Site-specific substituted cobalt(II) horse liver alcohol dehydrogenases. Preparation and characterization in solution, crystalline and immobilized state.

The specific substitution, using highly selective techniques, of catalytic and/or noncatalytic zinc ions by cobaltous ions in horse liver alcohol dehydrogenase (EC 1.1.1.1) has been studied with dissolved, crystalline and agarose-immobilised enzyme, in order to examine the effect of protein structure on the specificity of the metal exchange. The different binding sites can be clearly distinguished by the absorption spectra of their cobalt derivatives. In solution an anaerobic column chromatographic method made it possible to exchange half of the zinc in the enzyme by cobalt ions in a much shorter time than previous procedures. By raising the temperature in the exchange step, even the slowly exchanging zinc ions were substituted by cobalt, yielding products similar to cobalt alcohol dehydrogenases described earlier. Treatment of crystal suspensions of the enzyme with chelating agents (preferentially dipicolinic acid) gave an inactive protein with two zinc ions remaining bound. The enzyme could be reactivated by treatment of the crystalline protein with 5 mM zinc or cobaltous ions or by dialysis of dissolved inactive protein against 20 microM zinc or 1 mM cobaltous ions. Higher metal concentrations led to denaturation but the inactive protein could be crystallized from solution and then reactivated completely at higher metal concentrations. The preparation and absorption spectrum show that cobalt is bound specifically at the catalytic sites. Since metal substitution at these sites critically depends on the maintenance of the correct tertiary and quaternary structure, these must be preserved in the crystal lattice and partially altered in solution when the catalytic zinc ions are removed (or when excess of metal ions is applied), thus demonstrating the structure-stabilizing role of the catalytic metal ions. The enzyme immobilised on agarose, with unchanged content of active sites [Schneider-Bernl?hr et al. (1978) Eur. J. Biochem. 41, 475--484], was treated like the crystal suspensions. Although half of the zinc was removed, some activity remained. After reactivation with cobaltous ions, a loss of about 30% active sites was measured. Thus the apparently homogenous bound enzyme was rather heterogeneous in the properties of its catalytic metal binding sites. These results are taken as further proof for the dependence of the metal substitution on the proper tertiary and quaternary structure which is strained by multiple interactions in the covalently immobilised enzyme.     

The use of biochemical solid-phase techniques in the study of alcohol dehydrogenase. 1. Some studies on subunit interactions in alcohol dehydrogenase with subunits covalently bound to agarose

The EE and SS isozymes of horse liver alcohol dehydrogenase have been immobilized separately to weakly CNBr-activated Sepharose 4B. The resulting immobilized dimeric preparations lost practically all of their activity after treatment with 6 M urea. However, enzyme activity was regenerated by allowing the urea-treated Sepharose-bound alcohol dehydrogenase to interact specifically with either soluble subunits of dissociated horse liver alcohol dehydrogenase or soluble dimeric enzyme. The regeneration of steroid activity in the immobilized preparations after treatment of the bound S subunits with soluble E subunits seems to show that true reassociation of the enzyme had taken place on the solid phase, since only isozymes with an S-polypeptide chain are active when using 5 beta-dihydrotestosterone as substrate. The results presented in this paper indicate that immobilized single subunits of horse liver alcohol dehydrogenase are inactive and that dimer formation is a prerequisite for the enzymic activity.     

Effects in vitro of alloxan on the glucose metabolism of mouse pancreatic B-cells

To facilitate detailed studies of the B-cytotoxic action of alloxan we developed a model using isolated pancreatic islets of normal mice. An essential feature of this model is the low temperature employed during exposure to alloxan, which minimizes the degradation of the drug. The islets were incubated with alloxan for 30min at 4 degrees C and subsequently various aspects of their metabolism were studied. The O(2) consumption was measured by the Cartesian-diver technique. Islets exposed to 2mm-alloxan and control islets had the same endogenous respiration, whereas the O(2) uptake of the alloxan-treated islets was inhibited and that of the control islets stimulated when they were incubated with 28mm-glucose as an exogenous substrate. The islet glucose oxidation was estimated by measurement of the formation of (14)CO(2) from [U-(14)C]glucose at 37 degrees C. Compared with the controls, alloxan-treated islets showed a decrease in the glucose-oxidation rate in a dose-dependent manner. Pretreatment of the islets with 28mm-glucose for 30min at 37 degrees C completely protected against this effect, whereas preincubations at glucose concentrations below 16.7mm failed to exert any protective effect. The glucose utilization was estimated as the formation of (3)H(2)O from [5-(3)H]glucose. Alloxan (2mm) failed to affect islet glucoseutilization rate in the presence of either 2.8 or 28mm-glucose. In contrast, islets exposed to 5 or 10mm-alloxan exhibited lowered glucose utilization. It is concluded that in vitro alloxan has an acute inhibitory effect on the islet glucose metabolism, and that this action can be prevented by previous exposure to a high glucose concentration. The results are consistent with the idea that the B-cytotoxicity of alloxan reflects an interaction with intracellular sites involved in the oxidative metabolism of the B-cell.     

Surface glycoproteins of resting and activated human T lymphocytes

Summary This review summarizes some recent studies on the surface glycoproteins of human thymocytes and T lymphocytes. Purified cells were surface labeled by the galactose oxidase-NaB³H₄ or periodate-NaB³H₄ techniques. The radioactive membrane glycoproteins were separated by polyacrylamide slab gel electrophoresis and visualized by fluorography. Thymocytes and T lymphocytes show characteristic surface glycoprotein profiles which are easily distinguishable from those of the other main groups of human leukocytes. We observed specific changes in the surface glycoprotein patterns which correlate with the degree of maturation and functional activation of T cells. Surface molecules carrying T cell specific antigens were identified by immune-precipitation from lysates of surface labeled thymocytes and T lymphocytes using rabbit anti-human T cell antibodies. Finally we describe a leukocyte membrane glycoprotein which is a precursor of serum ₁ acid glycoprotein (orosomucoid).     

Effect of calcium ions on ethanol oxidation and drug glucuronidation in isolated hepatocytes.

The effect of extracellular Ca2+ concentration on ethanol oxidation and drug metabolism was studied in isolated rat hepatocytes. Both ethanol oxidation and drug glucuronidation showed similar dependence upon Ca2+, which was a stimulation of activity as Ca2+ was increased to physiological concentration, and inhibition at higher concentration.     

Prostaglandins contribute to the vasodilation induced by nicotinic acid

The significance of endogenously formed prostaglandins in the vasodilation induced by nicotinic acid (NIC) was investigated. The forearm venous plasma level of radioimmunoassayed PGE (R-PGE) and the forearm blood flow (FBF) were measured in 13 healthy male volunteers at rest and during infusion of NIC. Each subject was subsequently re-studied after pretreatment with the PG synthesis inhibitor, naproxen. In the absence of naproxen, NIC infusion resulted in an almost four-fold rise in the release of R-PGE and a 60% increase in FBF. Pretreatment with naproxen did not affect the basal release of R-PGE or the basal FBF but inhibited both the release of R-PGE and the increase in FBF following NIC. The data support the hypothesis that the vasolidating effect of NIC is largely dependent upon an increased vascular formation of PG.     

Clumping versus spacing out: Experiments on nest predation in fieldfares (Turdus pilaris)

Fieldfares (Turdus pilaris), which nest solitarily as well as in colonies, offer an opportunity to assess within one species the relative advantages of clumping versus spacing out. An experiment with artificial, egg-baited nests showed that predation in the absence of fieldfares was higher on clumped than on scattered nests. In another experiment, we tested whether artificial nests run a higher risk of predation near solitary fieldfare pairs than near fieldfare colonies. Nest predation was higher near solitary fieldfare pairs than outside and inside fieldfare colonies. The risk probably was higher outside than inside colonies. Communal antipredator attacks is the likely cause of reduced predation near colonies. Even solitary fieldfare pairs confer some degree of protection, because predation was lower at artificial nests near solitary fieldfare pairs than at nests without neighbouring fieldfares.     

Inside-out membrane vesicles isolated from spinach thylakoids.

Inside-out thylakoid vesicles have been separated from right-side-out material after press disruption of chloroplast lamellae. The sepration was obtained by partitionin an aqueous dextran-polyethylene glycol two-phase system, a method which utilizes differences in surface properties for separation of membrane particles. The isolated thylakoid vesicles showed the following inside-out properties: (1) light-induced reversible proton extrusion into the surrounding medium when supplied with the Photosystem II electron acceptor phenyl-p-benzoquinone; (2) a pH rise in the internal phase accompanying the external proton release, (3) sensitivity to trypsin treatment different from that of thylakoid membranes of normal orientation; (4) concave EF and convex PF freeze-fracture faces.     

Purification and characterization of a low molecular weight 1,4-beta-glucan glucanohydrolase from the cellulolytic fungus Trichoderma viride QM 9414

A low molecular weight 1,4-beta-glucan glucanohydrolase (endoglucanase) (1,4-(1,3;1,4)-beta-D-glucan 4-glucanohydrolase, EC 3.2.1.4) has been isolated from culture filtrates of the fungus Trichoderma viride QM 9414 by a two-step procedure of gel filtration and ion-exchange chromatography. The isolated enzyme appeared homogeneous upon polyacrylamide gel electrophoresis at pH 2.9, isoelectric focusing in a polyacrylamide gel slab, sedimentation equilibrium analysis and chromatography of the reduced and alkylated enzyme on a column of Sepharose 6B in 6 M guanidine - HCl. A molecular weight was calculated at approx. 20 000 and the isoelectric point was determined at pH 7.52. The purified enzyme was not a carbohydrate-containing protein.     

Cloning of murine transformed cell lines in suspension culture with efficiencies near 100%

Addition of 3 × 10⁶ thymus cells from either syngeneic, allogeneic or xenogeneic animals increases the cloning efficiencies of murine thymomas (EL-4, WC-2), B-lymphomas (McPC 1748, 38C-13), Abelson-virus transformed cell lines (F and K), mastocytomas (P815), myelomas (AbPC22, X63-AG8, 5563, MOPC 104 E, RFC 5, W 3469) and hybrids of myelomas and normal B-lymphocytes (Sp-1), all adapted to tissue culture, to near 100%. Thymus cells also increase the efficiencies of growth initiation in primary in vitro cultures of myeloma tumor cells (S117) transplanted in vivo, and of cells fused between the azaguanine-resistant X63-AG8 myeloma cell line and normal, LPS-stimulated B-lymphocyte blasts.