistant to the action of urea. The amino acid compositions of the two proteins revealed significant differences in several amino acids, particularly alanine, arginine, glycine, and leucine. The comparisons cited suggest that A. formicans and E. coli are not completely unrelated, for their beta-galactosidases show considerable structural similarity.     

A novel Arthrobacter beta-galactosidase with homology to eucaryotic beta-galactosidases.

An Arthrobacter beta-galactosidase has homology with the lysosomal acid beta-galactosidases from humans and mice and with a Xanthomonas manihotis enzyme. Phylogenetic analysis of the deduced amino acid sequence showed an unusual pattern, with this procaryotic enzyme clustering within the animal clade. The gene encodes a subunit of 52 kDa, and the enzyme appears to be active as a dimer. The enzyme hydrolyzed substrates with either a beta-1,4 or a beta-1,3 linkage.     

Intracellular distribution of beta-galactosidases in mucosal cells from hog small intestine.

1. Intracellular distribution of three types of beta-galactosidases (beta-D-galactoside galactohydrolase EC 3.2.1.12) i.e. hetero beta-galactosidase, lactase and acid beta-galactosidase, was studied by examining the properties of subcellular fractions isolated by a systematic fractionation of mucosal cells of the small intestine of the hog. Localization of hetero beta-galactosidase in cytosol could be shown. 2. Localization of lactase in the brush borders was shown by analyzing the purified brush borders prepared separately. 3. To domonstrate the lysosomal localization of acid belta-galactosidase, lysosomes were purified separately and their extract was chromatographed on a hydroxylapatite column. The activities of various enzymes in the purified lysosomes as well as in the intermediary fractions obtained during lysosome purification and the pattern of the hydroxylapatite chromatography led us to conclude that acid beta-galactosidase is a lysosomal enzyme.     

Glu-537, not Glu-461, is the nucleophile in the active site of (lac Z) beta-galactosidase from Escherichia coli.

The covalent intermediate formed during catalysis by the lac Z beta-galactosidase from Escherichia coli can be trapped by reaction of the enzyme with 2',4'-dinitrophenyl 2-deoxy-2-fluoro-beta-D-galactopyranoside, thereby inactivating the enzyme. Kinetic parameters for this inactivation process with the holo- and apo-enzymes have been determined. The intermediate so formed turns over only very slowly (t1/2 = 11.5 h) resulting in reactivation of the enzyme. The nucleophilic amino acid involved has been identified as Glu-537 by using a tritium-labeled inactivator to label the enzyme, then cleaving the labeled protein into peptides and purifying and sequencing the labeled peptide. This residue is conserved in five homologous beta-galactosidases and is different from that (Glu-461) proposed to be the nucleophile (Herrchen, M., and Legler, G. (1984) Eur. J. Biochem. 138, 527-531) on the basis of affinity labeling studies with conduritol C cis-epoxide. A role for glutamic acid residue 461 as the acid/base catalyst is proposed and justified.