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At least 19 recordsLinked to original sources
PubMed1977

Construction, isolation and implications of repressor-galactosidase - beta-galactosidase hybrid molecules.

Escherichia coli heterogenotes, which produce hybrid molecules between the chimaeric protein repressor-galactosidase and the enzyme beta-galactosidase, were constructed. Repressor-galactosidase in which fully active lac repressor is covalently linked to active beta-galactosidase, is an aggregate with a core structure of four beta-galactosidase parts and two peripheral lac repressor dimers. The lac repressor dimers, which are separated by tetrameric beta-galactosidase, retain all the biological activities of tetrameric lac repressor. Substitution of repressor-galactosidase subunits with beta-galactosidase subunits leads to hybrid molecules with y beta-galactosidase subunits aggregated with (4-y) repressor-galactosidase subunits (where y = 1, 2 or 3). A 2:2 hybrid, i.e. a tetrameric beta-galactosidase core with one lac repressor dimer grafted to it, binds at least 100 times less strongly to 32P-labelled lambdaplac DNA than pure lac repressor or repressor-galactosidase. The data suggest a model in which lac repressor binds with two subunits to lac operator and with the other two subunits elsewhere on the DNA, possibly on sequences like the lac operator.

Bacterial Proteins↗
PubMed1978

Acid glycohydrolase in Chinese hamster with spontaneous diabetes. I. Depressed levels of renal alpha-galactosidase and beta-galactosidase.

The activites of alpha-and Beta-galactosidases (alpha-D-galactoside galactohydrolase, EC 3.2.1.22; beta-D-galactoside galactohydrolase, EC 3.2.1.23) were significantly lower in the kidneys of diabetic XA line than those in the nondiabetic M line Chinese hamsters. The depression of these enzymes was found only in the kidney but not in liver, spleen, hind leg muscle, cheek pouch or spinal cord. In young XA animals before onset of glycosuria, renal alpha-galactosidase level was similar to that in age-matched M animals; whereas, their renal beta-galactosidase activity was about 90% of those in the M animals. Partial purification and separation of these enzymes were achieved by chromatography on DEAE-Sepharose CL-6B columns. beta-galactosidase was separated into two isozymes and depression of activity in the XA kidneys was evident in both. alpha-galactosidase was recovered in a single peak. The pH optima of these enzymes from XA and M animals were identical. With p-nitrophenyl glycosides as substrates, the Michaelis constants of these enzymes were also the same of XA and M animals. Molecular weight estimation by gel filtration on Sepharose 6B yielded similar results between M and XA samples: 2.4-10(5) for alpha-galactosidase and 1.6.10(5) and 1.9.10(5) for beta-galactosidase isozymes. The data suggest that the diabetic animals had lower concentrations of alpha-and beta-galactosidase in their kidneys, probably as a consequence of hyperglycemia.

Animals↗
PubMed1977

beta-Galactosidase in mucopolysaccharidoses and mucolipidoses. Deficiency of GM1 beta-galactosidase in liver and leukocytes.

beta-Galactosidase activities were studied in livers and leukocytes of mucopolysaccharidoses and mucolipidoses (I-cell disease and adult "beta-galactosidase deficiency" with macular cherry-red spots). Marked deficiency of hepatic 4-methylumbelliferyl (4MU) and GM1 beta-galactosidases was demonstrated in these diseases. Leukocyte GM1 beta-galactosidase was also deficient in mucolipidoses. The parents of the patients with I-cell disease and "beta-galactosidase deficiency" had normal beta-galactosidase activity in plasma and leukocytes, compared to the low enzyme activity in heterozygous carriers of GM1-gangliosidosis. The cause of this enzyme deficiency in these diseases is not clear at present. It seems to be affected seondarily by exgenous factors such as unknown stored materials in the cells. Mucopolysaccharides were not increased in the livers of two cases of I-cell disease and a case of "beta-galactosidase deficiency".

Adolescent↗
PubMed1978

beta-Galactosidase chimeras: primary structure of a lac repressor-beta-galactosidase protein.

A protein possessing both lac repressor and beta-galactosidase activities in a single polypeptide of about 155,000 daltons was purified from a deletion mutant of Escherichia coli in which the lacI and Z genes are fused. A 77-residue cyanogen bromide peptide containing the fusion joint was isolated. A radioimmunoassay with an antibody prepared against CNBr2 (residues 3-92) of beta-galactosidase was used to monitor its purification. The sequence of the joining peptide was determined by analysis of tryptic peptides and by automatic sequencer analysis. The site of joining is from residue 355 of lac repressor to residue 24 of beta-galactosidase (or 356 to 25), indicating that the last 4 residues at the carboxyl terminus of lac repressor and the first 23 residues at the amino terminus of beta-galactosidase are not essential for the activities of these two proteins. The exact site of the fusion is not known because lac repressor residue 356 and beta-galactosidase residue 24 are both leucine residues. Examination of the nucleotide sequences around the two end points of the deletion revealed a homology of 9 identities in a stretch of 11 base pairs.

Amino Acid Sequence↗
PubMed1978

Plasma alpha-galactosidase A:properties and comparisons with tissue alpha-galactosidases.

The human plasma form of alpha-galactosidase A (alpha-D-galactoside galactohydrolase, EC 3.2.1.22) was highly purified and exhibited apparent Km values of 1.9 mM with 4-methylumbelliferyl-alpha-D-galactopyranoside and 0.23 mM with globotriglycosylceramide. Its inhibition with myo-inositol (Ki = 0.29 M) was similar to that observed with alpha-galactosidase A from various tissues. The plasma form of this lysosomal enzyme has a lower molecular weight of 96 600, a lower pI of 3.7 and faster electrophoretic mobility in polyacrylamide gels than the enzyme obtained from human liver. These data and the increased pI obtained after neuraminidase treatment suggest that the plasma form is an isoenzyme with a more highly sialylated carbohydrate moiety than the tissue isoenzymes.

Galactosidases↗
PubMed1978

Probe of beta-galactosidase structure with iodoacetate. Differential reactivity of thiol groups in wild-type and mutant forms of beta-galactosidase.

Carboxymethylation with 14 C-labeled iodoacetate of cysteine residues in wild-type beta-galactosidase from Escherichia coli and in a defective beta-galactosidase from deletion mutant strain M15 was investigated in order to determine accessible positions in the tetrameric wild-type form and the dimeric mutant M15 protein. The extent of carboxymethylation, the effects on biological activity, antibody activation, physical stability, and the labeling of particular residues were studied. The results distinguish three groups of spatial relationships for cysteine residues in the protein, define possible regions for subunit interactions, and confirm that no cysteine residue is specifically involved in catalysis. Residue 1019 and to a lesser extent 498 are accessible in the tetrameric protein and probably represent exposed areas. In the M15 protein, these two, and three additional residues, at 76,387 and 600, were found to react significantly with reagent. One or more of the latter are suggested to be in the dimer-dimer interface. Complementation and activation by antibody are inhibited by carboxymethylation of M15 protein.

Binding Sites, Antibody↗
PubMed1976

Effects of methylation of the beta-galactosidase genome upon in vitro synthesis of beta-galactosidase.

A template DNA from phage lambdah80dlacp5 coding for the in vitro synthesis of beta-galactosidase was used to study the effect of DNA methylation by the alkylating agent, dimethyl sulfate (DMS). Increasing the levels of DMS up to 50 mM concentration in the incubation medium led to an increase of DNA methylation. When incubated for 10 min at 37 degrees C, 3-4% Of nucleotides were methylated. The increase was linear to about 0.6% nucleotide methylation level. A higher yield was obtained at 37 degrees C incubation temperature than at 20 degrees C. Methylation of lambdah80dlacp5 DNA alone without methylation of other factors in the incubation mixture caused inhibition of the synthesis of beta-galactosidase in vitro. Increasing levels of DNA methylation caused greater inhibition of the newly synthesized enzyme activity. Total protein and RNA synthesis was inhibited by the methylated DNA to a much lesser extent than the inhibition of enzyme activity. When the level of nucleotide methylation was 0.74%, only 2% of enzyme activity remained, but total protein and RNA synthetic activities were found to be 72% and 44%, respectively.

Coliphages↗
PubMed1976

Krabbe's globoid cell leucodystrophy. Studies on galactosylceramide beta-galactosidase and non-specific beta-galactosidase of leucocytes, cultured skin fibroblasts, and amniotic fluid cells.

Galactosylceramide beta-galactosidase (cerebrosidase) and nonspecific beta-galactosidase activities were measured in both cultured skin fibroblasts and leucocytes from a family with Krabbe's globoid cell leucodystrophy (GLD). The activities of these enzymes were also determined in cultured skin fibroblasts of a patient with GM1 gangliosidosis and in cultured amniotic fluid cells. While cerebrosidase activity was deficient in GLD fibroblasts and leucocytes, its activity in GM1 gangliosidosis fibroblasts was increased. Two forms of each enzyme were found on isoelectric focusing, but in the GM1 gangliosidosis fibroblasts, cerebrosidase activity occurred as a single but intermediate peak. The use of cultured cells in assessing isoenzyme abnormalities associated with certain neurolipidoses is discussed.

Amniotic Fluid↗
PubMed1976

Characterization of T and B antigen-binding cells for beta-galactosidase. I. beta-galactosidase-binding cells in the thymus and spleen of normal mice.

The results reported in this paper demonstrate that the enumeration of cells binding beta-galactosidase (Z) as an antigen, revealed by subsequent substrate hydrolysis, is an excellent method for the detection and study of antigen-binding cells (ZBC). The binding found is specific and is restricted to a small number of lymphocytes that bind a large number of Z molecules via surface receptors. Such ZBC were found at mean frequencies of 150 per 10(6) in the thymus and 200 to 300 per 10(6) in the spleen. The binding cells of both organs were heterogenous with individual ZBC binding from 10(5) to 10(6) molecules of enzyme as determined by substrate hydrolysis, although this might well be an overestimate of the number of actual receptors. The profiles for the frequency of ZBC binding different numbers of molecules were nearly identical for thymus and spleen, in contrast to descriptions of the binding of many other antigens. Receptors responsible for Z binding appear to be superficially located on the cell since they are trypsin-sensitive to a large extent and are not increased by fixation.

Animals↗