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PubMed2000

Biosynthesis, processing, and subcellular localization of rat spermbeta-D-galactosidase.

During spermatogenesis, spermatids synthesize constituent proteins present in mature spermatozoa; however, little information exists on the molecular processes involved. In previous studies, this laboratory reported the characterization of rat sperm beta-D-galactosidase. In this paper, we report the localization of this enzyme along with its biosynthesis and processing. An antibody against rat luminal fluid beta-D-galactosidase was used to immunolocalize the enzyme in the testis and in epididymal spermatozoa. We found that beta-D-galactosidase is localized within the acrosomal cap of spermatids and in the acrosome and cytoplasmic droplet of epididymal spermatozoa. A combination of germ cell radiolabeling, immunoprecipitation, SDS-PAGE, and autoradiography revealed that spermatids produce two forms of beta-D-galactosidase, 90 and 88 kDa. During pulse-chase analysis, a 56-kDa form appeared. Treatment of beta-D-galactosidase immunoprecipitates from testicular spermatozoa with N-glycanase or Endo H revealed that both the 90- and 88-kDa forms become a 70-kDa polypeptide on SDS-PAGE. Since Endo H or N-glycanase treatment provided similar results, the presence of extensive N-linked high mannose/hybrid-type glycans on these proteins is indicated. Treatment of the 56-kDa form of beta-D-galactosidase with Endo H or N-glycanase resulted in the appearance of 52- and 50-kDa forms, respectively. This result suggests that the 56-kDa form contains N-linked high mannose/hybrid as well as complex oligosaccharides. During epididymal maturation, the 90-kDa form of beta-D-galactosidase persists in caput epididymal spermatozoa and is gradually converted to a major 74-kDa form in cauda spermatozoa. In addition to the 90- to 74-kDa forms, cauda spermatozoa show a 56- to 52-kDa form on Western immunoblots. Since only the high-molecular weight forms of beta-D-galactosidase are present on immunoblots of isolated sperm heads, we suggest that they are acrosomal in origin and that the 56-kDa form, which is processed to 52 kDa in cauda spermatozoa, is associated with the cytoplasmic droplet.

Animals↗

PubMed1975

Chemical pathology of krabbe's disease. IV. Studies of galactosylceramide and lactosylceramide BETA-galactosidases in brain, white blood cells and aminotic fluid cells.

Galactosylceramide beta-galactosidase and lactosylceramide beta-galactosidase activities were investigated in normal human brain, leu-kocytes and amniotic fluid cells. The enzymatic assays were performed on brains from 11 patients with Krabbe's disease, on leukocytes from 16 patients and 18 obligate heterozygotes, and on amniotic fluid cells from 9 foetuses at risk. The brain enzyme was solubilized from a 900 g-100000 g pellet. With this enzyme preparation a profound deficiency of galactosylceramide beta-galactosidase activity in brain, approximately 1% of that in age-matched controls was shown. The lactosylceramide beta-galactosidase activity of brain was also strongly reduced, but not to the same extent as the other beta-galactosidase. Galactosylceramide beta-galactosidase activity in leukocytes from patients with Krabbe's disease was generally less than 5% of that in age-matched controls and there was no overlap between the patients and the obligate heterozygotes. Carrier detection by the leukocyte enzyme was, however, not possible because of considerable overlap between heterozygotes and normal controls. The lactosylceramide beta-galactosidase activity was only moderately reduced in leukocytes, but strongly reduced in cerebral tissue from patients with Krabbe's disease. The changes in the glycolipid pattern of cerebral tissue, recently described by us in patients with Krabbe's disease, offers an explanation to the serious glycolipid beta-galactosidase deficiency in CNS.

Amniotic Fluid↗

PubMed1975

Isolation and characterization of the newly evolved ebg beta-galactosidase of Escherichia coli K-12.

The ebg beta-galactosidase of Escherichia coli K-12 strain LC110 has been purified and characterized. Strain LC110 is a Lac+ revertant of a mutant with a deletion of the lacZ beta-galactosidase gene. Its new ebg beta-galactosidase activity was shown to be due to a discrete protein, immunologically unrelated to lacZ beta-galactosidase. Its kinetics of action conformed to those of a simple conventional enzyme. With o-nitrophenyl-beta-D-galactoside as substrate, the Vmax was 11,200 nmol/min per mg of enzyme, the Km was 5 mM, and the activation energy was 12,400 cal/mol. Corresponding values for lacZ beta-galactosidase of wild-type E. coli K-12 were 350,000 nmol/min per mg of enzyme, 1.3 mM, and 8,000 cal/mol. A series of sugars has been examined as competitive inhibitors of ebg beta-galactosidase. Kinetic analyses suggest that ebg beta-galactosidase has a particularly high affinity for galactosamine and gamma-galactonolactone, binds galatose more tightly than lactose, and shows a general preference for monosaccharides rather than beta-galactosides. We conclude that the ebg beta-galactosidase may have arisen by modification of a gene involved with the metabolism of a monosaccharide, possibly a 2-amino sugar.

Antigens, Bacterial↗

PubMed1966

Galactose repression of beta-galactosidase induction in Escherichia coli.

Beggs, William H. (University of Minnesota, Minneapolis), and Palmer Rogers. Galactose repression of beta-galactosidase induction in Escherichia coli. J. Bacteriol. 91:1869-1874. 1966.-Galactose repression of beta-galactosidase induction in Escherichia coli was investigated to determine whether the galactose molecule itself is the catabolite repressor of this enzyme system. Without exception, beta-galactosidase induction by cells grown in a synthetic salts medium with lactate or glycerol as the carbon source was more strongly repressed by glucose than by galactose. This relationship existed even when the organism was previously grown in the synthetic medium containing galactose as the source of carbon. Two observations suggested that the ability of galactose to repress beta-galactosidase formation by Escherichia coli depends directly upon the cells' capacity to catabolize galactose. First, galactose repression of beta-galactosidase synthesis was markedly enhanced in bacteria tested subsequent to gratuitous induction of the galactose-degrading enzymes with d-fucose. Second, galactose failed to exert a repressive effect on beta-galactosidase in a galactose-negative mutant lacking the first two enzymes involved in galactose catabolism. Glucose completely repressed enzyme formation in this mutant. This same mutant, into which the genes for inducible galactose utilization had been introduced previously by transduction, again exhibited galactose repression. Pyruvate was found to be at least as effective as galactose in repressing beta-galactosidase induction by cells grown in synthetic salts medium plus glycerol. It is concluded that the galactose molecule itself is not the catabolite repressor of beta-galactosidase, but that repression is exerted through some intermediate in galactose catabolism.

Colorimetry↗

PubMed1966

Role of the galactose pathway in the regulation of beta-galactosidase.

Paigen, Kenneth (Roswell Park Memorial Institute, Buffalo, N.Y.). Role of the galactose pathway in the regulation of beta-galactosidase. J. Bacteriol. 92:1394-1403. 1966.-Galactose and its metabolites, galactose-1-phosphate, uridine diphosphogalactose, and uridine diphosphoglucose, as well as metabolites derived from uridine diphosphoglucose, were tested for their role in the regulation of beta-galactosidase. In cultures of wild-type Escherichia coli strains K-12 and B, exogenous galactose was no more effective as a repressor than were other carbon sources. Exogenous galactose also did not repress beta-galactosidase when added to mutants which can accumulate intracellular galactose or galactose-1-phosphate, indicating that these compounds do not repress. In such strains, repression of beta-galactosidase formation did occur if galactose was added in the presence of another metabolizable carbon source. This repression is presumably a consequence of the growth inhibition which follows the accumulation of these compounds, and the general catabolite repression which develops during growth inhibition. Exogenous galactose did repress beta-galactosidase in a mutant which accumulates uridine diphosphogalactose. This appears to result from a combination of several factors. These include a general inhibition of protein synthesis through depletion of the uridine triphosphate pool, catabolite inhibition as a consequence of growth inhibition, as well as a specific inhibition of beta-galactosidase formation. Glucose repression of beta-galactosidase was normal in a mutant strain blocked in the formation of uridine diphosphoglucose from uridine triphosphate and glucose-1-phosphate, indicating that neither uridine diphosphoglucose nor any compound uniquely derived from it functions as the hypothetical catabolite repressor. It is concluded that at least two separate mechanisms exist for the endogenous repression of beta-galactosidase in E. coli. One is exerted by uridine diphosphogalactose or its metabolic product; the other, by the generalized catabolite repressor which is still formed in strains unable to make uridine diphosphogalactose or uridine diphosphoglucose.

Enzyme Repression↗

PubMed1992

Surface denaturation of proteins: the thermal inactivation of beta-galactosidase (Escherichia coli) on wall-liquid surfaces.

Irreversible inactivation of dilute beta-galactosidase (Escherichia coli) at relatively low temperatures was found to occur as a result of interactions of beta-galactosidase with wall-liquid surfaces. The rate of inactivation was directly proportional to the wall-liquid surface area, but independent of the air-liquid surface area, and the rate was also dependent on the wall composition. A small portion of the beta-galactosidase molecules was found to bind strongly to the surfaces of vessels in which the beta-galactosidase was stored. Bovine serum albumin eliminated the inactivation and it also eliminated the binding of beta-galactosidase to the wall. On the other hand, EDTA eliminated the inactivation, but it did not decrease the amount of beta-galactosidase bound. The addition of some transition metals increased the rate of inactivation. Protection of beta-galactosidase from surface inactivation by EDTA is not, therefore, a result of decreased binding of the enzyme to the walls of the vessels, but is probably a result of the ability of EDTA to scavenge certain trace metal ions present in solution, which are needed for the inactivation. The content of protein in the solution did not change as a result of the inactivation and, thus, the inactive enzyme does not accumulate at the surface. Since beta-galactosidase is often stored for long periods of time and since it is used to decrease the lactose content of milk for lactose intolerant individuals, this study may have practical significance. The presence of metal chelators and extraneous proteins should improve the stability of the enzyme, especially for processes that are carried out at elevated temperatures.

Absorption↗

PubMed2000

Senescence-associated (beta)-galactosidase reflects an increase in lysosomal mass during replicative ageing of human endothelial cells.

Senescence-associated (beta)-galactosidase is widely used as a biomarker of replicative senescence. However, it remains unknown whether this is a distinct enzyme active at pH 6, and differentially expressed in senescence, or a manifestation of an increase in the classic acid lysosomal (beta)-galactosidase. Here we have investigated the origin of senescence-associated-(beta)-galactosidase activity by modifying the intracellular and lysosomal pH of young and senescent human umbilical vein endothelial cells and examining the effect of these manipulations on the levels of activity, using a flow cytometric assay. Lysosomal alkalinisation with chloroquine or bafilomycin A(1), as well as equilibration of the intracellular milieu to pH 6 with nigericin, caused a profound (92-99%) inhibition of the total intracellular (beta)-galactosidase activity. However, independent of pH alterations, senescent cells showed levels of (beta)-galactosidase activity three- to sixfold higher than young cells. This increase in activity occurred in parallel to an increase in (beta)-galactosidase protein levels. Acridine Orange staining revealed an increase in lysosomal content with replicative age, which correlated with the increase in (beta)-galactosidase. These findings demonstrate that senescence-associated (beta)-galactosidase is a manifestation of residual lysosomal activity at a suboptimal pH, which becomes detectable due to the increased lysosomal content in senescent cells.

Acridine Orange↗

PubMed2003

beta-Galactosidase and its significance in ripening of "Saijyo" Japanese Persimmon fruit.

The fruit extracts of ripening cv. Japanese Persimmon, "Saijyo", contained a number of glycosidases and glycanases. Among them, beta-galactosidase appeared to be the most significant, and the activity increased in parallel with tissue ripening. Persimmon beta-galactosidase was presented in at least three isoforms, beta-galactosidase-I (pI = 4.88), beta-galactosidase-II (pI = 6.76), and beta-galactosidase-III (pI = 7.05). beta-Galactosidase-III had exo-type galactanase activity, while the others did not. The activity of endo-type glycanases was a maximum in immature green or yellow fruits. The firmness of the pulp tissue decreased dramatically, and the amount of water-soluble polysaccharide (WSS) increased. The enzyme activities of exo-type glycosidases, especially beta-galactosidase, appeared maximal in mature red fruits. The amount of extractable pectin remained unchanged, although the galactose content of the high-molecular-weight fraction in WSS decreased dramatically. These results suggest that the ripening of persimmon was caused by the solubilization of pectic polysaccharide by endo-type glycanases and digestion by exo-type glycosidases. beta-Galactosidase, in particular, seemed to play a major role in ripening the fruit.

Carbohydrates↗

PubMed1996

Endogenous beta-galactosidase activity in the larval, pupal, and adult stages of the fruit fly, Drosophila melanogaster, indicates need for caution in lacZ fusion-gene studies.

Beta-galactosidase activity is known to exist in Drosophila melanogaster, but a detailed analysis of the tissue-specific patterns of activity has not previously been reported. Such an analysis is of particular interest because Drosophila is commonly used for making transformants that carry fusion genes in which the E. coli beta-galactosidase gene, lacZ, is used as a reporter gene. When these transformants are analyzed for beta-galactosidase activity by using chromogen X-gal staining, the method does not distinguish true fusion-gene activity from endogenous beta-galactosidase activity or from the beta-galactosidase activity of bacterial contaminants. Therefore, detailed maps of endogenous beta-galactosidase activity in this organism would help to prevent errors in data interpretation and would indicate which stages were most appropriate for experiments with the lacZ transformants. We have constructed such maps by applying X-gal staining methods to serial frozen sections and whole mounts of larval, prepupal, pupal, and adult stages of D. melanogaster reared under axenic conditions. Results showed endogenous beta-galactosidase activity in a variety of organs including the larval intestine, spiracles, lymph glands, cellular epidermis, and eye-antenna imaginal discs; the pupal cellular epidermis, lymph glands, imaginal tissues, fat body, and spiracle; and the adult pericardial cells, thoracic nephrocytes, ventriculus, and reproductive system. The good correlation between staining and metamorphic remodeling and phagocytic activity indicates that endogenous beta-galactosidase is physiologically interesting.

Animals↗

PubMed2003

Purification and characterization of a beta-galactosidase from peach (Prunus persica).

A beta-galactosidase (EC 3.2.1.23) from peach (Prunus persica cv Mibackdo) was purified and characterized. The purified peach beta-galactosidase was 42 kDa in molecular mass and showed high enzyme activity against a the beta-galactosidase substrate, rho-nitrophenyl-beta-D-galactopyranoside. The Km and Vmax values of the enzyme activity of the peach beta-galactosidase were 5.16 and 0.19 mM for rho-nitrophenyl-beta-D-galactopyranoside mM/h, respectively. The optimum pH of the enzyme activity was pH 3.0, but it was relatively stable from pH 3.0-10.0. The temperature optimum was 50 degrees C. The enzyme activities were not improved in the buffers that contained Ca2+, Cu2+, Zn2+, and Mg2+, which indicates that the purified peach beta-galactosidase did not require these cations as co-factors. However, the enzyme was completely inhibited by Hg2+. The purified protein was cross-reacted with an antibody against the persimmon fruit beta-galactosidase. A further comparison of the N-terminal amino acid sequence of the purified protein showed high homologies to those of beta-galactosidase in apple (87%), persimmon (80%), and tomato (87%). Therefore, enzymatic, immunological, and molecular evidences in this study indicate that the purified 42-kDa protein is a peach beta-galactosidase.

Amino Acid Sequence↗

PubMed2006

Secretion and properties of a hybrid Kluyveromyces lactis-Aspergillus niger beta-galactosidase.

BACKGROUND: The beta-galactosidase from Kluyveromyces lactis is a protein of outstanding biotechnological interest in the food industry and milk whey reutilization. However, due to its intracellular nature, its industrial production is limited by the high cost associated to extraction and downstream processing. The yeast-system is an attractive method for producing many heterologous proteins. The addition of a secretory signal in the recombinant protein is the method of choice to sort it out of the cell, although biotechnological success is not guaranteed. The cell wall acting as a molecular sieve to large molecules, culture conditions and structural determinants present in the protein, all have a decisive role in the overall process. Protein engineering, combining domains of related proteins, is an alternative to take into account when the task is difficult. In this work, we have constructed and analyzed two hybrid proteins from the beta-galactosidase of K. lactis, intracellular, and its Aspergillus niger homologue that is extracellular. In both, a heterologous signal peptide for secretion was also included at the N-terminus of the recombinant proteins. One of the hybrid proteins obtained has interesting properties for its biotechnological utilization. RESULTS: The highest levels of intracellular and extracellular beta-galactosidase were obtained when the segment corresponding to the five domain of K. lactis beta-galactosidase was replaced by the corresponding five domain of the A. niger beta-galactosidase. Taking into account that this replacement may affect other parameters related to the activity or the stability of the hybrid protein, a thoroughly study was performed. Both pH (6.5) and temperature (40 degrees C) for optimum activity differ from values obtained with the native proteins. The stability was higher than the corresponding to the beta-galactosidase of K. lactis and, unlike this, the activity of the hybrid protein was increased by the presence of Ni2+. The affinity for synthetic (ONPG) or natural (lactose) substrates was higher in the hybrid than in the native K. lactis beta-galactosidase. Finally, a structural-model of the hybrid protein was obtained by homology modelling and the experimentally determined properties of the protein were discussed in relation to it. CONCLUSION: A hybrid protein between K. lactis and A. niger beta-galactosidases was constructed that increases the yield of the protein released to the growth medium. Modifications introduced in the construction, besides to improve secretion, conferred to the protein biochemical characteristics of biotechnological interest.

Journal Article↗

PubMed1989

Interspecific genetic complementation analysis of human and sheep fibroblasts with beta-galactosidase deficiency.

Interspecific somatic cell hybrids were analyzed by genetic complementation to determine if a lysosomal storage disease in sheep associated with deficiencies of beta-galactosidase and alpha-neuraminidase was homologous with any of four beta-galactosidase-deficient human diseases. Fibroblasts from beta-galactosidase-deficient sheep, cats, and human patients were fused and assayed histochemically for beta-galactosidase, with 5-bromo-4-chloro-3-indolyl beta-D-galactoside. We observed complementation in heterokaryons consisting of fibroblasts from beta-galactosidase-deficient sheep and fibroblasts from patients with galactosialidosis or mucolipidosis type II, but no complementation in heterokaryons consisting of fibroblasts from beta-galactosidase-deficient sheep and fibroblasts from human or feline GM1 gangliosidosis (type I) or from human mucopolysaccharidosis type IVB fibroblasts. We conclude that the ovine disease is due to a mutation at the genetic locus homologous with that of GM1 gangliosidosis and mucopolysaccharidosis type IVB, suggesting that the primary defect in the ovine disease is a mutation of the beta-galactosidase structural gene.

Animals↗

PubMed1978

I-Cell disease: isoelectric focusing, concanavalin A-Sepharose 4B binding and kinetic properties of human liver acid beta-D-galactosidases.

Isoelectric focusing of the acid beta-D-galactosidases (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in normal crude liver supernatant fluids demonstrated multiple isoelectric forms in the pH range 4.58-5.15, while corresponding I-cell disease samples showed an absence of isoelectric forms in the pH range 4.99-5.15. Concanavalin A-Sepharose 4B chromatography of the I-cell disease mutant C.A. demonstrated a 31% and 37% decrease in the binding of 4-methyl-umbelliferyl-beta-D-galactosidase and GM1 beta-D-galactosidase activities, respectively, when compared to normal samples. Isoelectric focusing profiles of the concanavalin A-Sepharose 4B alpha-methyl-D-mannoside effluents containing normal and I-cell disease acid beta-D-galactosidase were generally similar, but the unadsorbed I-cell disease enzyme from concanavalin A-Sepharose 4B demonstrated more activity in the pH range 4.21-4.49 than normals. Normal and I-cell disease acid beta-D-galactosidase "A" and "B", separated by gel column chromatography were found to have similar properties with respect to apparent molecular weights pH vs. activity profiles and apparent Km values for the 4 methylumbelliferyl-beta-D-galactopyranoside, GM1-ganglioside and asialofetuin (ASF) substrates. However, the apparent V values for the ICD samples were consistently reduced when compared to the results obtained with the corresponding normal fractions. The greatest decreases in apparent V were obtained for acid beta-D-galactosidase activities in I-cell disease crude supernatant fluids, and for the separated I-cell disease "B" enzyme. The differences in the isoelectric focusing profiles, the altered binding to concanavalin A-Sepharose 4B, and the reduced V values with natural and synthetic substrates may be related to changes in carbohydrate composition of I-cell disease acid beta-D-galactosidase.

Chromatography, Affinity↗

PubMed1978

Characterization of purified human liver acid beta-D-galactosidases A2 and A3.

1. Human liver acid beta-galactosidase A2 and A3 were isolated by chromatography on concanavalin A-Sepharose 4B, Sepharose 6B, and Sepharose 4B-6-aminohexyl 1-thio-beta-D-galactopyranoside. beta-Galactosidase A2 and A3 were purified to final specific activities of 45.5 and 20.6 mumol/min per mg respectively with 4-methylumbelliferyl beta-D-galactopyranoside as substrate. 2. Form A2 had a mol.wt. of 150000 +/- 15000 (gel filtration) and appeared as a single band of protein (mol.wt 65000 +/- 1000) on electrophoresis in the presence of sodium dodecyl sulphate. 3. Form A3 had a mol.wt. (gel filtration) of 660000 +/- 66000. On electrophoresis in the presence of sodium dodecyl sulphate, form A3 appeared as a major band of protein (72% of total) of mol.wt. 65000 +/- 1000 and minor protein bands of mol.wt. 44000 +/- 1000 and 26,000 +/- 1000 and 22000 +/- 1000. 4. Gel-filtration chromatography of purified beta-galactosidase A3 generated approximately equal amounts of forms A3 and A2. beta-Galactosidase A1 was not detected by gel-filtration chromatography of partially or highly purified preparations of forms A2 and A3. 5. Both forms A2 and A3 had identical isoelectric points of 4.42 +/- 0.02. The data suggest that forms A2 and A3 are dimeric and multimeric forms of beta-galactosidase A1. 6. Amino acid analysis of beta-galactosidase A2 gave a ratio of acidic to basic amino acids of 2.6:1. 7. beta-Galactosidase A2 contained 7.5% carbohydrate by weight and sialic acid, D-galactose, D-glucosamine and D-mannose were present in the molar proportions 1.1:1.0:1.7:2.7.

Amino Acids↗

PubMed1981

Characterization of a membrane-associated receptor from bovine liver that binds phosphomannosyl residues of bovine testicular beta-galactosidase.

A receptor that binds the phosphomannosyl recognition marker of bovine testicular beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) was isolated from bovine liver membranes. The receptor was extracted from crude plasma membrane preparations with Triton X-100 and immunoprecipitated as a receptor--beta-galactosidase complex with anti-beta-galactosidase. The receptor was dissociated from the precipitate with mannose 6-phosphate, labeled with 125I, and purified on a beta-galactosidase-Sepharose 4B affinity matrix. A quantitative binding assay employing anti-beta-galactosidase and IgGsorb (formalin-fixed Staphylococcus aureus) was devised to study the binding of 125I-labeled receptor to beta-galactosidase. Maximal binding of receptor to enzyme occurred at pH values between 5.7 and 6.5. Divalent cations were not required for binding. The values of the dissociation constant obtained for beta-galactosidase varied between 200 nM observed with "lower uptake" forms and 20 nM for "higher uptake" forms of the enzyme. A number of phosphorylated monosaccharides were tested as inhibitors of binding of enzyme to receptor; mannose 6-phosphate and fructose 1-phosphate served as inhibitors and exhibited Ki values of 0.064 mM and 0.24 mM, respectively. The receptor has a subunit molecular weight of 215,000. Similar receptors were also demonstrated in Triton X-100 extracts of human skin fibroblasts, Chinese hamster ovary cells, and rat hepatocytes. These cell types are known to assimilate lysosomal enzymes containing covalently bound mannose 6-phosphate residues.

Animals↗

PubMed2002

Localization and measurement of extracellular plant galactosidases.

A simple, rapid and sensitive procedure for the identification and determination of plant extracellular alpha-galactosidase and beta-galactosidase is described using callus cultures and seedlings from tomato. Synthetic substrates (1-naphthyl- and p-nitrophenyl-alpha-D- and beta-D-galactopyranosides) were used for the identification and determination of intracellular and extracellular activity of alpha-galactosidase and beta-galactosidase, respectively. Many iminosugars or azasugars are strong glycosidase inhibitors and some of them show promising chemotherapeutic effects against viral diseases, and are potentially antidiabetic agents, as well as antitumor agents. These facts initiated our interest in a rapid and sensitive assay to determine activity of alpha-galactosidase and beta-galactosidase in plant tissues. The results presented here show the potential of the assay of the activity of intracellular and extracellular galactosidases of plant origin in inhibitory and/or biotechnological studies.

Cells, Cultured↗

PubMed1982

Genetically controlled variation of "acid" beta-galactosidase detected in Rattus norvegicus by isoelectric focusing.

Two genetically variant forms of rat "acid" beta-galactosidase were found to differ in isoelectric point and pH dependence, but not in thermostability or sensitivity to inhibition by p-mercuribenzoate (PMB). The results of two backcrosses and an intercross indicated that the isoelectric focusing phenotypes are controlled by two codominant alleles at a single autosomal locus, for which we propose the name Glb-1. No significant linkage between Glb-1 and albino (LG I), brown (LG II), or hooded (LG VI) was observed. Strain-specific differences in total levels of kidney beta-galactosidase were detected, but it is not yet known whether the variation is controlled by genes linked to Glb-1. Experiments in which organ homogenates were incubated with neuraminidase indicated that the genetically variant forms do not result from differences in sialylation, though sialylation does appear to be largely responsible for the presence of multiple bands within each phenotype and for differences in the banding patterns of beta-galactosidases derived from different organs. The beta-galactosidase present in the bands used for Glb-1 typing resembles human GM1 gangliosidase (GLB1) with respect to pH optimum, substrate specificity, and susceptibility to inhibition by PMB. It also appears that Glb-1 is homologous with the Bgl-e locus of the mouse. In rats as in mice the genetically variant bands of beta-galactosidase are active at acid pH and have relatively high isoelectric points. In both species these bands are readily detectable in kidney homogenates, and can be revealed in homogenates of liver or spleen following treatment with neuraminidase. The presence of the same beta-galactosidase bands in homogenates of rat kidney and small intestine as well as in neuraminidase-treated homogenates of liver and spleen suggests that the Glb-1 variants differ by one or more point mutations in the structural gene for "acid" beta-galactosidase.

Alleles↗

PubMed1979

Genetic determination of the developmental program for mouse liver beta-galactosidase: involvement of sites proximate to and distant from the structural gene.

The identification and mode of action of genetic loci that program gene expression during development are important for understanding differentiation in higher organisms. Previous work from this laboratory has identified two patterns for the postnatal development of liver beta-galactosidase among inbred mouse strains: type I, where activity levels remain constant after about 30 days of age, is found in strains DBA/2J, CBA/J, and BALB/cJ, among others; type II, where activity levels increase between 25 and 50 days of age to reach a new adult level, is found in strain C57BL/6J and related strains. It has been shown that the type I vs. type II developmental difference between strains C57BL/6J and DBA/2J is due to variation at a locus, Bgl-t, that maps with the beta-galactosidase complex, [Bgl], on chromosome 9. In the present study, we have confirmed the existence of Bgl-t as a temporal locus within [Bgl] by analysis of both a congenic strain carrying the beta-galactosidase complex of strain CBA/J in the C57BL/6J genetic background and a cross of strains CBA/J and C57BL/6J. The existence of additional temporal loci for beta-galactosidase that segregate independently of the structural gene and participate in determination of the type I vs. type II difference was revealed by analysis of: (1) a congenic strain containing the beta-galactosidase complex of strain BALB/cJ in the C57BL/10Sn background; (2) recombinant inbred lines derived from progenitor strains C57BL/6ByJ and BALB/cByJ; and (3) a genetic cross between strains C57BL/6ByJ and BALB/cByJ. Thus, for these pairs of strains, the type I vs. type II developmental difference is due to variation at a temporal locus (or loci) unlinked to the enzyme structural gene, and not at Bgl-t. These facts, together with information gathered from an examination of the distribution of beta-galactosidase phenotypes among over 100 inbred strains (Breen, Lusis and Paigen 1977), have led us to conclude that the postnatal developmental pattern for liver beta-galactosidase is determined by a set of interacting temporal genes. One of these, Bgl-t, is located within [Bgl], and one or more are separable from [Bgl] by recombination. A possible mode of interaction among the temporal and instructural loci is suggested.

Animals↗