Properties of polyphenol oxidase from tubers of the yam Dioscorea bulbifera

The subunit MW of Dioscorea bulbifera polyphenol oxidase (MW 115 000 ± 2000) determined by SDS-PAGE is ca. 31 000 indicating that the enzyme is an oligomeric protein with four subunits. K_i values of various inhibitors and their modes of inhibition have been determined with catechol and pyrogallol as substrates. p-Nitrophenol, p-cresol, quinoline and resorcinol are competitive inhibitors of catechol binding while only orcinol and p-nitrophenol behave in the same way towards pyrogallol as substrate. From the effect of pH on V_max, groups with pK values ca. 4.7 and 6.8 have been identified to be involved in catalytic activity. The Arrhenius activation energy (E_a) at pH 4.0 is 8.9 kcal/mol between 40–65°. At pH 7.0, the value is 22.1 kcal/mol between 40 and 60°. The enthalpies (ΔH) at pH 4.0 and pH 7.0 are 2.3 kcal/mol and 32.4 kcal/mol respectively. The results are discussed considering the conformational changes of the enzyme during substrate binding.     

Induction of 3-Hydroxybenzoate 2-Hydroxylase in a Pseudomonas testosteroni Mutant

Benzoate was established as the inducer of a unique 3-hydroxybenzoate 2-hydroxylase activity found in a Pseudomonas testosteroni mutant which is unable to grow on m-hydroxybenzoate as its sole source of carbon and energy.     

Cell-mediated immunity in aged mice: an underlying lesion in IL 2 synthesis

It is well established that immune potential declines with age and that aged animals are unable to mount vigorous cell-mediated immune responses. Production of the lymphokine, Interleukin 2 (IL 2) in response to the T cell mitogen concanavalin A or allogeneic stimulation is also diminished in aged individuals. This lesion in IL 2 synthesis in aged mice underlies their deficiency in cell-mediated responses. Mixed lymphocyte responses, cell-mediated lympholysis, and antigen-induced T cell proliferation are all markedly enhanced by supplementation with this lymphokine.     

Development in ontogeny of the effect of ACTH fragment 4-7 on rat learning

Studies have been made of the effect of ACTH fragment 4-7 on learning in rats in early postnatal ontogenesis, as well as of the possibility of preservation of early learning during administration of this peptide to adult animals. It was shown that conditioned reaction of active avoidance practically cannot be formed in normal 13-15-day animals; however, administration of ACTH 4-7 increases the number of animals exhibiting adequate reactions. Weak effect of ACTH 4-7 in 12-15-day animals, in older ones (20-24 days) is changed by a significant stimulating effect.     

Aminoglycoside-3'-phosphotransferase from Actinomyces fradiae. Its isolation, purification and properties]

Aminoglycoside phosphotransferase was isolated from the mycelium of Act. fradiae, the neomycin-producing organism, with paromomycin, neomycin and to a less extent ribostamycin being substrates of aminoglycoside-phosphotransferase. It was purified to homogenous state. The maximum activity of the enzyme preparations was observed at pH 7.7--7.8;KM for neomycin and paromomycin was about 20 micron and KM for ATP was 150 micron. Mg2+ ions were necessary for the enzyme activity. None of the divalent cations tested could replace the magnesium ions in the reaction of phosphorylation catalyzed by the enzyme. High sensitivity to the ionic strength of the buffer was characteristic of the enzyme. It lost about 80 per cent of the initial activity at a concentration of KC1 equal to 1.0 M. The molecular mass of the enzyme from the mycelium of Act. fradiae was determined by the method of gel-filtration through sefadex G-100. It was about 22,000. High stability was characteristic of the enzyme. The fingings indicate that aminoglycoside phosphotransferase from Act. fradiae differs from the described aminoglycoside-3'-phosphotransferases isolated from antibiotic resistant bacteria.     

Studies of bovine liver glutamate dehydrogenase by analytical affinity chromatography on immobilized AMP analogs.

Bovine liver glutamate dehydrogenase has been studied by analytical affinity chromatography on two immobilized AMP analogs, i.e., N⁶-(6-aminohexyl)-AMP and 8-(6-aminohexyl)-amino-AMP. The existence of various enzyme-coenzyme and enzyme-effector complexes has been verified. Also the cooperative formation of two ternary complexes, i.e., glutamic dehydrogenase (GHD)-NADP-glutamate and GDH-ADP-leucine, has been shown. The results of this study have been rationalized by the “ligand exclusion theory.” which has been proposed for the regulation of the glutamic dehydrogenase. It has been shown that the active site and the ADP-binding effector site are oriented close to each other on the enzyme. Furthermore, the data suggest that the adenylic site is not identical to the nonactive coenzyme binding site. A mechanism based on electrostatic interactions is suggested for the cooperative binding of oxidized coenzyme and substrate. Dissociation constants for complexes between the enzyme and two coenzyme fragments (P-ADPR and 2′,5′-ADP) have been estimated.     

A spectroscopic study of metal ion and ligand binding to β-lactamase II

β-Lactamase II has two metal-binding sites. The electronic spectra of Cd(II)- and Co(II)-substituted β-lactamase II have been investigated. It is suggested that a thiol ligand is involved in metal binding at the first site. The stoichiometric dissociation constants for Co(II) binding to β-lactamase II were estimated to be 0.13 and 2.66 mM (pH 6.0, 4°C, 1 M NaCl) by equilibrium dialysis. Competition between Zn(II) and Co(II) for the first metal binding site suggests a value of 0.7 μM (pH 6.0, 30°C, 1 M NaCl) for the dissociation constant o Zn(II).The electronic spectra of the Co(II) enzyme lead to the suggestion that the coordination geometries around the metal ions in the first and second sites are related to those of a distorted tetrahedron and octahedron, respectively.