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Biomedical subjects

B Rotman

Publications and source records attributed to B Rotman.

At least 55 records · Page 3Linked to original sources
PubMed1979

Covalent attachment of enzyme as a membrane-label for viable eucaryotic cells.

An enzyme, beta-D-galactosidase, was covalently coupled to mammalian cells by means of a bifunctional reagent. The coupling procedure did not cause appreciable loss of cell viability (less than 6%) as measured by plating efficiently and membrane integrity. After 24 h in culture, the cells exhibited an average of 2.6 x 10(4) molecules of beta-D-galactosidase per cell. Histological evidence indicated that the enzyme was localized on the cell surface and distributed uniformly among the cell population. Considerations for choosing enzyme-label include sensitivity of assay by enzymatic, immunologic and histochemical methods, and the possibility of isolating labeled membrane components by enzyme-specific affinity chromatography.

Animals↗
PubMed1978

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↗
PubMed1978

Genetic control of immunologic unresponsiveness to adjuvant-free solutions of beta-D-galactosidase. I. Inheritance of the Ir-Z1 and ir-Z2 loci in mice.

Two genetic loci, Ir-Z1 and ir-Z2, controlling the immune response to adjuvant-free bacterial beta-D-galactosidase (Z) are present in inbred mouse strains SJL/J and CE/J, respectively. Each locus segregates as a single, autosomal gene: Ir-Z1 as dominant and ir-Z2 as recessive. The response is characterized by production of activating and precipitating IgG. Maximal levels of circulating IgG occur between 16 and 20 days after immunization with a single, 50-microgram dose of enzyme. Failure of proteins other than Z to elicit an immune response indicates that the Ir-Z control is specific for determinant(s) of this enzyme. The immunogenicity of beta-D-galactosidase preparations cannot be attributed to either the catalytic activity of the enzyme or adjuvant contamination. Non-responder mice acquire immunologic memory without detectable increase in circulating specific IgG under the same conditions that elicit antibody production in responder strains.

Adjuvants, Immunologic↗
PubMed1977

On the rate limiting step in downhill transport via the LacY permease of Escherichia coli.

Strains of Escherichia coli K12 were constructed for the specific purpose of evaluating the inducibility of the influx mechanism controlled by the lacY gene. These strains are heteromerodiploids characterized by a high and relatively constant level of beta-D-galactosidase which is not affected significantly by induction of the Lac operon. These properties were obtained by introducing episomal lacI+,Oc,Z+,Y-genes into the cells. In these merodiploids the rate of o-nitrophenyl-beta-D-galactopyranoside (ONPG) hydrolysis of extracted cells is 50-times that of intact cells. This difference indicates that the rate limiting step in the ONPG hydrolysis by intact cells is influx. Using a set of merodiploids with and without the LacY transport system, we were able to demonstrate a specific induction of ONPG influx. However, the increase in influx due to induction was only 3.5-fold as compared to the 40-fold increase observed when the LacY permease was measured by intracellular accumulation of [14C]TMG.

Biological Transport↗
PubMed1976

Roles of individual mgl gene products in the beta-methylgalactoside transport system of Escherichia coli K12.

Previous findings showed that galactose-binding protein defective mutants (mgl B-,A+,C+) of Escherichia coli K12 are still capable of growth on methyl-beta-D-galactopyranoside, while mgl A- and mgl C- mutants are not. When assayed by previous methods, none of these mutants exhibited methylgalactoside transport system activity. In this study, we present a modified assay developed for measuring low levels of transport. Using this assay, we found that mgl B-,A+,C+ mutants defective in galactose-binding protein accumulate methyl-beta-D-galactopyranoside up to six times the concentration gradient while mgl A- and mgl C- mutants failed to accumulate this substrate. Similar results were obtained using D-glyceryl-beta-D-galactopyranoside, another substrate of the methylgalactoside transport system. In contrast, all sugars tested which are not substrates of this system were transported equally by all mgl- mutants. The kinetic parameters of transport in mgl B- mutants were compared to those of the isogenic mgl+ strain which accumulates methyl-beta-D-galactopyranoside against a 10,000-fold concentration gradient. The apparent Km of methyl-beta-D-galactopyranoside influx was 1,000 times greater in mgl B- than in mgl+ strains. In contrast, there was no significant difference between these strains in either the Vmax of substrate influx or the rate of substrate exit. D-Galactose competitively inhibited methyl-beta-D-galactopyranoside influx into both mgl B- and mgl+ strains; the Ki of inhibition in mgl B- cells was 2,000-fold greater than that in mgl+ cells.

Biological Transport, Active↗
PubMed1976

Effect of uncoupler on "downhill" substrate efflux of Escherichia coli is dependent on (Mg2+, Ca2+). Adenosine triphosphatase.

Previous studies have shown that mutations in the unc gene of Escherichia coli K12 cause defects in energy transduction as well as a membrane-bound (Mg2+, Ca2+)-adenosine triphosphatase. We studied the effect of this mutation on the "downhill" efflux of methyl-beta-D-galactopyranoside, a suboli K12 did not show significant differences in substrate influx of efflux, a differential effect of an uncoupler, 2,4-dinitrophenol was demonstrated. In contrast to the unc+, dinitrophenol failed to inhibit significantly the rate coefficient of efflux in the unc- strain. Analysis of spontaneous unc+ revertants of the unc- mutant provided additional evidence that a functional unc gene is necessary for dinitrophenol inhibition of efflux. Other uncouplers tested in the unc+ strain showed different effects on efflux. While arsenate, azide and carbonyl cyanide p-trifluoromethoxyphenulhydrazone caused little or no effect, 2,4-dibromophenol and pentachlorophenol increased efflux by a considerable factor.

Adenosine Triphosphatases↗
PubMed1976

A galactosidase immunosorbent test for human immunoglobulin E.

We report here the development of a galactosidase-immunosorbent test (GIST) for immunoglobulin E (IgE) antibodies in which the amount of galactosidase adsorbed to a cellulose disc is a single valued function of IgE concentration in human serum. Rabbit anti-IgE immunoglobulin insolubilized on cellulose discs is incubated sequentially with human serum, sheep anti-IgE serum, and a covalent conjugate of rabbit antisheep immunoglobulin with the enzyme beta-D-galactoside galactohydrolase (E.C.) 3.2.1.23). Colorimetric assay of enzyme conjugate adsorbed to discs permits quantitation of 1.0 to 25 ng of IgE per test. Concentrations of IgE in 48 sera as measured by the GIST gave a linear correlation coefficient of 0.97 with IgE concentrations as determined by radioimmunoassay. Preliminary studies indicate that the GIST makes possible nonisotopic measurement of ragweed-specific IgE antibiotics in human serum. The GIST for IgE is simple to perform and requires neither short-lived radioisotopes, expensive scintillation detection equipment, nor scarce, purified IgE.

Allergens↗
PubMed1975

Inactivation of normal beta-D-galactosidase by antibodies to defective forms of the enzyme.

A counterpart of the antibody-mediated activation of genetically defective enzymes is reported here. Antibodies elicited by certain mutant forms of beta-D-galactosidase (EC 3.2.1.23) of Escherichia coli were found to inactivate the normal form of the enzyme. (Antibodies elicited by normal beta-D-galactosidase do not affect the enzyme's catalytic activity.) We present evidence that the inactivating antibodies are directed against one or a few determinants of the enzyme. The level of inactivation caused by the antibodies was independent of temperature below 25 degrees and increased with temperature above 25 degrees. The inactivation was proportional to the concentration of antiserum until a maximum level of 50% inactivation was reached. Antibodies capable of inactivating up to 87% of the activity were obtained after the antiserum was partially absorbed in an affinity column. This antibody preparation showed a 10-fold enrichment of inactivating antibodies over other antibodies direct against the enzyme. The antibody-mediated inactivation caused a reduction in the Vmax of beta-D-galactosidase without affecting the apparent Km of the enzyme. In contrast to antibodies to normal beta-D-galactosidase, inactivating antibodies changed the response of the enzyme to cations. To explain these results, we present a model in which there is a temperature-dependent equilibrium between two active forms of beta-D-galactosidase. Inactivation results from a conformational change induced by the binding of inactivating antibodies to only one of these two forms.

Antibodies↗
PubMed1975

Evidence for binding protein-independent substrate translocation by the methylgalactoside transport system of Escherichia coli K12.

Three genes, mgl A, B, and C, are required for active transport of substrate by the methylgalactose permease of E. coli K12. We report here that only two of these genes are required for substrate translocation, as seen by the ability or inability of isogenic mgl mutants (referred to as Tra+ and Tra minus, respectively) to grow on methyl-beta-D-galactopyranoside, supplied as sole carbon source. Individual mutants of both the Tra+ and Tra minus classes exhibited no detectable intracellular accumulation of methyl-beta-D-galactopyranoside; thus, the Tra+ phenotype cannot be explained by the mutants' levels of residual active transport. The phosphotransferase (Pts), the beta-galactoside (LacY), and the arabinose (Ara E and Ara F) transport systems are not required for substrate translocation by Tra+ cells. The Tra+ phenotype was identified with mutants defective in the mgl B, locus of the galactose-binding protein, by genetic complementation; the Tra minus phenotype was observed with both mgl A and mgl C mutants. The conclusion that the galactose-binding protein is not required for substrate translocation was supported by direct assays of the mgl mutants' binding protein activity. Mutants capable of translocation all showed reduced galactose-binding protein activity; mutants incapable of translocation exhibited binding protein activity equal to that of the mgl+ parent.

Bacterial Proteins↗
PubMed1972

Inhibition of methylgalactoside transport in Escherichia coli upon the cessation of unsaturated fatty acid biosynthesis.

The activity of the methylgalactoside transport system of E. coli is impaired upon treatment with 3-decynoyl-N-acetylcysteamine, an inhibitor of unsaturated fatty-acid synthesis. Treated cells are unable to be induced for permease activity, while transport sites synthesized before treatment show a regular loss of activity. The inhibition of methylgalactoside transport occurs at a step after translation of the galactose-binding protein, a component of the permease, and appears to be highly specific, since drug-treated cells show normal viability, protein synthesis, and membrane integrity when transport activity is greatly reduced. A second transport system, the galactoside permease, shows significantly less sensitivity to the inhibitor. That the activity of this permease is maintained in the presence of this inhibitor suggests that the inhibitor does not impair energy coupling.

Biological Transport, Active↗
PubMed1972

Antibody-mediated modification of the binding properties of a protein related to galactose transport.

A galactose-binding protein related to the mglP transport system of Escherichia coli increases its affinity and binding capacity for the substrate when exposed to both d-galactose and specific antibodies. For this increase to occur, the binding protein has to be in contact with d-galactose for at least 2 min prior to the addition of the antibodies. This reaction was used to show that other substrates of the mglP transport system compete with galactose for a site(s) of the binding protein and that the degree of competition is comparable to that observed in vivo. A model for substrate translocation is presented postulating a cellular component that can induce conformational changes in the galactose-binding protein similar to those caused by antibodies.

Animals↗
PubMed1971

Antibody-mediated activation of a defective beta-D-galactosidase. II. Immunological relationship between the normal and the defective enzyme.

Two closely related protein antigens were used to study immunogenic competition. Namely, normal beta-D-galactosidase of Escherichia coli (Z) and a genetically defective beta-D-galactosidase (AMEF) which seems to differ from the normal in one amino acid substitution. A unique characteristic of this pair of antigens is that, although they are indistinguishable in precipitation and absorption tests with antibodies, the enzymatic activity of AMEF is specifically increased several-hundredfold in the presence of antibodies directed against Z. The following results show that Z and AMEF also differ in their immunogenic ability: (a) antibodies directed against Z activated AMEF; antibodies directed against AMEF did not activate, but competed specifically with activating antibodies. (b) Animals immunized with AMEF failed to produce activating antibodies when they were subsequently challenged with Z, although the presence of some cells primed to produce activating antibodies could be demonstrated by adoptive transfer. (c) Animals preimmunized with Z were stimulated in their production of activating antibodies by AMEF challenge, although not as efficiently as with Z. A model explaining these observations by competition for the immunogenic site among antigen-sensitive cells carrying cross-reacting receptors is presented.

Adoptive Transfer↗
PubMed1971

Specific detection of antigen-binding cells by localized growth of bacteria.

A new method for the enumeration of lymphoid cells with specific surface-receptors for antigen is described, based on the use of beta-D-galactosidase (EC 3.2.1.23), either directly as an antigen or as a conjugated antigen. Binding of beta-D-galactosidase is revealed by its activity in releasing riboflavin from a synthetic substrate, riboflavin-beta-D-galactopyranoside. The riboflavin, inactive as a vitamin in the galactosidic form, becomes active when released by the enzyme, and can be detected by bioassay. Hence, lymphoid cells with receptors for beta-D-galactosidase on their surface can be detected after they have been exposed to the enzyme, washed, and then plated in agar containing riboflavin-beta-D-galactopyranoside, streptomycin, riboflavin-deficient medium, and a streptomycin-resistant strain of Streptococcus faecalis that requires riboflavin. Release of riboflavin is signalled by the growth of characteristic bacterial colonies over the cell that bound beta-D-galactosidase.

Animals↗