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Cell lineage analyses of epithelia and blood vessels in chimeric mouse embryos by use of an embryonic stem cell line expressing the beta-galactosidase gene.

We have established an embryonic stem (ES) cell line, MS1-EL4, which has the potential to make various tissues in chimeric embryos and, at the same time, expresses the beta-galactosidase gene which was introduced as a good cell marker. To examine cell behavior and lineage during embryogenesis, we injected MS1-EL4 cells into host blastocysts and recovered chimeric embryos at various developmental stages. We examined the distribution of the MS1-EL4 cell derivatives by staining whole embryos with X-gal and by making serial paraffin sections. So far we have obtained the following results: (1) the MS1-EL4 cell line is useful for studying cell lineages because of its ubiquitous expression at least until the mid-gestation stage; (2) cells of the primitive ectoderm and its derivative epithelial tissues continue to intermingle with each other until the late primitive streak stage. Then, at early somite stages, cells of various epithelia stop intermingling and give rise to small coherent clones; (3) blood vessels of the yolk sac are formed by local aggregation of the ancestor cells and those of the embryo proper by proliferation and sprouting from fewer angiogenic cells.

Animals↗

Comparison of beta-galactosidase production by two inducible promoters in Myxococcus xanthus.

The inducibility of two promoter systems, one heterologous and one homologous, has been assessed in the Gram-negative bacterium Myxococcus xanthus. The heterologous system involved the hybrid tac promoter and the presence of lacIq, the lac repressor from Escherichia coli. This system is inducible in its natural host with isopropyl-beta-D-thiogalactopyranoside (IPTG). The homologous promoter system involves the light-inducible carQRS promoter, which is normally involved in the expression of the regulators of the light-inducible light-protective carotenoid synthesis regulon in M. xanthus. In each case, promoter activity and strength was assayed using the E. coli gene lacZ. In our constructs, which were present in a single copy in the M. xanthus chromosome, the carQRS promoter yielded at least a 47-fold increase in beta-galactosidase production upon light induction, whilst IPTG increased by 8-fold the amount of enzyme produced under the control of the ptac-lacIq system. Regulation by the latter was significantly higher than that obtained with the unmodified lacZ promoter.

Enzyme Induction↗

The discovery of beta-galactosidase.

The enzyme beta-galactosidase was first mentioned in the literature by Beijerinck exactly a hundred years ago. The Department of Microbiology and Enzymology of the Delft University of Technology keeps the memory of Beijerinck, its first professor, alive by maintaining a 'Beijerinck-room' in the attic of the building. In addition to manuscripts and laboratory notebooks, this room contains some of his chemicals and biological preparations, and it was here that we recently found a 90-year old lactase preparation. Even after storage under suboptimal conditions, the preparation still exhibited measurable enzyme activities.

Galactosidases↗

The stereochemistry of the addition of glycerol to D-galactal, catalyzed by beta-D-galactosidase.

On incubation with beta-D-galactosidase, D-galactal-2-d (1) plus glycerol yielded 1-deoxyglycerol-1-yl 2-deoxy-beta-D-lyxo-hexopyranoside-2(S)-d. By 1H-n.m.r. analysis, it was shown that the hydrogen atom introduced on C-2 is trans-related to the aglycon moiety. In contrast to this stereospecific, enzyme-catalyzed addition, the reaction of phenol with peracetylated 1, catalyzed by p-toluenesulfonic acid, which yields phenyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-lyxo-hexopyranoside-2-d, was shown to entail both a trans and a cis addition.

Catalysis↗

The ineffectiveness of analogs of D-galactal as competitive inhibitors of, and substrates for, beta-D-galactosidase from Escherichia coli.

2,6-Anhydro-3-deoxy-aldehydo-D-lyxo-hept-2-enose (7) and 2,6-anhydro-3-deoxy-D-lyxo-hept-2-enitol (8) were synthesized as half-chair analogs of D-galactal (1). As 1 is a strong inhibitor of, as well as a substrate for, beta-D-galactosidase from Escherichia coli, the same properties were expected for 7 and 8; however, both were ineffective. This result, together with those of other authors, allows speculative conclusions on the tight binding of 1 to the enzyme only, when water or an alcohol is bound as a co-substrate.

Escherichia coli↗

Rapid procedures for determination of endo-N-acetyl-alpha-D-galactosaminidase in Clostridium perfringens, and of the substrate specificity of exo-beta-D-galactosidases.

Culture fluid of Clostridium perfringens hydrolyzed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OPh and beta-Gal-(1 leads to 3)-alpha-GalNAc-1 leads to OC6H4-NO2-o or -p to beta-Gal-(1 leads to 3)-GalNAc and the aglycon. Such assays facilitated the characterization and purification of this endo-N-acetyl-alpha-D-galactosaminidase activity. This activity was purified 1200-fold by fractionation with ammonium sulfate and chromatography on columns of Sephadex-G200, DEAE-Sephadex, and hydroxylapatite. The final preparation showed activity over a broad range of pH, with an optimum at 9.0, but less-pure material had two pH optima, 4.0 and 9.0. Another assay method, which employed the synthetic, chromogenic substrates beta-Gal-(1 leads to 3)-beta-GlcNAc-1 leads to OC6H4NO2-p, beta-Gal-(1 leads to 4)-beta GlcNAc-1 leads to OC6H4NO2-p, and beta-Gal-(1 leads to 6)-beta-GlcNAc-1 leads to OC6H4NO2-p, was developed for the rapid identification of the linkage specificity of exo-beta-D-galactosidases from any source via a coupled reaction with N-acetyl-beta-D-hexosaminidase.

Acetylgalactosamine↗

Purification and thermostability of beta-galactosidase (lactase) from an autolytic strain of Streptococcus salivarius subsp. thermophilus.

beta-Galactosidase from an autolytic strain of Streptococcus salivarius subsp. thermophilus was purified 109-fold to near homogeneity. The yield of purified enzyme was 41% and the specific activity was 592 o-nitrophenyl beta-D-galactopyranoside U/mg at 37 degrees C. Two isozymes were present, but only one subunit was detected, having a mol. wt of 116,000. Enzyme stability was 37-83 times greater in milk than in buffer in the range 60-65 degrees C. At 60 degrees C the half-life in milk was 146 min. Denaturation in buffer was first-order, but in milk the overall reaction order with respect to enzyme concentration was approximately 0.5. The activation energy for denaturation was 453 kJ/mol in milk and 372 kJ/mol in buffer. In milk the activation energy for lactose hydrolysis was 35.1 kJ/mol.

Animals↗

Cis-active control of mouse beta-galactosidase biosynthesis by a systemic regulatory locus.

Several higher organisms have been reported in which enzyme levels are determined genetically by sites located in close proximity to the corresponding structural genes. In several cases, these sites have been shown to act by controlling the rates of enzyme synthesis. Cis compared with trans action has been tested for those proximate regulatory sites controlling enzymes for which appropriate structural variants exist. The rate of synthesis of beta-galactosidase in mouse tissues is under the control of a regulatory locus; Bgl-s, that is tightly linked to the enzyme structural gene; we have tested the cis/trans nature of Bgl-s action by analysis of the electrophoretic mobility of the enzyme from animals heterozygous for the appropriate regulatory and structural alleles. Our results indicate that Bgl-s acts cis, controlling the expression of the structural gene located on the same chromosome.

Animals↗

Comparative studies of two acid beta-galactosidases from rabbit and bovine kidney.

Comparative studies of two acid beta-galactosidases from rabbit and bovine kidney have been made. The enzyme from rabbit (enzyme I) was purified 450-fold, and the activity of the bovine enzyme (enzyme II) was enriched 250-fold using conventional purification methods. Both purified enzymes were characterized and their properties were compared. Enzyme I showed a lower optimal temperature and was less pH stable. Enzyme II appeared to be homogeneous in charge, in contrast to the heterogeneity observed for enzyme I. Studies on their specificity using natural substrates showed that enzyme I hydrolyzed GM1, asialofetuin and lactose. However, enzyme II was only able to cleave galactose from the disaccharide. Some of the carbohydrates tested acted as activators for enzyme II, suggesting a mechanism of transglycosilation. Using lactose as substrate we confirmed the ability of enzyme II to transfer galactose residues to D-maltose and N-acetylgalactosamine.

Animals↗

Affinity labelling with a deaminatively generated carbonium ion. Kinetics and stoicheiometry of the alkylation of methionine-500 of the lacZ beta-galactosidase of Escherichia coli by beta-D-galactopyranosylmethyl-p-nitrophenyltriazene.

1. beta-D-Galactopyranosylmethyl-p-nitrophenyltriazene is an active-site-directed irreversible inhibitor of Mg2+-bound and Mg2+-free lacZ beta-galactosidase from Escherichia coli. 2. The Mg2+-enzyme binds the inhibitor more tightly but the complex then decomposes less rapidly than is the case with Mg2+-free enzyme. 3. Loss of enzyme activity is a linear function of the fraction of enzyme protomers to which are attached beta-D-galactopranosyl[14C]methyl residues: complete inactivation of fully active enzyme results in incorporation of 0.91 equivalent of carbohydrate label per enzyme protomer. 4. When the beta-galactopyranosylmethyl cation is generated in the active site of Mg2+-enzyme, it is captured essentially completely by the protein, but in the active site of Mg2+-free enzyme it is only captured with an efficiency of 25%. 5. Labelled enzyme was carboxymethylated and digested with trypsin; acidic hydrolysis of the isolated tryptic peptide, and field-desorption mass spectrometry of the isolated radioactive derivative, showed it to be 2,5-dioxo-3[2-(beta-D-galactopyranosylmethylthio)ethyl]-1,6-trimethylenepiperazine. 6. This is considered to have arisen from labelling of the sulphur atom of a methionine residue adjacent to a proline residue. 7. The complete amino acid sequence of the molecule [Fowler & Zabin (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 1507-1510] enables the labelled methionine residue to be identified as either Met-421 or Met-500. 8. Sequence data [Fowler, Zabin, Sinnott & Smith (1978) J. Biol. Chem. in the press] show the site of attack to be Met-500.

Affinity Labels↗

The necessity of magnesium cation for acid assistance aglycone departure in catalysis by Escherichia coli (lacZ) beta-galactosidase.

1. Removal of Mg2+ from Escherichia coli (lacZ) beta-galactosidase slightly increases the rate of hydrolysis of galactosyl pyridinium salts, but decreases the rate of hydrolysis of arylgalactosides. 2. Fair correlation of logkcat. and log (Km) with the pKa of aglycone is now observed for arglygalactosides, as well as for glycosyl pyridinium salts. 3. Degalactosylation of Mg2+-free enzyme is the rate-limiting step in the hydrolysis of 2,4-dinitrophenyl galactoside. 4. alpha-Deuterium kinetic isotope effects for both sets of substrates are consistent with the rate-determining generation of a glycosyl cation. 5. The pH-independent, SNl hydrolysis of 3,4-dinitrophenyl galactoside has been measured: it is as fast as that of the galactosyl 3-chloropyridinium ion. 6. Hydrolysis of these two substrates by Mg2+-free enzyme proceeds at very similar rates. 7. It is concluded that loss of both types of aglycone takes place, without acid catalysis, from the first ES complex of substrate and apoenzyme. 8. Data for galactosyl azide and thiopicrate confirm that neither charge nor change of atom is the cause of the differences in behavior between aryl galactosides and galactosylpyridinium salts.

Apoenzymes↗

Interaction of the lacZ beta-galactosidase of Escherichia coli with some beta-D-galactopyranoside competitive inhibitors.

1. The location of the bivalent metal cation with respect to bound competitive inhibitors in Escherichia coli (lacZ) beta-galactosidase was investigated by proton magnetic resonance. 2. Replacement of Mg(2+) by Mn(2+) enhances both longitudinal and transverse relaxation of the methyl groups of the beta-d-galactopyranosyltrimethylammonium ion, and of methyl 1-thio-beta-d-galactopyranoside; linewidths are narrowed by increasing temperature. 3. The Mn(2+) ion is located 8-9A (0.8-0.9nm) from the centroid of the trimethylammonium group and 9A (0.9nm) from the average position of the methylthio protons. 4. The effective charge at the active site was probed by measurement of competitive inhibition constants (K(i) (o) and K(i) (+) respectively) for the isosteric ligands, beta-d-galactopyranosylbenzene and the beta-d-galactopyranosylpyridinium ion. 5. The ratio of inhibition constants (Q=K(i) (+)/K(i) (o)) obtained with 2-(beta-d-galactopyranosyl)-naphthalene and the beta-d-galactopyranosylisoquinolinium ion at pH7 with Mg(2+)-enzyme was identical, within experimental error, with that obtained with the monocyclic compounds. 6. The variation of Q for Mg(2+)-enzyme can be described by Q=0.1(1+[H(+)]/4.17x10(-10))/1+[H(+)]/10(-8)). 7. This, in the theoretical form for a single ionizable group, is ascribed to the ionization of the phenolic hydroxy group of tyrosine-501. 8. The variation of Q for Mg(2+)-free enzyme is complex, probably because of deprotonation of the groups normally attached to Mg(2+) as well as tyrosine-501.

Binding Sites↗

Proteinase activity in chondroitin lyase (chondroitinase) and endo-beta-D-galactosidase (keratanase) preparations and a method to abolish their proteolytic effect on proteoglycan.

Significant amounts of proteinase activity have been found in chondroitin ABC lyase (EC 4.2.2.4), chondroitin AC II lyase and endo-beta-D-galactosidase (keratanase) from commercial sources. It would appear, therefore, that certain earlier biochemical and histochemical studies, which employed these commercial enzyme preparations for their presumed ability to degrade only glycosaminoglycans, may require re-evaluation. A mixture of EDTA, N-ethylmaleimide, phenylmethanesulphonyl fluoride and pepstatin abolishes the effect of the contaminating proteinases on proteoglycan with less significant effect on the chondroitin lyase or keratanase activity.

Animals↗

The binding of human liver acid beta-galactosidase to wheat-germ lectin is influenced by aggregation state of the enzyme.

At low ionic strength and pH 6.0 human liver acid beta-galactosidase exists in two aggregation states, monomeric and multimeric. These species can be separated on wheat-germ lectin-Sepharose, Cellogel electrophoresis and gel filtration on Sephadex G-200, and are not normally interconvertible. On re-application of either form to wheat-germ lectin-Sepharose the equilibrium is re-established and the two forms are interconverted.

Binding Sites↗

A solvent-isotope-effect study of proton transfer during catalysis by Escherichia coli (lacZ) beta-galactosidase.

1. Michaelis-Menten parameters for the hydrolysis of 4-nitrophenyl beta-D-galactopyranoside and 3,4-dinitrophenyl beta-D-galactopyranoside Escherichia coli (lacZ) beta-galactosidase were measured as a function of pH or pD (pL) in both 1H2O and 2H2O. 2. For hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-free enzyme, V is pL-independent below pL 9, but the V/Km-pL profile is sigmoid, the pK values shifting from 7.6 +/- 0.1 in 1H2O to 8.2 +/- 0.1 in 2H2O, and solvent kinetic isotope effects are negligible, in accord with the proposal [Sinnott, Withers & Viratelle (1978) Biochem. J. 175, 539-546] that glycone-aglycone fission without acid catalysis governs both V and V/Km. 3. V for hydrolysis of 4-nitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme varies sigmoidally with pL, the pK value shifting from 9.19 +/- 0.09 to 9.70 +/- 0.07; V/Km shows both a low-pL fall, probably due to competition between Mg2+ and protons [Tenu, Viratelle, Garnier & Yon (1971) Eur. J. Biochem. 20, 363-370], and a high-pL fall, governed by a pK that shifts from 8.33 +/- 0.08 to 8.83 +/- 0.08. There is a negligible solvent kinetic isotope effect on V/Km, but one of 1.7 on V, which a linear proton inventory shows to arise from one transferred proton. 4. The variation of V and V/Km with pL is sigmoid for hydrolysis of 3,4-dinitrophenyl beta-D-galactopyranoside by Mg2(+)-enzyme, with pK values showing small shifts, from 8.78 +/- 0.09 to 8.65 +/- 0.08 and from 8.7 +/- 0.1 to 8.9 +/- 0.1 respectively. There is no solvent isotope effect on V or V/Km for 3,4-dinitrophenyl beta-D-galactopyranoside, despite hydrolysis of the galactosyl-enzyme intermediate governing V. 5. Identification of the 'conformation change' in the hydrolysis of aryl galactosides proposed by Sinnott & Souchard [(1973) Biochem. J. 133, 89-98] with the protolysis of the magnesium phenoxide arising from the action of enzyme-bound Mg2+ as an electrophilic catalyst rationalizes these data and also resolves the conflict between the proposals and the 18O kinetic-isotope-effect data reported by Rosenberg & Kirsch [(1981) Biochemistry 20, 3189-3196]. It should be noted that the actual Km values were determined to higher precision than can be estimated from the Figures in this paper.(ABSTRACT TRUNCATED AT 400 WORDS)

Catalysis↗

Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins.

Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.

Amino Acid Sequence↗

Studies on metatherian sex chromosomes. XII. Sex-linked inheritance and probable paternal X-inactivation of alpha-galactosidase A in Australian marsupials.

An investigation of genetic variation in the electrophoretic mobility of the enzyme alpha-galactosidase A (EC 3.2.1.22) has been carried out for 33 species of Australian metatherian (marsupial) mammals. The results are compatible with the enzyme being sex-linked in macropodids (kangaroos and wallabies) and probably in dasyurids (marsupial 'mice', etc.), as it is in eutherian (placental) mammals. The results also suggest that the mode of dosage compensation for this locus is the same as for other sex-linked loci in kangaroos, i.e. paternal X inactivation, rather than the random X inactivation system of eutherian mammals. The bearing of the enzyme mobility data on phylogenetic relationships among macropodid species is discussed.

Animals↗

Antibody-mediated activation of a defective beta-D-galactosidase: dimeric form of the activatable mutant enzyme.

Sedimentation analyses of AMEF, an activatable mutant beta-D-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23), and the products of its reaction with Fab fragments of activating antibody show that this enzyme exists mainly as 10S dimers. Activation of AMEF by purified antibody resulted in formation of 16S tetramers. A unifying hypothesis postulating a dimer--tetramer equilibrium accounts for this observation as the counterpart of inactivation, which was shown to involve the breakdown of tetramers into inactive subunits [Roth, R. A. & Rotman, B. (1975) Biochem. Biophys. Res. Commun. 67, 1382--1390]. Conditions are described under which AMEF loses the specific antigenic determinant(s) responsible for binding activating antibody, allowing its subsequent use as an absorption to obtain immunologically purified activating antibody,

Antibodies↗