Hydrophobic residues 382-386 of antithrombin III, Ala-Ala-Ala-Ser-Thr, serve as the epitope for an antibody which facilitates hydrolysis of the inhibitor by thrombin.

In the presence of a monoclonal antibody raised against the human thrombin-antithrombin III complex, the reaction between thrombin and antithrombin III proceeded to form preferentially a two-chain form of the inhibitor rather than to follow the major pathway of stable acyl complex formation. We thus propose the term "switching antibody" for an antibody that switches the enzyme-inhibitor reaction (Asakura, S., Matsuda, M., Yoshida, N., Terukina, S., and Kihara, H. (1989) J. Biol. Chem. 264, 13736-13739). By analyzing a CNBr fragment of the thrombin-antithrombin III complex that reacts with the antibody we localized the epitope for the antibody to a strongly hydrophobic residue 382-386 peptide segment, Ala-Ala-Ala-Ser-Thr, of the inhibitor, which is also contiguous with a hydrophobic amino acid Ala at its carboxyl terminus. This particular region should be cryptic in nascent antithrombin III, but could have been exposed to provide the reactive site for the antibody at an early stage of the reaction. Thereby a conformational change may have been induced at or near the reactive site of the complex, facilitating hydrolysis of the inhibitor by the enzyme. Interestingly, this hydrophobic region is highly conserved among members of the serpin family.     

Biosynthesis of xyloglucan in suspension-cultured soybean cells. Occurrence and some properties of xyloglucan 4-beta-D-glucosyltransferase and 6-alpha-D-xylosyltransferase

A particulate enzyme fraction that catalyzes the transfer of glucose from UDP-[14C]glucose and of xylose from UDP-[14C]xylose into a xyloglucan has been isolated from suspension-cultured soybean cells. The incorporation of radioactivity from [14C]xylose into the polysaccharide was dependent on the presence of UDP-glucose in the incubation mixture, and that from [14C]glucose was dependent on the concentration of UDP-xylose in the mixture. Mn2+ was required for the incorporation of xylose and the optimum concentration of Mn2+ was about 10 mM. This reaction showed a pH optimum at 6.5 to 7.0 in 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid buffer and was inhibited by phosphate buffer and Tris buffer. On hydrolysis with Trichoderma endoglucanase, the polysaccharide synthesized in vitro gave a pentasaccharide, a hepatasaccharide, and a small amount of non-asaccharide. Based on the results from fragmentation and methylation analyses, the following structures were proposed for the penta- and the heptasaccharides from the xyloglucan synthesized in vitro: (formula, see text).     

Biosynthesis of Xyloglucan in Suspension-Cultured Soybean Cells. Synthesis of Xyloglucan from UDP-Glucose and UDP-Xylose in the Cell-Free System

When UDP-[¹⁴C]glucose or UDP-[¹⁴C]xylose was incubated witha particulate fraction from soybean cells, radioactive polymerswere synthesized. On digestion with Aspergillus oryzae enzymes,these polymers gave ¹⁴C-monosaccharides and a ¹⁴C-disaccharidewith chromatographic and electrophoretic mobilities indistinguishablefrom those of authentic isoprimeverose (6-O--D-xylopyranosyl-D-glucopyranose).The disaccharide consisted of xylose and glucose, and the latterwas located at the reducing end. Evidence that the disaccharideis isoprimeverose was provided by methylation analysis. Hydrolysisof the methylated disaccharide yielded 2,3,4-tri-O-methyl-D-xyloseand 2,3,4-tri-O-methyl-D-glucose. Thus, incorporation of radioactivityinto isoprimeverose, the smallest structural unit of xyloglucan,suggests that xyloglucan is synthesized in vitro from UDP-glucoseand UDP-xylose. (Received November 20, 1980; Accepted February 14, 1981)     

Semisynthesis and properties of some insulin analogs

The trypsin-catalyzed coupling of bovine (Boc)₂-desoctapeptide (B23-B30)-insulin with synthetic octapeptides, H-Gly-X₂-X₃-X₄-Thr-Pro-Lys(Boc)-Thr-OH (X₂ = Phe or Ala, X₃ = Phe or Ala, X₄ = Tyr or Ala), followed by deprotection and purification produced the [Ala^B24, Thr^B30]-, [Ala^B25, Thr^B30]-, and [Ala^B26, Thr^B30]-analogs of bovine insulin in yields of 32, 35, and 32%, respectively. The biological activity of these analogs decreased in the order, normal insulin ([Thr^B30]-bovine insulin) = Ala^B26-insulin > Ala^B25-insulin > Ala^B24-insulin, as assayed for receptor binding and some other biological effects, in contrast with the corresponding Leu-analogs of human insulin, in which the activity decreased in the order, normal insulin > Leu^B24-insulin > Leu^B25-insulin. The affinity to insulin antibodies greatly diminished in both Ala^B24-insulin and Leu^B24-insulin but not in the B25-substituted analogs. The CD spectra of the Leu- and the Ala-analogs were compared with those of normal insulins to show that no apparent correlation seems to exist between the decrease in biological activity and the conformational changes observed in solution. The effects of organic solvents on the peptide-bond equilibrium and on the stability of trypsin are also discussed.     

Interaction of thymidylate synthetase with 5-nitro-2'-deoxyuridylate

5-Nitro-2'-deoxyuridylate (NO2dUMP) is a potent mechanism-based inhibitor of dTMP synthetase. After formation of a reversible enzymeìnhibitor complex, there is a rapid first order loss of enzyme activity which can be protected against by the nucleotide substrate dUMP. From studies of model chemical counterparts and the NO2dUMPdTMP synthetase complex, it has been demonstrated that a covalent bond is formed between a nucleophile of the enzyme and carbon 6 of NO2dUMP. The covalent NO2dUMPènzyme complex is sufficiently stable to permit isolation on nitrocellulose membranes, and dissociates to give unchanged NO2-dUMP with a first order rate constant of 8.9 x 10(-3) min-1. Dissociation of the complex formed with [6-3H]NO2dUMP shows a large alpha-secondary isotope effect of 19%, verifying that within the covalent complex, carbon 6 of the heterocycle is sp3-hybridized. The spectral changes which accompany formation of the NO2dUMPènzyme complex support the structural assignment and, when used to tritrate the binding sites, demonstrate that 2 mol of NO2dUMP are bound/mol of dimeric enzyme. The interaction of NO2dUMP with dTMP synthetase is quite different than that of other mechanism-based inhibitors such as 5-fluoro-2'-deoxyuridylate in that it neither requires nor is facilitated by the concomitant interaction of the folate cofactor, 5,10-CH2-H4folate, and that the covalent complex formed is unstable to protein denaturants.     

A high-potential nonheme iron protein (HiPIP)-linked,thiosulfate-oxidizing enzyme derived fromChromatium vinosum

A thiosulfate-oxidizing enzyme was partially purified fromChromatium vinosum, and some of its properties were studied. The enzyme rapidly reducede HiPIP (high-potential nonheme iron protein) in the presence of thiosulfate. Cytochromesc of yeast and tuna and ferricyanide also acted well as electron acceptors for the enzyme; horse cytochromec was a poor electron acceptor. Cytochromec-552, cytochromec′, and cytochromec-553 did not act as electron acceptors. The enzyme was inhibited by cyanide and sulfite. On the basis of the stoichiometry in reduction of ferricyanide catalyzed by the enzyme in the presence of thiosulfate, the oxidized product of thiosulfate was inferred to be tetrathionate.     

The amino acid sequences of the tryptic, chymotryptic and peptic peptides from the L-2 light chain of rabbit skeletal muscle myosin.

The amino acid sequences of the tryptic peptides from the aminoethylated L-2 light chain of rabbit skeletal muscle myosin were determined by various enzymatic hydrolyses, partial hydrolysis with dilute acetic acid and Edman degradation. The amino acid sequences of the chymotryptic and peptic peptides from the carboxymethylated L-2 light chain were partially analysed in the same manner as the tryptic peptides. The primary structure of the L-2 light chain of rabbit skeletal muscle myosin was deduced from the above results.     

Inhibition of the synthesis of microbial cellulose by coumarin

Coumarin, a specific inhibitor of the biosynthesis of celluloseof higher plant cell walls, inhibited the cellulose formationof Acetobacter xylinum. The degree of inhibition reached 55%in the presence of 1 mM coumarin, which causes 70% inhibitionin the case of plant cellulose. (Received April 12, 1976; )     

Purification and properties of the extracellular dextransucrase from Leuconostoc mesenteroides NRRL B-1299.

Dextransucrase [EC 2.4.1.5] activity from cell-free culture supernatant of Leuconostoc mesenteroides NRRL B-1299 was purified by (NH4)2SO4 fractionation, adsorption on hydroxyapatite, chromatography on DEAE-cellulose and gel filtration on Sephadex G-75. The extracellular enzyme was separated into two principal forms, enzymes I and N, and the latter was shown to be an aggregated form of the protomer, enzyme I. Enzymes I and N were both electrophoretically homogeneous and their relative activities reached 820 and 647 times that of the culture supernatant, respectively. On sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis, enzyme N dissociated into the protomer enzyme I, with a molecular weight of 48,000. Enzyme I was gradually converted into enzyme N upon aging, and this conversion was stimulated in the presence of NaCl. The optimum pH and temperature of enzyme I activity were pH 6.0 and 40 degrees, respectively, while those of enzyme N were pH 5.5 and 35 degrees. The Km values of enzymes I and N were 13.9 and 13.1 mM, respectively. Ca2+, Mg2+, Fe2+, and Co2+ stimulated the activity of enzyme N, and EDTA showed a potent inhibitory effect on this enzyme. Moreover, the activity of enzyme N was more effectively stimulated by exogenous dextrans as compared with enzyme I.