Canadian Science Publishing

Acetamide and N-methylurea have been shown for the first time to be substrates for jack bean urease. In the enzymatic hydrolysis of urea, formamide, acetamide, and N-methylurea at pH 7.0 and 38 °C, k_cat has the values 5870, 85, 0.55, and 0.075 s⁻¹, respectively. The urease-catalyzed hydrolysis of all these substrates involves the active-site nickel ion(s). Enzymatic hydrolysis of the following compounds could not be detected: phenyl formate, p-nitroformanilide, trifluoroacetamide, p-nitrophenyl carbamate, thiourea, and O-methylisouronium ion. In the enzymatic hydrolysis of urea, the pH dependence of k_cat between pH 3.4 and 7.8 indicates that at least two prototropic forms are active. Enzymatic hydrolysis of urea in the presence of methanol gave no detectable methyl carbamate. A mechanism of action for urease is proposed which involves initially an O-bonded complex between urea and an active-site Ni²⁺ ion and subsequently an O-bonded carbamato–enzyme intermediate.

An enzyme activity that catalyzes conversion of N⁶-(Δ²-isopentenyl)adenosine (i⁶Ado) to adenosine was detected in cultured tobacco tissue by Pačes et al. (1971) (Plant Physiol. 48, 775–778). Purification and characteristics of this enzyme in corn kernels have been studied. Only the naturally occurring cytokinins i⁶Ado and ribosylzeatin serve as substrates; the nucleoside or free base works equally as well. The reaction requires oxygen. An unstable intermediate appears to be the primary reaction product. This product decomposes to adenosine.The enzyme activity is greatest at pH 5–7. It is independent of added magnesium. The molecular weight of the enzyme is about 88 000. The activity of the enzyme in corn kernels increases from the time of pollination to about 21 days.A nucleoside hydrolase is isolated with i⁶Ado oxidase. The activities can be partially separated by G-150 gel filtration. The hydrolase is less stable than the i⁶Ado oxidase and frozen preparations lose their activity after 2 months, whereas i⁶Ado oxidase is stable under these conditions.

The purified high molecular weight serogoup Y meningococcal polysaccharide contains equimolar proportions of D-glucose and N-acetylneuraminic acid and is partially O-acetylated. Carbon-13 nuclear magnetic resonance (NMR) studies, together with other chemical data, have indicated that the polysaccharide is linked only at C-6 of the D-glucose and C-4 of the sialic acid residues, all the linkages being in the α-configuration. The ¹³C NMR data also indicated that the Y polysaccharide is composed of an alternating sequence of these two different residues, and this was confirmed by its autohydrolysis where the major product was 4-O-α-D-glucopyranosyl-β-D-N-acetylneuraminic acid. The W-135 polysaccharide differs from that of Y only in the absence of O-acetylation and in the configuration of one hydroxyl group of the disaccharide repeating unit. In this case autohydrolysis yielded 4-O-α-D-galactopyranosyl-β-D-N-acetylneuraminic acid as the major product. Structural evidence indicates that the BO and Y polysaccharides are identical.Methanolysis of the Y polysaccharide yielded in addition to the methyl glycosides of glucose and sialic acid, a 9-O-acetyl derivative of the latter. This derivative was formed during the re-N-acetylation process and its formation was mainly due to the presence of sodium ions in the original polysaccharide.

A procedure for the quantitative measurement of tryptamine in mammalian tissues is described. The amine is isolated by ion-exchange chromatography, converted to its dansyl derivative, further purified by thin-layer chromatography, and quantitated by the mass-spectrometric integrated ion current technique using an isotopically labelled internal standard.The concentrations of tryptamine in some tissues of male Wistar rats were (ng/g ± S.D.): brain 0.50 ± 0.07, heart 0.62 ± 0.10, kidney 8.04 ± 2.10, liver 0.73 ± 0.07, lung 0.54 ± 0.18, and spleen 0.43 ± 0.14. In the brain, the hypothalamus contained 0.94 ± 0.22, the cerebellum 0.27 ± 0.02, the stem 0.24 ± 0.06, the caudate nucleus 2.93 ± 1.14, and the "rest" 0.32 ± 0.05 ng/g (mean ± mean deviation).

Kinetic, spectral, and other studies establish that hydroxamic acids bind reversibly to active-site nickel ion in jack bean urease. Equilibrium ultracentrifugation studies establish that the molecular weight of native urease is 590 000 ± 30 000 while that of the subunit formed in 6 M guanidinium chloride in the presence of β-mercaptoethanol is ~95 000. Essentially the same subunit molecular weight (~93 000) is found by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, subsequent to denaturation in a guanidinium chloride – β-mercaptoethanol system at various temperatures. Coupled with an equivalent weight of 96 600 for binding of the inhibitors acetohydroxamic acid and phosphoramidate, these results establish securely that urease is a hexamer with one active site per 96 600-dalton subunit. Consistent values for the equivalent weight are obtained by a routine spectrophotometric titration of the active site of freshly prepared urease with trans-cinnamoylhydroxamic acid. General equations are derived which describe spectrophotometric titrations of binding sites of any enzyme with a reversible inhibitor. These equations allow the evaluation of the difference spectrum of the protein–inhibitor complex even when the binding sites cannot readily be saturated with the inhibitor or vice versa.

A modified Yemm and Cocking ninhydrin–cyanide reagent has been developed which gives quantitative color yields with free amino acids, but much lower yields with most peptides tested. Using this reagent, a more sensitive assay for peptidase activity has been developed, and its use in column fractionation of peptidase activity is described.The ϵ values for peptides, obtained with the modified reagent, are compared to those obtained with the conventional ninhydrin–hydrindantin reagent. The formation of Ruhmann's Purple by the modified reagent is inhibited by Tris buffer. No inhibition occurs with the ninhydrin–hydrandantin reagent. The absorption spectra of the two reaction mixtures indicate that Tris reacts in a different manner with the two reagents.

Two protein kinases, designated NI and NII, have been isolated from rat liver nuclei. These enzymes have a similar pH optimum and phosphorylate phosvitin and casein more readily than histone. Both enzymes require magnesium for activity. In the absence of Mg²⁺, other divalent cations such as Ca²⁺, Co²⁺, and Mn²⁺ can substitute partially for Mg²⁺ when the reaction is catalyzed by NI. With NII, only Co²⁺ showed any activity in the absence of Mg²⁺. Magnesium decreased the apparent K_m for ATP of protein kinase NI without changing the V_max of the reaction, and decreased the apparent K_m's for both ATP and casein, while increasing the V_max of the reaction threefold with protein kinase NII. Both enzymes are stimulated about twofold by low concentrations (0.1–0.3 M) of NaCl, KCl, and sodium acetate, whereas higher concentrations (> 0.5 M) inhibit their activities. Both enzymes are inhibited by low concentrations of NaF (0.02 M) and (NH₄)₂SO₄ (0.1 M). NI and NII were found to have sedimentation coefficients of 3.6 S and 10.8 S, respectively. The nuclear protein kinases are not activated by cyclic AMP or cyclic GMP, and are not inhibited by the heat-stable cyclic AMP-dependent protein kinase inhibitor.

The three-dimensional structures of the bacterial serine proteases SGPA, SGPB, and α-lytic protease have been compared with those of the pancreatic enzymes α-chymotrypsin and elastase. This comparison shows that ~60% (55–64%) of the α-carbon atom positions of the bacterial serine proteases are topologically equivalent to the α-carbon atom positions of the pancreatic enzymes. The corresponding value for a comparison of the bacterial enzymes among themselves is ~84%. The results of these topological comparisons have been used to deduce an experimentally sound sequence alignment for these several enzymes. This alignment shows that there is extensive tertiary structural homology among the bacterial and pancreatic enzymes without significant primary sequence identity (<21%). The acquisition of a zymogen function by the pancreatic enzymes is accompanied by two major changes to the bacterial enzymes' architecture: an insertion of 9 residues to increase the length of the N-terminal loop, and one of 12 residues to a loop near the activation salt bridge. In addition, in these two enzyme families, the methionine loop (residues 164–182) adopts very different conformations which are associated with their altered substrate specificities.

Commercially obtained pure human serum albumin (HSA) was shown to contain molecular aggregates and was significantly contaminated with Cu(II). A solution of commercial HSA was first passed through a Sephadex G-200 column to obtain pure monomeric HSA. The monomer of HSA was subsequently passed through Chelex-100 resin to free it from Cu(II). All Cu(II)-binding studies were conducted with monomeric and copper-free HSA. The first Cu(II)-binding site on HSA appears to be stronger than the second and the subsequent binding sites. Significant amounts of L-histidine and L-threonine were bound to HSA when Cu(II) was added in the form of Cu(II) – amino acid complexes. In the absence of Cu(II), free L-histidine or L-threonine do not bind to HSA at pH 7.4. It is concluded that, in the presence of either L-histidine or L-threonine, ternary complex formation is involved both at the first and the subsequent binding sites for Cu(II) on HSA. In view of this finding, it appears that the equilibrium between HSA–Cu(II) and Cu(II) – amino acid complex is mediated through a ternary complex HSA – Cu(II) – amino acid.

A study was made of the amounts and fatty acid compositions of the cholesterol esters, phospholipids, and triglycerides present in the yolk of the fertile unincubated egg and in the yolk, liver, and extrahepatic tissues of the chick embryo at various stages of development. Esterification of cholesterol, mainly with oleic acid, occurred in the yolk during incubation. There appeared to be a preferential absorption from the yolk sac of phospholipids rich in docosahexaenoic acid. Considerable amounts of cholesterol esters, of which 80% was cholesterol oleate, accumulated in the embryonic liver. The liver phospholipids contained more stearic, arachidonic, and docosahexaenoic acids, and less palmitic and oleic acids, than did the yolk phospholipids. Docosahexaenoic acid occurred in a surprisingly high concentration in the liver triglycerides. The extrahepatic triglycerides contained more palmitic and C₁₈polyunsaturated acids, but less docosahexaenoic acid, than did the liver triglycerides. The concentration of oleic acid in the extrahepatic cholesterol esters was much less than in the liver cholesterol esters. The extrahepatic phospholipids contained more arachidonic and docosahexaenoic acids, but less oleic acid, than did the yolk phospholipids. The implications of these findings are discussed in relation to the general lipid metabolism of the chick embryo.