Cobalt(II)-substituted class III alcohol and sorbitol dehydrogenases from human liver

The catalytic zinc atoms in class III (chi) alcohol dehydrogenase (ADH) and sorbitol dehydrogenase (SDH) from human liver have been specifically removed and replaced by cobalt(II) with a new ultrafiltration technique. The electronic absorption spectrum of class III cobalt ADH (epsiolon 638 = 870 M-1 cm-1) is nearly identical with those of active site substituted horse EE and human class I (beta 1 beta 1) cobalt ADH. Thus, the coordination environment of the catalytic metal is strictly conserved in these enzymes. However, significant differences are noted when the spectra of class III ADH-coenzyme complexes are compared to the corresponding spectra of the horse enzyme. The spectrum of class III ADH.NADH is split into three bands, centered at 680, 638, and 562 nm. The class III ADH.NAD+ species resembles the alkaline form of the corresponding horse enzyme complex but without exhibiting the pH dependence of the latter. These spectral changes underscore the role of the coenzymes in differentially fine tuning the catalytic metal for its particular function in each ADH. The noncatalytic zinc of class III ADH exchanges with cobalt at pH 7.0. While 9 residues out of 15 in the loop surrounding the noncatalytic zinc of class III ADH differ from those of the class I ADH, the electronic absorption spectra of cobalt in the noncatalytic metal site of class III ADH establish that the coordination environment of this site is conserved as well. The spectrum of cobalt SDH differs significantly from those of cobalt ADHs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Alcohol dehydrogenase genes: restriction fragment length polymorphisms for ADH4 (π-ADH) and ADH5 (χ-ADH) and construction of haplotypes among different ADH classes

Of the five human alcohol dehydrogenase (ADH) genes located in the region q21–25 of chromosome 4, genetic markers have been reported previously only for class I enzymes, ADH1-3. Here, new restriction fragment length polymorphisms (RFLPs) are described for the genes of two other classes, ADH4 () and ADH5 ( or formaldehyde dehydrogenase, FDH). The frequencies and modes of inheritance of these RFLPs were determined with DNA both from unrelated individuals and from families. A polymorphic PstI site is assigned to the fourth intron of the ADH4 gene. Pairwise linkage disequilibrium calculations for these new RFLPs and already known RFLPs at the ADH2 and ADH3 loci establish strong linkage disequilibria between polymorphic MspI and BstXI sites in the ADH5 gene as well as between XbaI and MspI sites in the ADH3 gene. Furthermore, linkage disequilibria were detected between RFLPs of the ADH2 and ADH3 genes as well as between those of the ADH4 and ADH5 genes. The latter disequilibrium implies a hitherto unknown physical proximity of two genes belonging to different ADH classes. The RFLPs were used to construct chromosomal haplotypes that include three ADH classes. Of the 16 possible haplotypes for four RFLP markers used here, 10 were experimentally detected. The potential application of the ADH RFLPs and haplotypes in linkage or association studies of inherited diseases such as familial alcoholism is discussed.     

Influence of anions and pH on the conformational change of horse liver alcohol dehydrogenase induced by binding of oxidized nicotinamide adenine dinucleotide: binding of chloride to the catalytic metal ion

The conformational change of horse liver alcohol dehydrogenase induced by binding of NAD+ was studied by electronic absorption spectroscopy using cobalt as a spectroscopic probe in the active site. The complex of the enzyme with NAD+ exists in an acidic and an alkaline form. The transition between the two forms proceeds through several intermediates and is controlled by an apparent pKa of 6.9. Only at pH values below this pKa can a complex between enzyme, NAD+, and Cl- be formed. The spectral changes indicate that chloride displaces the cobalt-bound water molecule in a tetracoordinate structure. We conclude that a negative charge at the active site is necessary to stabilize the closed conformation of the enzyme in the presence of NAD+. Spectral correlations are given which strongly support the postulation of a metal-bound alkoxide in the closed structure of the enzyme as an essential feature of the catalytic mechanism of horse liver alcohol dehydrogenase.     

Binding of ligands to horse liver alcohol dehydrogenase lacking zinc ions at the active sites

The zinc-deficient enzyme binds the fluorescence probes for the enzyme substrate pocket (auramine O, 13-ethylberberine, chlorprothixene and acridine orange) more tightly than the native enzyme, whereas 1-anilinonaphthalene 8-sulphonic acid is bound with comparable affinity. The use of fluorescence probes as reporter ligands revealed that the formation of binary complexes between the zinc-deficient enzyme and aldehydes is possible (as with the native enzyme) and confirmed an increased affinity of coenzymes to the modified enzyme. The absence of catalytic zinc ions brings about a loss of the essential stabilization effect in simultaneous NADH and aldehyde binding to liver alcohol dehydrogenase. 2,2'-Bipyridine, which chelates the active-site zinc ion in the native enzyme, is bound rather loosely to the same site as aldehydes, auramine O and ethylberberine in the case of the zinc-depleted enzyme. The stopped-flow measurements showed that the pH dependence of auramine O and ethylberberine binding to native and zinc-depleted enzyme is essentially similar. These data are compatible with the presence of ionizable groups in the surroundings of the bound probes. This group might be either His-67, bound to the zinc ion, or the zinc-liganding water molecule in the case of the native enzyme (pK close to 9), or the free His-67 residue in the case of the zinc-deficient enzyme (pK about 8).     

Validity of lymphoid cell line for enzymatic studies of GM2-gangliosidosis variant 0 (Sandhoff disease)

A lymphoid cell line established by Epstein-Barr virus (EBV)-transformation of peripheral blood B-lymphocytes from a patient with Sandhoff disease showed a severe deficiency of beta-N-acetylhexosaminidase activity (residual activity around 10% of that in lymphoid cell lines from normals or other lipidotic patients). This residual beta-N-acetylhexosaminidase was completely heat-labile in contrast to that of normals. The molecular forms of residual beta-N-acetylhexosaminidase from Sandhoff lymphoid cell line were separated by Con A-sepharose and electrofocusing. Their properties and electrofocusing profiles were compared to those from Sandhoff fibroblasts and from fetal brain: this comparison permitted to identify the residual molecular forms with Hex S and Hex C. The microheterogeneity of Hex S and Hex C, demonstrated by electrofocusing, was discussed. 2-Acetamido-2-deoxy-D-galactonolactone (GalNAcLone) showed a strong inhibitory effect on lysosomal Hex A, B and S, but only a very slight effect on Hex C. Studies of the inhibition type (competitive on Hex A, B and S and mixed on Hex C) gave some informations about the enzymatic site. Elsewhere, differences in affinity of GalNAcLone for the various isoenzymes could be utilized to define optimal assay conditions for specifically determining Hex C (standard assay containing 400 mumol/l of GalNAcLone). These results demonstrated that EBV-transformed lymphoid cell lines represent an accurate model system for enzymatic studies of Sandhoff disease.     

Pinocytotic uptake of protein from the reservoir inEuglena

Alveolate vesicles in the anterior regions ofEuglena gracilis are shown to function in pinocytosis, being capable of incorporating exogenous horseradish peroxidase through the reservoir wall. It is thought that this represents a mechanism for recovering large molecules otherwise lost during contractile vacuole discharge.     

THE FINE STRUCTURE OF TWO PHOTOSYNTHETIC SPECIES OF DINOPHYSIS (DINOPHYSIALES,DINOPHYCEAE)1

Two closely related, photosynthetic species belonging to the genus Dinophysis were examined, D. acuminata Claparède et Lachmann and D. fortii Pavillard. Typical dinoflagellate features include the amphiesmal covering enclosing the cells and the structure of the nucleus and mitochondria. Many other characteristics seem to be specific to the order Dinophysiales. Many rhabdosomes are present, and complex mucocysts are found beneath the amphiesma. The thecal pores are unusual with the base of the pore occluded by a thin disc that is continuous with the main amphiesmal plate. The structure of the apical pore is also distinctive. Chloroplasts are grouped together in chromatospheres, enclosed by a double membrane, and contain paired thylakoids with electron dense contents in the lumen. The two pusules are extensive, each branching off the flagellar canal, and consisting of a large antechamber and a number of convoluted sacs. The entrance of each antechamber, and site of an emerging flagellum, is surrounded by a striated fibrous collar. Near the flagellar pore is a prominent microtubular/microbody complex which penetrates deep into the cell cytoplasm. Consideration is given to taxonomic position of the Dinophysiales and also to the nature and origins of the chloroplasts.     

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.     

Biochemical and ultrastructural findings in Epstein-Barr virus-transformed lymphoid cell lines from type 1 Gaucher disease

Lymphoid cell lines established by Epstein-Barr Virus (EBV)-transformation of peripheral blood B-lymphocytes from patients affected with type 1 Gaucher disease showed a severe deficiency of glucosylceramide-beta-glucosidase activity (residual activity around 15%-30% of control activity). Ultrastructural investigations showed, in these lymphoid cell lines from type 1 Gaucher disease, the presence of numerous membrane-bound inclusion bodies characteristic of Gaucher cells.