Hormone, serum, and methylxanthine interactions in the regulation of DNA synthesis in organ-cultured rat mammary tumors.

Previous studies have shown that the growth of MTW9, a hormone-dependent rat mammary tumor, is stimulated by the presence of a mammosomatotropic tumor coimplant (MtTW10) but is inhibited by the administration of dibutyryl cyclic AMP (DBcAMP). This study examines the direct effect of serum taken from MtTW10-bearing WF rats (MtT serum) and of substances that increase cellular levels of cyclic AMP (cAMP) on DNA synthesis in organ-cultured MTW9. Explant DNA synthesis was significantly greater in the presence of MtT serum than in either normal female or male rat serum. Preparations of hormones known to be present in MtT serum failed to simulate the activity of MtT serum. Whereas 1 mM cAMP 1 mM did not alter DNA synthesis in MTW9 explants, inhibitors of cyclic nucleotide phosphodiesterases substantially suppressed the rate of [3H]thymidine incorporation into DNA. However, addition of MtT serum to cultures completely prevented the effects of these substances. This activity of MtT serum could not be duplicated by any of the following additions to the culture medium: normal female or male rat serum, National Institutes of Health (NIH) rat prolactin (2.5 micrograms/ml), NIH rat growth hormone (2.5 micrograms/ml), insulin (5 micrograms/ml), 17 beta-estradiol (5 micrograms/ml), or a hormone combination simulating the known endocrine constituents of MtT serum. The results suggested that the serum of the MtT-bearing animal contained an activity, unidentified at present, that could antagonize the growth-inhibitory action of the cAMP system and that may participate in the regulation of mammary tumor growth.

The origin and composition of multiple forms of dextransucrase from Streptococcus sanguis.

Multiple forms of purified dextransucrase have been observed in the presence of low detergent concentrations ( Luzio , G.A., Grahame , D. A. and Mayer, R.M. (1982) Arch. Biochem. Biophys. 216, 751-757). We now show these forms to arise partly as a result of proteolysis, and partly due to incomplete dissociation of the enzyme. Upon 25 degrees C incubation of the crude enzyme, several new bands appeared with little or no change in total activity. The electrophoretic pattern of aged, crude enzyme was similar to that of partially purified enzyme. Specific detection of dextransucrase on SDS gels revealed a single polypeptide of 174 kDa, which is converted to a 156 kDa protein during the aging process. The observation indicates the occurrence of proteolysis. The polypeptide composition of several of the enzyme forms was determined by two-dimensional electrophoresis. Forms Ia and IIa are composed exclusively of 174 kDa polypeptides. Forms III and IVa consist of 156 kDa units, as does the newly observed form Ic. It is likely that form Ib contains both 174 and 156 kDa polypeptides. The results indicate that incomplete dissociation of aggregates of the 174 kDa unit accounts for all of the bands observed on native gels run on fresh culture extracts. Additional enzyme forms result from aggregation of the 156 kDa proteolysis product alone, and from aggregation with unproteolyzed units to form hybrid aggregates.

Intestinal mucins and cholesterol uptake in vitro.

A mucus material, secreted by intestinal segments, with a high affinity for cholesterol, has been isolated and chemically characterized. The mucin contained 11% carbohydrate, largely as glucosamine, galactose and N-acetylneuraminic acid, and 19% lipid, of which 86% was unesterified fatty acid. The isolated material readily bound cholesterol in a stoichiometric manner. Conditions known to enhance cholesterol absorption in vivo also decreased mucin complexing to cholesterol in vitro. This association of cholesterol and intestinal surface mucin also occurred during incubations of intestinal segments with dispersed cholesterol, resulting in a high level of intestinal adsorption, with little or no cellular absorption of the sterol. However, when cholesterol was solubilized in simple or complex micelles containing bile salts, surface adsorption of cholesterol was reduced and net absorption was increased. The results suggest that surface mucin binding of cholesterol may represent at least one major diffusion limitation to cholesterol absorption in the intestine.

Purification, and comparison, of two forms of dextransucrase from Streptococcus sanguis.

A procedure has been developed whereby native and proteolyzed forms of dextransucrase have been purified; it involves gel filtration, and hydroxylapatite chromatography in the presence of 0.10% sodium dodecyl sulfate. This procedure is highly reproducible, and permits approximately 30% recovery of high purity (94% homogeneous) enzyme as an inactive, SDS complex that can be reactivated by the addition of Triton X-100. The purified enzymes have been compared with regard to amino acid compositions, and isoelectric and catalytic properties. An analysis of the structure of their product D-glucans was also made. Although the structural characteristics of the enzyme forms differ, proteolysis does not cause alterations in their catalytic properties.

Interaction of deoxyhalosucrose derivatives with dextransucrase.

Members of a series of deoxyhalosucrose analogs substituted at one, two, or three primary carbon atoms with bromine or chlorine were prepared. Dextransucrase isolated from Streptococcus sanguis was separately treated with 6-bromo-6-deoxysucrose, 6,6'-dibromo-6,6'-dideoxysucrose, 6,1',6'-tribromotrideoxysucrose, and 6,6'-dichlorodideoxysucrose, in order to determine if they were inactivators. Variation in time of exposure, and in the concentration of the sucrose analogs, did not yield significant irreversible inactivation. In supplementary studies, it was found that the compounds serve as weak, reversible inhibitors.

Structural characteristics of native and enzymically formed dextran of S. sanguis ATCC 10558.

The structure of the native dextran produced by Streptococcus sanguis ATCC 10558 was analyzed by g.l.c.-m.s. of the methylated alditol acetates derived from the polymer. The results indicate that the polymer contains D-glucosyl residues substituted at C-6 or C-3, or both, as well as unsubstituted D-glucosyl residues. These data aially purified dextransucrase on sucrose. The proportion of D-glucosyl residues substituted at C-3 is diminished in this case. It is concluded that several enzymes are involved in the dextran synthesis.

Photolabeling of dextransucrase from Streptococcus sanguis with p-azidophenyl alpha-D-glucopyranoside.

Dextransucrase from Streptococcus sanguis ATCC 10558 was photolabeled using p-azidophenyl alpha-D-glucopyranoside with an apparent rate constant of inactivation of 1.40 min-1. The dissociation constant for this compound, which acts as an acceptor molecule in the enzymatic reaction, is 90 microM. Apparently two acceptor binding sites exist on dextransucrase as shown by (i.) photolabeling the enzyme with p-azidophenyl-alpha-D-[5,6-3H]glucopyranoside and (ii.) fluorescence titration experiments.

Formation of alpha-(1-->6), alpha-(1-->3), and alpha-(1-->2) glycosidic linkages by dextransucrase from Streptococcus sanguis in acceptor-dependent reactions.

Dextransucrase from Streptococcus sanguis 10558 was found to synthesize alpha-(1-->6), alpha-(1-->3), and alpha-(1-->2) linkages during an acceptor-dependent glucosyl transfer reaction. Normally, new glucosyl residues are added at C-6 of monosaccharide acceptors. However, sugars blocked at C-6 also can serve as good acceptors. The disaccharide and trisaccharide products formed when methyl 6-bromo-6-deoxy-alpha-D-glucopyranoside was used as acceptor were isolated and characterized. Both were found to contain only alpha-(1-->3) glycosidic bonds. This supports the hypothesis that when C-6 is blocked the acceptor binds to the enzyme in a flipped orientation, resulting in an approximate exchange in space of the C-3 and C-6, thereby putting C-3 adjacent to the active site. The second alpha-(1-->3) links in the trisaccharide are formed by a single-chain mechanism without release of the intermediate disaccharide. With maltose as acceptor, new glucosyl residues are added at C-6'. However, if that position is blocked with a bromine atom, the resulting compound, 6'-bromo-6'-deoxy-maltose, can still serve as an acceptor. The product in this case was isolated and characterized. The new glycosidic link was found to be alpha-(1-->2).

The purification and properties of dextransucrase from Streptococcus sanguis ATCC 10558.

Dextransucrase has been purified from the culture fluids of S. sanguis 10558 by a combination of hydroxylapatite, ion-exchange, and gel-filtration steps. Two active proteins were isolated with specific activities approaching one order of magnitude higher than other preparations reported. The enzymes have mol. wt. on the order of 100 000 and exhibit pH optima between 5,8 and 6.2. In addition, detailed analysis of one of the enzymes indicates that the enzyme undergoes two ionizations that are important for activity. One pK is at 4.4 and the second at 7.4. The structures of dextrans produced by the two enzymes have been examined by p.m.r. spectroscopy, and a substantial degree of similarity was observed, with only minor differences in the proportion of alpha-(1 leads to 3) and alpha-(1 leads to 6) bonds. No evidence could be obtained that either of the enzymes was capable of catalyzing a rearrangement of alpha-(1 leads to 6) to alpha-(1 leads to 3) bonds.

Stereochemistry and mechanism of the GDP-mannose dehydratase reaction.

The reaction catalyzed by bacterial GDP-mannose dehydratase (E.C. 4.2.1.47), the conversion of GDP-D-mannose to GDP-4-keto-6-deoxymannose (GDP-6-deoxy-D-lyxo-hexos-4-ulose), was studied with (6R)- and (6S)-GDP-D-[4-2H1,6-3H]mannose. Conversion of these stereospecifically labeled substrates in the presence of excess unlabeled GDP-mannose into the 4-keto-6-deoxy derivatives followed by Kuhn-Roth oxidation gave acetic acid samples which were subjected to configurational analysis of the isotopically chiral methyl group. The observed F values of 64 for the material from the (6S) substrate and 31 for that from the (6R) isomer, corresponding to 48% e.e. R and 66% e.e. S configuration, respectively, of the methyl group indicate that (a) the oxidoreductase reaction involves transfer of H-4 to C-6, (b) the transfer is predominantly intramolecular, and (c) the transfer is stereospecific, H-4 replacing the C-6 hydroxyl group with inversion of configuration. A mechanism for the reaction is proposed on the basis of these results.

A D-glucosylated form of dextransucrase: demonstration of partial reactions.

A D-glucosylated form of dextransucrase, whose preparation and characteristics have just been reported in Carbohydr. Res., was employed in a series of studies designed to explore the question of whether the bound sugars participate in the reactions catalyzed by the enzyme. When exposed to maltose, a good acceptor-substrate, monomeric D-glucosyl groups were rapidly transferred to the disaccharide, affording a trisaccharide. In the absence of an acceptor, monomeric D-glucose was released from the enzyme by hydrolysis. In a reaction with D-fructose, the charged enzyme catalyzed the formation of sucrose. Finally, in the presence of unlabeled sucrose, monomeric D-glucosyl groups were chased into enzyme-associated oligomers. Evidence is also presented which indicates that the various pathways for the bound D-glucosyl groups are competitive. The significance of these observations is discussed.

Structural determination of alginic acid and the effects of calcium binding as determined by high-field n.m.r.

The nature of the solution conformations of the alginic acid components D-mannuronan (poly-ManA) and L-guluronan (poly-GulA) from Azotobacter vinelandii were investigated by both one- and two-dimensional n.m.r. methods. Unequivocal proton assignments for both polymers as well as their constituent monomer units were made based on chemical-shift theory, coupling constant analysis, and nuclear Overhauser enhancement measurements. These data were used to investigate the interactions of poly-GulA and poly-ManA with Ca2+ ion in aqueous medium. Based on relative crosspeak integrals measured in two-dimensional phase-sensitive NOESY spectra of free and calcium-bound polymer, a model for calcium binding is proposed.

A D-glucosylated form of dextransucrase: preparation and characteristics.

Dextransucrase was treated with [14C]sucrose, and the product applied to gel-permeation columns. In the absence of the detergents SDS and Triton X-100, poor recovery of enzyme was observed; however, that enzyme which was recovered was labeled. In the presence of detergents, recovery was increased, but the material appeared to be a large aggregate (mol. wt. greater than 5 X 10(6) ). In addition, the ratio of D-glucose to enzyme suggested that a polymer had been formed. Disc-gel electrophoresis in the presence of a mixture of SDS and Triton X-100 showed similar results, and indicated that the aggregate was disrupted upon treatment with dextranase. Native enzyme that had been immobilized on hydroxylapatite could also be labeled with [14C]sucrose, and the labeling followed saturation kinetics. The labeled protein could be released from the gel with 8M urea, but was aggregated. Radioactive sugars, free from protein, could be released by heating the labeled enzyme. The sugars released consisted of a mixture of D-glucose with oligosaccharides having an average chain-length of 17 D-glucosyl residues. The significance of these observations is discussed.