Binding of simple carbohydrates and some of their chromophoric derivatives to soybean agglutinin as followed by titrimetric procedures and stopped flow kinetics

The number of carbohydrate-binding sites of the GalNAc-specific lectin is four per tetramer. The binding parameters of N-acetyl-D-galactosamine and methyl-N-acetyl-alpha-D- galactosaminide , were determined by titrating the perturbation in the absorption spectrum of the protein. For D-galactosides, it was necessary to use p-nitrophenyl-N-acetyl-beta-D- galactosaminide as an indicator in substitution titrations. The association constants K were determined at several temperatures yielding 2.4 X 10(4) M-1 at 25 degrees C with delta H degree' = -45 kJ mol-1 and delta S degree' = -67 J X K-1 mol-1 for methyl-N-acetyl-alpha-D- galactosaminide and 1.0 X 10(3) M-1 at 25 degrees C, delta H degree' = -38 kJ mol-1 and delta S degree' = -69 J X K-1 mol-1 for methyl-alpha-D-galactoside. The increase in K by a factor of 25 caused by the acetamido group is largely enthalpic . Whenever different methods were used to determine the association constant of a given compound, the agreement was excellent. The observed changes in absorption or fluorescence of all chromophoric carbohydrate derivatives used are specific for the binding of carbohydrates. For large aromatic beta- aglycons such as p-nitrophenyl or 4-methylumbelliferyl groups, the increase in K of the N-acetyl-D- galactosaminide moiety is by a factor of 2 or less, but for a large N-5-dimethylaminonaphthalene-1-sulfonyl (dansyl) group this factor is about 20 as compared with the acetyl group. The concomitant 10-fold increase in dansyl fluorescence, also observed with four other GalNAc-binding lectins together with a favorable and large delta S degree' = +60 J X K-1 mol-1 strongly point at the presence of a hydrophobic region in the vicinity of the carbohydrate-binding site. The results of stopped flow kinetics with 4-methylumbelliferyl-N-acetyl-beta-D- galactosaminide and the lectin are consistent with a simple mechanism for which k+ = 1.1 X 10(4) M-1 S-1 and k- = 0.4 S-1 at 25 degrees C. This k- is slower than for any monosaccharide-lectin complex reported so far.     

Initiation of DNA synthesis by human thrombin: relationships between receptor binding, enzymic activity, and stimulation of 86Rb+ influx

Stimulation of amiloride-sensitive sodium (Na+) influx and the subsequent activation of NA+, K+-ATPase by serum or growth factors have been implicated as early events leading to initiation of cell proliferation. We recently demonstrated that amiloride inhibits thrombin-initiated DNA synthesis not by inhibiting an early event occurring during the first 8 hr, but rather by inhibiting some later event 8 to 12 hr after thrombin addition. To further probe the relationship between stimulation of ion influx and initiation of cell proliferation, human alpha-thrombin was converted to gamma-thrombin, nitro-alpha-thrombin, and diisopropylphospho (DIP)-alpha-thrombin. These derivatives retain either the capacity to bind cell surface alpha-thrombin receptors or thrombin esterase activity, but they do not initiate DNA synthesis. At low concentrations of alpha-thrombin or the various thrombin derivatives, only alpha-thrombin stimulates 86Rb+ influx, suggesting a correlation between stimulation of influx and the ability of these derivatives to initiate DNA synthesis. Concentrations of a DIP-alpha-thrombin that saturate the alpha-thrombin receptors (up to 2 micrograms/ml) do not stimulate either the early or late influx of 86Rb+, indicating that DIP-alpha-thrombin binding alone is not sufficient to stimulate ion fluxes. High concentrations of either gamma-thrombin or nitro-alpha-thrombin, however, stimulate both early and late 86RB+ uptake but do not initiate DNA synthesis. These results demonstrate that events leading to both the early and late stimulation of 86Rb+ influx by themselves are not sufficient to initiate cell proliferation. Thus, initiation may require a combination of events that can be independently regulated by different transmembrane signals.     

Heterogeneity among microtubules of the cytoplasmic microtubule complex detected by a monoclonal antibody to alpha tubulin

Three monoclonal antibodies specific for tubulin were tested by indirect immunofluorescence for their ability to stain cytoplasmic microtubules of mouse and human fibroblastic cells. We used double label immunofluorescence to compare the staining patterns of these antibodies with the total microtubule complex in the same cells that were stained with a polyclonal rabbit antitubulin reagent. Two of the monoclonal antitubulin antibodies bound to all of the cytoplasmic microtubules but Ab 1-6. 1 bound only a subset of cytoplasmic microtubules within individual fixed cells. Differential staining patterns were observed under various fixation conditions and staining protocols, in detergent-extracted cytoskeletons as well as in whole fixed cells. At least one physiologically defined subset of cytoplasmic microtubules, those remaining in cells pretreated for 1 h with 5 microM colcemid, appeared to consist entirely of Ab 1-6. 1 positive microtubules. The same was not true of the microtubules that remained in either cold-treated cells or in cells that had been exposed to hypotonic medium. The demonstration of antigenic differences among microtubules within single fixed cells and the apparent correlation of this antigenic difference with at least one "physiologically" defined subset suggests that mechanisms exist for the differential assembly or postassembly modification of individual microtubules in vivo, which may endow them with different physical or functional properties.     

A low-cost antifoam additive for agar-based culture media

A highly effective, low-cost silicone-based antifoam emulsion is described which, at a final concentration of 100 ppm, prevents bubble formation during the preparation and dispensing of agar media. The compound is heat-stable, has a long shelf-life and offers considerable savings in cost by reduction in wastage of time and materials. It has no demonstrable deleterious effect on the growth of a wide range of pathogenic and commensal bacteria, or on antibiotic disc susceptibility testing, when examined using a range of different media.     

Preliminary characterization of a Chinese hamster ovary cell glycosylation mutant isolated by screening for low intracellular lysosomal enzyme activity

A novel screening procedure was developed for isolating Chinese hamster ovary cell mutants altered in the early steps of the biosynthesis of asparagine-linked glycoproteins. This procedure identifies cells with low intracellular levels of two lysosomal hydrolases, beta-glucuronidase and alpha-iduronidase. One mutant cell line isolated in this way, CHB 11-1-3, has low intracellular levels of seven lysosomal enzymes as compared to wild-type cells. Although CHB 11-1-3 synthesizes mannosylphosphoryldolichol and [Man]₅[NAcG1cNH₂]₂-P-P-lipid, it fails to utilize these lipid intermediates to make normal amounts of [Glc]₃[Man]₉[NAcG1cNH₂]₂P-P-lipid. As a consequence of this glycosylation defect, this mutant transfers oligosaccharides of a different structure than wild type to the lysosomal enzyme beta-hexosaminidase. In addition, it underglycosylates its proteins.     

Binding of 4-methylumbelliferyl beta-D-galactosyl-(1 leads to 3)-N-acetyl-beta-D-galactosaminide to peanut agglutinin. Characterisation and application in substitution titrations

Binding of 4-methylumbelliferyl-2-acetamido-2-deoxy-3-O-(beta-D-galactopyranosyl) beta-D-galactopyranoside [MeUmb beta Gal(beta 1 leads to 3)GalNAc] to peanut agglutinin was characterized by equilibrium dialysis and by measurement of the increase in ultraviolet absorption or fluorescence of the chromophoric glycoside upon continuous titration with excess of the lectin. All data in the 4-30 degrees C range correspond to delta G = -(26.5 +/- 0.1) kJ mol-1, delta H = -(58.4 +/- 2) kJ mol-1 and delta S = -(107 +/- 8)J mol-1 K-1. Values of the association constants are e.g. K = 2.5 X 10(5) M-1 at 4 degrees C and K = 4.5 X 10(4) M-1 at 25 degrees C. MeUmb beta Gal(beta 1 leads to 3)GalNAc was used as an indicator ligand to determine K values for nonchromophoric carbohydrates by continuous displacement titrations, measuring either fluorescence or difference in absorption of the indicator. The data were analyzed in terms of the general expression for a non-ideal indicator system (as detailed in the appendix). Thus, the values of K are not underestimated. They are K = 4.8 X 10(3) M-1 for methyl alpha-D-galactopyranoside [Me alpha Gal], 2.0 X 10(3) M-1 for methyl beta-D-galactopyranoside [Me beta Gal] and 4.7 X 10(3) M-1 for lactose [Gal(beta 1 leads to 4)Glc], all at 14.5 degrees C. The MeUmb difference absorption spectra resulting from binding of the lectin with MeUmb beta Gal(beta 1 leads to 3)GalNAc and MeUmb beta Gal(beta 1 leads to 4)Glc are larger than for MeUmb beta Gal and MeUmb alpha Gal. These observations are consistent with the extended nature of the combining site of peanut agglutinin.     

Transformation of Clostridium acetobutylicum Protoplasts with Bacteriophage DNA

Techniques for the transformation of Clostridium acetobutylicum protoplasts with bacteriophage DNA are described. Transformation required regeneration of protoplasts and a 2-h eclipse period.     

Hypoxanthine-guanine phosphoribosyltransferase in human erythroid cells: Properties of the isozymes

We have previously given evidence that the hypoxanthine-guanine phosphoribosyltransferase (HGPRT; EC 2.4.2.8) isozymes in human erythroid cells result from posttranslational modifications of a single gene product [Johnson, G. G., et al. (1982). Biochemistry 21:960]. In the present work we compare the properties of the unmodified and two major modified isozymes, which collectively account for 90% of the HGPRT enzyme activity in cell lysates. The modified isozymes differ from the parent molecule in the pH dependence of activity and in the relative utilization of the two purine base substrates, hypoxanthine and guanine. In contrast to the changes in the catalytic properties of the enzyme, the modifications have no detectable effects on the heat stability or on the equilibrium between enzyme dimers and enzyme tetramers.This work was supported by United States Public Health Service Grant 5 RO1 CA 16754-03 and by the San Diego State University Foundation.     

Synthesis of 2,4-diacetamido-2,4,6-trideoxy-L-altrose, -L-idose, and -L-talose from benzyl 6-deoxy-3,4-O-isopropylidene-beta-L-galactopyranoside

Acetylation of benzyl 6-deoxy-3,4O-isopropylidene-β-L-galactopyranoside gave benzyl 2-O-acetyl-6-deoxy-3,4-O-isopropylidene-β-L-galactopyranoside (1). Removal of the isopropylidene group afforded benzyl 2-O-acetyl-6-deoxy-β-L-galactopyranoside (2), which was converted into benzyl 2-O-acetyl-6-deoxy-3,4-di-O-(methyl-sulfonyl)-β-L-galactopyranoside (3). Benzyl 2,3-anhydro-6-deoxy-4-O-(methyl-sulfonyl)-β-L-gulopyranoside (4) was obtained from 3 by treatment with alkali. Reaction of 4 with sodium azide in N,N-dimethylformamide gave a mixture of two isomeric benzyl 2,4-diazido-2,4,6-trideoxy hexoses, the syrupy diazido derivative 5 and the crystalline benzyl 2,4-diazido-2,4,6-trideoxy-β-L-idopyranoside (6). Acetylation of 6 afforded a compound whose n.m.r. spectrum was completely first order and in agreement with the structure of benzyl 3-O-acetyl-2,4-diazido-2,4,6-trideoxy-β-L-idopyranoside (7). Lithium aluminium hydride reduction of 5, followed by acetylation, afforded a crystalline product (8), shown by n.m.r. spectroscopy to be benzyl 2,4-diacetamido-3-O-acetyl-2,4,6-trideoxy-β-L-altropyranoside. Similar treatment of the diazido derivative 6 afforded benzyl 2,4-diacetamido-3-O-acetyl-2,4,6-trideoxy-β-L-idopyranoside (9). Compounds 8 and 9 could also be obtained from 4 by treatment of the crude diazido mixture with lithium aluminium hydride, with subsequent N-acetylation. The syrupy benzyl 2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranoside (10) and the crystalline benzyl 2,4-diacetamido-2,4,6-trideoxy-β-L-idopyranoside (11) thus obtained were then O-acetylated to give 8 and 9 respectively. Benzyl 2,4-diacetamido-2,4,6-trideoxy-β-L-talopyranoside (15) was obtained from 11 by treatment with methanesulfonyl chloride and subsequent solvolysis. Compound 15 was O-acetylated to yield benzyl 2,4-diacetamido-3-O-acetyl-2,4,6-trideoxy-β-L-talopyranoside (16). the n.m.r. spectrum of which was in full agreement with the assigned structure. The mass spectra of compounds 8–11, 15, and 16 were also in agreement with their proposed structures. Removal of the benzyl groups from 10, 11 and 15 afforded the corresponding 2,4-diacetamido-2,4,6-trideoxyhexoses 12, 13, and 17, having the L-altro, L-ido, and L-talo configurations, respectively.