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PubMed1984

Intracellular targeting and import of an F1-ATPase beta-subunit-beta-galactosidase hybrid protein into yeast mitochondria.

The gene coding for the yeast mitochondrial F1-ATPase beta subunit (ATP2) has been fused to the Escherichia coli lacZ gene. The chimeric ATP2-lacZ gene codes for a hybrid protein consisting of some 350 amino acids of the F1-ATPase beta subunit at its amino terminus and a large enzymatically active portion of the lacZ gene product, beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), at its carboxyl terminus. The beta-subunit-beta-galactosidase hybrid protein is expressed in both E. coli and yeast. In yeast, this hybrid molecule is targeted to the mitochondrion and is protected in isolated mitochondria from added protease under conditions in which an outer membrane enzymatic marker is digested. Yeast cells carrying the ATP2-lacZ gene fusion on plasmid p beta Z1 are unable to grow on a nonfermentable carbon source. Upon loss of the p beta Z1 plasmid, growth of the cured host strain on the nonfermentable substrate is restored. In the presence of the beta-subunit-beta-galactosidase hybrid protein, the energy-transducing capacity of the mitochondrial membrane as measured by the 32Pi-ATP exchange reaction is only 9% of that measured in the absence of the gene fusion product. The results indicate that it is the presence of the beta-subunit-beta-galactosidase hybrid protein within mitochondria that interferes with function(s) essential for respiratory growth. These observations open up the prospect of genetic characterization of the signals and cellular machinery responsible for mitochondrial protein delivery.

Amino Acid Sequence↗
PubMed1983

Immunocytochemical localization of lysosomal beta-galactosidase in rat liver.

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta-galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta-galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.

Animals↗
PubMed1989

Viable starter culture, beta-galactosidase activity, and lactose in duodenum after yogurt ingestion in lactase-deficient humans.

Ten lactose malabsorbers were intubated and given fresh or heated yogurt to which polyethylene-glycol (PEG) and spores of Bacillus stearothermophilus (SBS) had been added as internal standards. In duodenal samples taken after fresh yogurt ingestion, viable starter culture was detected for 60 min in 6 of 7 subjects and the ratio of microbial beta-galactosidase activity to SBS remained similar during this period to its value in the preingested yogurt. In the two groups ingesting fresh and heated yogurt respectively, ratios of lactose to PEG remained similar to preingested values for 90 min and duodenal pH remained less than 5.1. In vitro, at pH 5.0, beta-galactosidase activity in yogurt dropped by 80%. These data clearly show that after fresh yogurt ingestion, viable starter culture reaches the duodenum and contains beta-galactosidase activity. However, the buffering capacity of the yogurt that protects bacteria from acidic gastric secretion also prevents microbial beta-galactosidase from hydrolyzing lactose in the duodenum.

Adult↗
PubMed1990

Retroviral mediated gene transfer into bone marrow progenitor cells: use of beta-galactosidase as a selectable marker.

Recombinant retroviruses have been utilized as vectors for gene transfer in model systems of gene therapy. Since many of these model systems require the transplantation of genetically modified primary cells it is important to devise methods which will allow the rapid and efficient selection for transplantation of only the cells which are capable of expressing high levels of the transferred gene. This report describes the use of beta-galactosidase as such a selectable marker. Bone marrow progenitors are infected with a recombinant retrovirus encoding beta-galactosidase. Using a fluorescence assay for beta-galactosidase we demonstrate that it is possible to use cell sorting to enrich for cells which will form bone marrow colonies that express high levels of beta-galactosidase. This rapid and non-toxic selection of bone marrow cells may facilitate attempts to achieve gene therapy in a variety of model systems.

Animals↗
PubMed1986

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.

Animals↗
PubMed1986

Cloning, expression and location of the Streptococcus lactis gene for phospho-beta-D-galactosidase.

Genes for lactose catabolism and proteinase production in Streptococcus lactis 712 are encoded by a 56.5 kb metabolic plasmid, pLP712. A lactose mini-plasmid of only 23.7 kb, pMG820, was constructed by introducing two deletions into pLP712, and was cloned as two segments of DNA into the Escherichia coli vector pAT153 using restriction endonuclease PstI. The lactose genetic region of pLP712, which has been defined by deletion and restriction mapping, was cut into two parts by this process. When the smaller 10.8 kb segment of pMG820 DNA was present, the key lactic streptococcal lactose splitting enzyme, phospho-beta-D-galactosidase, was expressed in E. coli. The gene for phospho-beta-D-galactosidase was more precisely located by introducing a series of deletions into cloned DNA by in vitro manipulations and then assaying for enzyme activity. The presence of this phospho-beta-D-galactosidase activity was correlated with the production of a 58 kDa 35S-labelled protein both by E. coli minicells and after coupled transcription and translation of cloned DNA. The product of a second gene, a 37 kDa protein ('protein X'), and a possible truncated phospho-beta-D-galactosidase protein of 16 kDa were also detected in minicells.

Cloning, Molecular↗
PubMed1979

beta-Galactosidase from Aspergillus niger. Separation and characterization of three multiple forms.

The enzyme beta-galactosidase (EC 3.2.1.23) from Aspergillus niger was purified and resolved into three multiple forms, using molecular sieving, ion-exchange, an hydrophobic chromatography. The isolated enzyme forms accounted for 83%, 8%, and 9% of the total beta-galactosidase activity, respectively. They were glycoproteins with estimated molecular weights of 124,000, 150,000 and 173,000, isoelectric points of about 4.6, and pH optima between 2.5 and 4.0. Amino acid and carbohydrate analyses showed that multiplicity was mainly due to dissimilar carbohydrate contents (about 12.5%, 20.5% and 29% neutral carbohydrates, respectively). The multiple form pattern might depend on the culture conditions. The beta-galactosidase forms were heat-stable up to about 60 degrees C. The Km values for lactose ranged from 85 mM to 125 mM, whereas those for the synthetic substrate o-nitrophenyl-beta-D-galactopyranoside were equal to about 2.4 mM. The V values obtained at 30 degrees C for lactose and o-nitrophenyl-beta-D-galactopyranoside were 104 units/mg enzyme protein and 121 units/mg enzyme protein, respectively (weighted averages for the three enzyme forms). The slight reactional dissimilarities between the three enzyme forms are unlikely to be physiologically relevant. The biological significance of A. niger beta-galactosidase multiplicity might be related to the observed differences in carbohydrate content, as suggested by recent reports on other microbial glycoprotein enzymes.

Amino Acids↗
PubMed1986

Characterization of a beta-galactosidase hybrid protein carrying the catalytic domain of Escherichia coli adenylate cyclase.

A hybrid protein of Escherichia coli, exhibiting both adenylate cyclase and beta-galactosidase activities, was purified and characterized. This protein, obtained by genetic engineering, contained the first 556 amino acids of adenylate cyclase connected to the eighth-residue of beta-galactosidase through a pentapeptide Val-Gly-Asp-Pro-Val. The fusion protein was less stable than the native beta-galatosidase. Trypsin cleaved preferentially the adenylate cyclase moiety of the hybrid protein at a ratio of 1/50 (w/w). The kinetic properties of the hybrid protein were comparable, with a few exceptions, to those of native adenylate cyclase and beta-galactosidase. 'Truncated' adenylate cyclase was no longer sensitive to inhibition by excess ATP, which seems to indicate a second nucleotide binding site of wild-type adenylate cyclase. Photoirradiation of the hybrid protein with 8-azidoadenosine 5'-triphosphate inactivated the adenylate cyclase activity, leaving intact the beta-galactosidase activity. A radiolabeled ATP analog was incorporated after photoirradiation into the adenylate cyclase moiety of the fusion protein as shown by limited digestion with trypsin.

Adenosine Triphosphate↗
PubMed1979

Purification and properties of an inducible beta-galactosidase isolated from the yeast Kluyveromyces lactis.

beta-Galactosidase (EC 3.2.1.32) was purified 80-fold from the yeast Kluyveromyces lactis induced for this enzyme by growth on lactose. When the purified enzyme was subjected to electrophoresis on an acrylamide gel in the presence of sodium dodecyl sulfate, one protein with an apparent molecular weight of 135,000 was observed. The enzyme has a sedimentation coefficient of 9.6S. This beta-galactosidase and the one from Escherichia coli are not antigenically related. Maximal enzyme activity requires Na+ and Mn2+ and a reducing agent. beta-Galactosidase has Km values of 12 to 17 and 1.6 mM for lactose and o-nitrophenyl-beta-D-galactoside, respectively. The hydrolase and transgalactosylase activities of the enzyme are similar to those of E. coli beta-galactosidase.

Antigens, Fungal↗
PubMed1979

Effects of 8-substituted analogs of cyclic adenosine 3',5'-monophosphate on in vivo and in vitro syntheses of beta-galactosidase in Escherichia coli.

Several 8-substituted alkylthio and alkylamino cyclic adenosine 3',5'-monophosphate (cAMP) derivatives were tested for their ability to stimulate beta-galactosidase synthesis in Estherichia coli in vivo and in vitro and to inhibit the cAMP phosphodiesterase activity of E. coli. Stimulation of beta-galactosidease synthesis in vivo by cAMP derivatives decreased with increasing length of the unbranched carbon chain of the substituent. On the other hand, the stimulation in vitro was increased as the carbon chain elongated. The 8-decylthio- and 8-dodecylthio-cAMP compounds stimulated beta-galactosidase synthesis almost eight-fold compared with cAMP, whereas 8-undecyl-, 8-dodectyl-, and 8-tridecylamino-cAMP stimulated beta-galactosidase synthesis about threefold. However, in in vitro experiments with a phosphodiesterase-deficient strain of E. coli, the Crooks strain, the stimulatory effects of the derivatives disappeared, except for 8-dodecylthio cAMP which stimulated beta-galactosidase about 1.4- to 1.6-fold. All derivatives were quite resistant to hydrolysis by phosphodiesterase. Most derivatives competitively inhibited the hydrolysis of cAMP by phosphodiesterase.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1979

Lactose metabolism involving phospho-beta-galactosidase in Klebsiella.

Klebsiella strain RE1755A is a Lac- Gal- mutant which has lost both of its lac operons, but possesses a gene specifying beta-galactosidase III, an enzyme which hydrolyzes o-nitrophenyl-beta-D-galactopyranoside but does not hydrolyze lactose. Selective pressure was applied to isolate mutants able to utilize lactose. The lactose-utilizing mutants obtained were shown to possess an unaltered beta-galactosidase III. Lactose utilization was shown to result from a pleiotropic mutation which also (i) permits galactose utilization and (ii) prevents induction of beta-galactosidase III synthesis by lactose. Evidence is presented suggesting that a phospho-beta-galactosidase enzyme is involved in lactose metabolism.

Galactosephosphates↗
PubMed1987

Characterization of the promoter, signal sequence, and amino terminus of a secreted beta-galactosidase from "Streptomyces lividans".

The gene for a secreted 130-kilodalton beta-galactosidase from "Streptomyces lividans" has been cloned, its promoter, signal sequence, and amino terminal region have been localized, and their nucleotide sequence has been determined. The signal sequence extends over 56 amino acids and shows the characteristic-features of signal sequences, including a hydrophilic amino terminus followed by a hydrophobic core near the signal cleavage site. The secretion of beta-galactosidase depends on the presence of the signal sequence. beta-Galactosidase is the major protein in culture supernatants and extracts of strains expressing the cloned beta-galactosidase gene and represents a valuable tool in the study of protein secretion in Streptomyces spp.

Base Sequence↗
PubMed1983

Fusion of the Saccharomyces cerevisiae leu2 gene to an Escherichia coli beta-galactosidase gene.

The promoter and translation initiation region of the Saccharomyces cerevisiae leu2 gene was fused to the Escherichia coli beta-galactosidase gene. This fusion located the control region of the leu gene and orientated its direction of expression. When the fusion was placed into yeast cells, beta-galactosidase was expressed under the same regulatory pattern as the original leu2 gene product: its synthesis was repressed in the presence of leucine and threonine. Sensitive chromogenic substrates for beta-galactosidase were used to detect expression in isolated colonies growing on agar medium. Mutant yeast cells with increased beta-galactosidase activity were identified by the color of the colonies they formed. One class of mutants obtained appeared to affect ars1 plasmid maintenance, and another class appeared to affect beta-galactoside uptake.

3-Isopropylmalate Dehydrogenase↗
PubMed1979

Effect of carbon sources on the rates of cyclic AMP synthesis, excretion, and degradation, and the ability to produce beta-galactosidase in Escherichia coli.

We have determined the rates of adenosine 3',5'-cyclic monophosphate (cAMP) synthesis, excretion, and degradation, and the cAMP pool size in Escherichia coli grown on various carbon sources. We have found that the cAMP pool size increases in approximate proportion to increases in the cAMP synthetic rate. Although the combined rate of excretion and degradation of cAMP is in approximate proportion to the cAMP pool size, no such regular relationship is seen between the cAMP pool size and either the excretion rate or the degradation rate. Using a method which we have developed for determining the cellular efficiency of enzyme production (termed 'cellular' rate), we have reexamined the relationship between cAMP pool size and the rate of beta-galactosidase production. Although there exists an overall trend of increasing rate of beta-galactosidase production with increasing cAMP pool size, large variations in the rates of beta-galactosidase production are seen even under culture conditions which yield similar cAMP pool sizes. This suggests that the intracellular level of cAMP cannot be the unique regulator of beta-galactosidase production.

Culture Media↗
PubMed1984

Human placental beta-galactosidase: structural and immunological observations.

beta-Galactosidase purified to apparent homogeneity from human placenta occurred in two separable fractions. A low molecular mass form (relative mass (Mr) 170 000) is composed of a single polypeptide chain (Mr 70 000). This was derived from a larger form by molecular sieve chromatography at both low (10 mM) and high (500 mM) NaCl concentration. The larger form of beta-galactosidase also contains small amounts of two polypeptides with apparent Mr values of 23 000 and 35 000 daltons. Both forms of the enzyme hydrolyze synthetic aryl galactosides and natural glycolipid substrates at comparable rates. Antibodies raised in rabbits against the low Mr beta-galactosidase also cross-reacts with the high Mr enzyme. The antibody preparation also cross-reacted with beta-hexosaminidase even though the latter was found at very low levels in the antigen, as judged by lack of detection of representative protein bands on sodium dodecyl sulfate - polyacrylamide gel electrophoresis and enzyme activity measurements. A portion of this cross-reactivity (35%) against beta-hexosaminidase could not be absorbed from the preparation without the simultaneous loss of beta-galactosidase activity, suggesting that the two enzymes show a degree of antigenic identity.

Amino Acids↗
PubMed1984

Evaluation of beta-D-galactosidase from Escherichia coli and horseradish peroxidase as labels by sandwich enzyme immunoassay technique.

beta-D-galactosidase from Escherichia coli and horseradish peroxidase were evaluated as labels of Fab' in dose-response curves for human alpha-fetoprotein and human chorionic gonadotropin by sandwich enzyme immunoassay technique using fluorogenic substrates for enzyme assay. The non-specific binding of Fab'-peroxidase conjugates to IgG-coated polystyrene balls was less than that of Fab'-beta-D-galactosidase conjugates, and the affinity-purified Fab'-peroxidase conjugates gave more sensitive dose-response curves for these antigens than the corresponding beta-D-galactosidase conjugates. However, a large quantity of Fab'-peroxidase conjugates was required and a longer incubation was necessary for the peroxidase assay, since the peroxidase assay was much less sensitive than the beta-D-galactosidase assay. Other advantages and disadvantages of the two enzymes are discussed.

Antigen-Antibody Reactions↗
PubMed1984

Isolation of a beta-galactosidase from chicken liver.

In search of a beta-galactosidase which specifically hydrolyses beta 1----3 bound galactose residues in galacto-glycoconjugates, an acid beta-galactosidase from chicken liver was investigated. The isolation procedure involved ammonium sulphate precipitation followed by lectin chromatography on Con A-Sepharose 4B, ion-exchange chromatography on DEAE-cellulose, gel filtration on Sepharose 6B and affinity chromatography on p-aminophenyl-thio-beta-D-galactoside-agarose. The beta-galactosidase was purified 3000-fold with 11% recovery of enzyme activity. The purified protein showed an apparent molecular mass of above 200000 in SDS-polyacrylamide gel electrophoresis. A few minor bands were also present. The reduced and denatured beta-galactosidase migrated as a single major band with an apparent molecular mass of 67000. The enzyme released galactose from lactose and from the synthetic substrates Gal beta 1----3Gal, Gal beta 1----6Gal and Gal beta 1----3Ara. However, the enzyme did not release galactose from the snail gland galactans and the high molecular weight galacto-glycoconjugates and it did not hydrolyse the peanut agglutinin receptor of the red blood cell membrane.

Ammonium Sulfate↗
PubMed1982

Serum and urinary beta-galactosidase and beta-glucuronidase activity in patients with acute myocardial infarction.

To determine the effect of acute myocardial infarction (AMI) on serum and urinary activity of beta-galactosidase and beta-glucuronidase (lysosomal enzymes) 40 patients were studied. Eighteen patients had acute myocardial infarction and 22 were assigned as controls. Three of the 18 patients with acute myocardial infarction died within 5 to 10 days after hospitalization. Although the serum and urinary beta-glucuronidase and serum beta-galactosidase activity was higher in patients with acute myocardial infarction when compared with the control subjects these differences did not achieve statistical significance. However, the mean values of urinary beta-galactosidase in the control and acute MI groups were 158.68 and 333.3 nmol/mg creatinine/hr, respectively (p less than 0.046). These findings indicate that there is a significant increase in the urine beta-galactosidase activity during the early phases of acute myocardial infarction.

Female↗