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.     

Stimulation of glycosaminoglycan sulfotransferase from chick embryo cartilage by basic proteins and polyamines

A soluble glycosaminoglycan sulfotransferase (3'-phosphoadenylylsulfate:chondroitin 4'-sulfotransferase, EC 2.8.2.5) from chick embryo cartilage has been prepared free from endogenous acceptor. The reaction with this enzyme preparation was stimulated by basic proteins and polyamines, the degree of stimulation being dependent on the chemical nature of both basic compounds and acceptor glycosaminoglycans. A maximum stimulation was obtained when protamine (basic compound) and chondroitin (acceptor) were involved in the reaction mixture at a molar ratio of protamine to repeating disaccharide units of chondroitin, 1:100. The stimulation of sulfotransferase activity by basic substances was much higher than that by Mn2+. However, increasing the Mn2+ concentration immediately reduced the stimulation by basic substances. The Km value for 3'-phosphoadenosine 5'-phosphosulfate of the sulfotransferase, when chondroitin was used as acceptor, was 1 . 10(-6) M in the presence of 25 microgram/ml protamine, compared to 2 . 10(-5) M in the absence of protamine. These observations indicate that the basic proteins and polyamines may interact with acceptor polysaccharide, thereby causing an increase in the affinity of the enzyme toward 3'-phosphoadenosine 5'-phosphosulfate.     

The seminiferous epithelium in the guinea fowl (Numida meleagris)

Summary Spermiogenesis and cellular associations in the seminiferous epithelium of the guinea fowl were studied and described in sexually active adult birds. PAS stain was found to be useful in the recognition of steps of spermatid differentiation only in the first early stages. Nuclear morphological changes were subsequently found to be more reliable in tracing steps of spermiogenesis. It was observed that haematoxylin-eosin stained tissue can be used in the study of spermiogenesis in the bird. Various stages of the seminiferous epithelium were observed in any cross-section of the seminiferous tubules. Distinct cellular associations were observed, but intermix of adjacent germ cells or heterogenous cellular associations were frequently encountered.     

Isolation and reconstitution of two electron transfer components of tryptophan side chain oxidase.

The isolation and reconstitution of two electron transfer components of tryptophan side chain oxidase from Pseudomonas (ATCC 29574) are described. The dehydrogenase component abstracts electrons from the substrate and transfers them to oxidation-reduction dyes such as potassium ferricyanide and 2,6-dichlorophenolindophenol but not to molecular oxygen. It is composed of a single polypeptide chain with a molecular weight of 72,000 and exhibits the absorption spectrum of a reduced b-type cytochrome with maxima at 563, 532, 433, 323, and 278 nm. The oxidase component transfers electrons, derived from the former component, to oxygen, and has a molecular weight of 48,000. The absorption spectrum exhibits broad peaks at 680, 438, and 358 nm, and a peak at 280 nm. On sucrose gradient centrifugation and polyacrylamide gel electrophoresis, these two components are shown to form a molecular complex, which has the reconstituted oxidase activity. The turnover number of the reconstituted enzyme is comparable to that of the native enzyme.     

Histamine-releasing activity (HRA). I. Production by mitogen- or antigen-stimulated human mononuclear cells.

Supernatants from 1- to 2-day cultures of human mononuclear cells induced the release of histamine from basophils. Generation of this histamine-releasing activity (HRA) was stimulated by addition of concanavalin A to the cell cultures. Mononuclear cells were also cultured with SKSD and Candida albicans antigens. Stimulation of HRA production by these antigens was correlated with positive delayed skin reactions. Serial dilutions of supernatants assayed for HRA provided a semiquantitative determination of the level of HRA in mitogen- or antigen-stimulated samples. Antigen increased HRA production when added during the first or second day of culture. Generation of HRA probably requires active protein synthesis, since puromycin was inhibitory, and since preformed HRA could not be recovered from lysed cells. HRA was detected in supernatants after 4 hr, and the effects of antigen stimulation were apparent after 8 hr of culture. Replacement of supernatants with fresh culture medium allowed continued synthesis of substantial quantities of HRA during the second day of culture. A linear correlation was observed between the amount of HRA produced and the mononuclear cell concentration. Our findings provide evidence for the interaction of lymphocytes and basophils via a soluble mediator.     

DEVELOPMENT OF ACETYLCHOLINESTERASE MULTIPLE MOLECULAR FORMS IN CHICKEN MUSCLES

Abstract— AChE activity and protein content in chicken ALD and PLD muscles was studied during pre- and postnatal development. Protein content in both muscles increased whereas AChE activity increased in ALD and decreased in PLD during development. All studied values reached the steady-state 3 weeks after hatching.
Electrophoretic separation of the samples showed three molecular forms of AChE present in both adult ALD and PLD muscles. Two molecular forms in ALD muscle increased slowly, one form quickly. On the other hand, the activity of AChE forms in PLD muscle decreased with different rates. It appears from these results that the multiple molecular forms of AChE in muscles are not of the same physiological importance.     

A new type of semi-empirical molecular orbital method for large molecules.

A new type of molecular orbital method is proposed. It is applicable to large molecules containing large conjugated substructures. Only π-electrons in the conjugated part, but all-valence electrons in the non-conjugated part of a molecule, are taken into account explicitly. The Fock matrix elements are evaluated from the semi-empirical values employed in the existing all-valence-electron methods. The examples presented here suggest that the new type of MO method predicts electronic structures which are quite similar to those obtained by complete semi-empirical MO calculations. This new method may make it possible to reasonably well describe the electronic structure of, and interaction between, large molecules using considerably less computation time and core storage than the complete calculation analogs.