Association of nucleosidediphosphate kinase with microtubules.

A nucleosidediphosphate kinase activity (EC 2.7.4.6) which phosphorylates GDP to GTP is present in bovine brain microtubule protein prepared by cycles of assembly-disassembly. This activity persists through 5 cycles of assembly-disassembly and sediments with microtubules in sucrose density gradients, but is not associated with the tubulin dimer. It is proposed that the kinase is an integral part of the microtubule and is therefore a microtubule associated protein (MAP). Several isozymes of nucleosidediphosphate kinase exist in our preparations with a pI 7.6 form predominant. It may be speculated that this enzyme affects tubulin assembly $\text{in vivo}$ by modulating the $\text{GTP}\text{GDP}$ ratio in the microtubule environment.     

Acid-catalyzed isomerization and dehydration of dl-glyceraldehyde and 1,3-dihydroxy-2-propanone

The interconversion of 2,3-dihydroxypropanal (1) and 1,3-dihydroxy-2-propanone (3) and their dehydration to pyruvaldehyde (5) has been studied in 0.5m sulfuric acid at 100°. The conversion of 1 and 3 into 5 under these conditions, in tritiated water, was monitored by isolation of 5 and determination of the distribution of carbon-bound tritium (namely, that at the aldehyde and methyl groups). This was accomplished by conversion of 5 into the phenylosazone and counting, and by conversion of 5 into pyruvic acid (isolated as the p-nitrophenylhydrazone), counting, and a difference calculation. In all cases negligible activity (about 0.8 % the activity of the solvent) was found at the aldehyde carbon of 5, derived from either 1 or 3, and parallel chromatographic studies indicated only a small extent of interconversion. As 5 incorporated tritium under conditions of its formation, plots of incorporation versus time were made and the curves extrapolated to zero time, in order to determine initial activity at the methyl group of 5, derived from either 1 or 3. This method showed that the 5 derived from 3 contained 10 % of the activity of the solvent at the methyl group as it was produced, whereas the 1-derived 5 contained no activity. In another experiment, [2-³H]1 was prepared and converted into 5. The resulting 5 was found to be labeled at C-3, indicating at C-2 → C-3 intramolecular, hydrogentransfer reaction during the conversion of 1 into 5.     

Effects of nonuniform column packing in analytical gel chromatography. II. Associating solutes

Effects of nonuniform column packing on boundary profiles for selfassociating systems have been investigated by computer simulation. Migration rate of each of the interconverting solute species changes along the column as a result of nonuniform packing, and the difference in velocity of monomer and n-mer is not constant as the sample moves down the column. A greater amount of overall axial dispersion results, as compared to the constant-column case. Procedures developed in this study can be applied to any experimentally measured column nonuniformity.     

Reversible inactivation of avian lysozymes by dimethyl (2-hydroxy-5-nitrobenzyl)-sulfonium bromide

The reaction of hen egg white lysozyme with a 4 molar excess of dimethyl (2-hydroxy-5-nitrobenzyl)-sulfonium bromide at pH 6.0 leads to total loss of enzymatic activity within 5 minutes. Upon standing, the inactivated enzyme spontaneously regains activity, leveling off at 60% of the original activity after 72 hours. Under the same conditions, turkey egg white lysozyme is reduced to less than 5% of its original activity within 5 minutes, then spontaneously reactivates to 85% of its original activity after 24 hours. Human lysozyme shows no dramatic loss of activity when treated under these conditions. The presence of the substrate, chitotetraose, prevents the initial inactivation of both hen and turkey enzymes.     

Essential carboxyl residues in yeast enolase.

Yeast enolase (EC 4.2.1.11) is rapidly inactivated at pH 6.1 by three different water-soluble carbodiimides — 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride, 1-cyclohexyl-3-(2-morpholinoethyl)-carbodiimide metho-p-toluenesulfonate, and 1-ethyl-3-(4-azonia-4,4-dimethylpentyl)-carbodiimide iodide. Inactivation is most likely due to the modification of essential carboxyl residues at the enzyme active site.     

The structure of the type-specific polysaccharide of Pneumococcus type XIX

The structure of the capsular polysaccharide of Type XIX Streptococcus pneumoniae (S-XIX) has been elucidated by ¹H- and ¹³C-n.m.r. spectroscopy. Mild hydrolysis of S-XIX with acid yielded a major oligosaccharide, the repeating unit of S-XIX, which was shown to be O-2-acetamido-2-deoxy-β-d-mannopyranosyl-(1→4)-O-α-d-glucopyranosyl-(1→2)-l-rhamnose 4′′-phosphate. Phosphoric acid forms a diester linkage in the S-XIX molecule, which explains the instability of S-XIX towards acid or alkali. The phosphodiester linkages in S-XIX join HO-1 of α-l-rhamnose and HO-4 of the 2-acetamido-2-deoxy-d-mannopyranosyl residue in the next repeating-unit. Treatment of S-XIX with alkali or alkaline-NaBH₄ produced the repeating units in a lower yield. The proposed structure of S-XIX is

     

Conformation of 1- and 3-deazaadenosines in solution as studied by 1H nuclear magnetic resonance spectroscopy.

¹H nuclear magnetic resonance spectra of 1 - (II) and 3-deazaadenosines (III) together with adenosine (I) in dimethylsulfoxide have been examined. Features of coupling constants indicate that the furanose rings of I, II, and III have similar conformational preferences and that conformations about the 4′-C–5′-C bond are preferentially $\text{gauche-gauche}$. Nuclear Overhauser effect and spin-lattice relaxation-time measurements demonstrate that II predominantly adopts the $\text{syn}$-conformation similar to that of I, whereas that of III has a greater $\text{anti}$ (freely rotating) component. The results suggest that the $\text{syn}$-conformation in II as well as I is stabilized presumably through a hydrogen bond between the 3-N and 5′-hydroxyl group.     

Synthesis of potential antiprogestogens.

Acylated derivatives of 17 alpha-hydroxyprogesterone were prepared in order to test the hypothesis that dialkylamino alkyl moieties have the effect of transforming progestogens into antiprogestogens. This approach has been successful with certain estrogens. Compounds with other functional groups were synthesized to determine whether these might exert binding influence outside the area occupied by progesterone itself. The compounds were tested for competitive affinity against tritiated progesterone and receptor from rabbit uterus cytosol. The low affinity of all derivatives makes it unlikely that they would be active as antiprogestational agents.     

Inactivation of β-lactamase I from B. cereus569H with phenylglyoxal,an arginine-selective reagent

β-Lactamase (EC 3.5.2.6: penicillin amido-β-lactam hydrolase) I from B. cereus$\text{569}\text{H}$ is inactivated by treatment with phenylglyoxal. Inactivation depends on the pH and the presence of bicarbonate in a manner which suggests that it is due to the modification of arginyl residues. Total inactivation correlates with the loss of 8 arginines per β-lactamase, and a competitive inhibitor provides full protection from inactivation and protects ca. 2 arginines from modification.     

2-amino-2,5-anhydro-2-deoxy-DL-ribitol: an amino sugar derivative having nitrogen in the ring

A direct, high-yielding route for synthesis of a pyrrolidine analog of a 2-deoxy-erythro-pentose is reported. The synthesis involves modification of pyrrole-2-carboxylic acid by reduction followed by a hydroxylation step. The structure and stereochemistry of 2,5-anhydro-2-deoxy-2-p-toluenesulfonamido-DL-ribitol (5a) was established by chemical transformations and by ¹³C n.m.r. data.     

Anticytokinin activity of 4-furfurylamino-7-(beta-D-ribofuranosyl)pyrrolo(2,3)pyrimidine

4-Furfurylamino-7-(β-D-ribofuranosyl)pyrrolo[2,3-d]-pyrimidine, the 7-deaza analog of kinetin riboside, has been synthesized and found to be a potent anticytokinin in the tabacco callus bioassay.     

An improved method for the quantitative determination of deoxyribonucleoside triphosphates in cell extracts

The analysis of deoxyribonucleoside 5'-triphosphates (dNTPs) in cell extracts by high-pressure liquid chromatography [C. Garrett , and D.V. Santi (1979) Anal. Biochem. 99, 268-273] requires the prior, selective degradation of ribonucleoside 5'-triphosphates ( rNTPs ) that are present in the extracts in large quantities. When this method was used for quantifying the dNTPs in mammalian cell extracts, the presence of an interfering peak in the HPLC between the peaks for dTTP and dATP was observed. This unwanted peak sometimes overlapped with that of dATP, depending on the pH of the eluant. It was found that the material which gave this peak was formed during the periodate oxidation of rNTPs in the presence of methylamine, and that it could be removed by changing the order of addition of the reagents in the procedure, i.e., the methylamine was added only after the excess periodate was decomposed, instead of adding it together with periodate, as given in the original procedure. Furthermore, an addition of deoxyguanosine to the reaction mixture was found to be effective in preventing the partial loss of dGTP in the oxidation procedure. By using this improved method, the dNTP contents of the extracts of Ehrlich ascites tumor cells have been measured in an accurate and reproducible manner. The analysis requires about 10(6) cells, and all four dNTPs can be quantified in 2.5 h, starting from the harvest of the cells.     

Conformations of the d-glucarolactones and d-glucaric acid in solution

The conformations of d-glucaric acid (1), d-glucaro-1,4-lactone (2), d-glucaro-6,3-lactone (3), and d-glucaro-1,4:6,3-dilactone (4) in solution were investigated by ¹H-n.m.r. and ¹³C-p.F.t., n.m.r. spectroscopy. The solvents used were deuterium oxide, methanol-d₄, and dimethyl sulfoxide-d₆, and praseodymium chloride was employed as a lanthanide shift-reagent. For 2, it was found that the conformational equilibrium ³E(d)

E₃(d) exists in solution, and that the OH-5 group tends to occupy the position over the lactone ring in the favored E₃(d),gg conformation. The n.m.r. data for 3 indicated that the conformational equilibrium is shifted in favor of the ⁴E(d)

E₄(d),gt conformation in solution. The dienvelope conformation ³E:E₄(d) was found to be the favored conformation of 4. For 1, a conformational equilibrium between one planar, zigzag form and two sickle forms was indicated by the n.m.r. data observed. ¹³C-N.m.r. spectroscopy proved to be a convenient method for monitoring the lactonization of 1, and the hydrolysis of its lactones. Lactones other than 2–4 were not found in solutions prepared from 1–4, either during their mutarotation or after equilibration at 30°.