Electron-nuclear double resonance spectroscopy of the desulfo-inhibited molybdenum(V) center in bovine milk xanthine oxidase

The "desulfo-inhibited" Mo(V) center of bovine milk xanthine oxidase has been investigated by electron-nuclear double resonance spectroscopy. Comparison of spectral data obtained from samples prepared with [1H4]ethylene glycol and with [2H4]ethylene glycol allowed assignment of proton resonance lines due to the methylene protons of the coordinated ethylene glycol (AH = 3.6 MHz). Deuterium resonance lines were observed with the deuterated sample (AD = 0.4 MHz). No spectral evidence was obtained for any weakly coupled nitrogen nuclei to the Mo center under a variety of conditions. Dissolution of the sample in D2O had little effect on the resonance lines centered about the proton Zeeman frequency, which shows they are not due to exchangeable protons and suggests the Mo center does not have contact with bulk solvent. A deuterium delta m = +/- 2 "forbidden" transition is observed at high radio-frequency power levels, which suggests either an exchangeable proton on a Mo ligand or a coordinated solvent. Weakly coupled, nonexchangeable proton lines are observed about the free proton frequency, which exhibit properties characteristic of alpha-protons. A number of arguments are presented to support the proposal that these protons originate from the C(1') and C(2') positions on the side chain of the molybdopterin cofactor.     

Morphological variation in Kellicottia longispina

The lengths of the body, the posterior spine and the three longest anterior spines were measured for 25 specimens of Kellicottia longispina from each of the eight lakes distributed from Imikpuk at Point Barrow, Alaska (latitude 71° 15) to Lake Washington (latitude 47° 38). Collections were available for more than two dates from six of the lakes. Temperature ranged from 1.2° to 18 °C. Mean lengths and ratios were examined in relation to latitude and temperature. Each population differed from the others in some aspect of absolute size, variability, or shape as expressed by the ratios of the dimensions. The population from Point Barrow is similar but not identical to Olofsson's var. heterospina.     

Astrotactin: a novel neuronal cell surface antigen that mediates neuron- astroglial interactions in cerebellar microcultures

A microculture system for mouse cerebellar cells has been used to identify an immune activity, raised in rabbits against postnatal cerebellar cells, that blocks neuron-glial interactions in vitro. In the presence of blocking antibodies, stable neuron-glial contacts did not form and neuronal induction of glial process outgrowth did not occur. Subsequently, neurons were randomly arranged in the cultures rather than organized along the arms of astroglia. We have named the immune activity that blocks neuron-astroglial interactions anti-astrotactin. Partial purification of the anti-astrotactin blocking antibodies was obtained by cellular absorption with PC12 cells, a clonal cell line which expresses both the N-CAM and NILE (Ng-CAM, L1) glycoproteins. Subsequent absorption with purified cerebellar granule cells, but not with astroglial cells, removed the blocking activity, suggesting that the antigen(s) bound by blocking antibodies are neuronal. Immunoprecipitation of [35S]methionine- or [3H]fucose-radiolabeled Triton extracts of early postnatal cerebellar cells showed that the unabsorbed antiserum recognized a large number of proteins. Among these were bands with apparent molecular masses of N-CAM (180 and 140 kD) and NILE (230 kD). After absorption of the immune serum with PC12 cells, the number of bands recognized by the antiserum was reduced to a prominent band at 100 kD and a diffuse smear of material between 80 and 90 kD. The prominent band at 100 kD was removed by subsequent absorption of the immune serum with granule cells, a step which removed the blocking activity in the cerebellar microculture assay. Further evidence suggests that the astrotactin activity is missing or defective on granule cells from the neurological mutant mouse weaver, an animal that suffers a failure of glial-guided neuronal migration. When anti-astrotactin Fab fragments were pre-absorbed with weaver cerebellar neurons and then tested in the functional assay of neuron-glial interactions, the immune blocking activity was not removed. In contrast, wild-type cerebellar neurons removed the anti-astrotactin blocking activity under the same conditions. Subsequently, when [3H]fucose-radiolabeled Triton extracts of weaver and normal cells were immunoprecipitated with whole or PC12-absorbed anti-astrotactin antiserum, the intensity of the band at 100 kD was reduced by 95% in weaver cells.     

pKa values of the 8 alpha-imidazole substituents in selected flavoenzymes containing 8 alpha-histidylflavins

Difference absorption spectroscopy as a function of pH is described as a probe to determine the pKa values of the 8 alpha-imidazole substituent in flavoenzymes containing 8 alpha-histidylflavin coenzymes. Reversible absorption difference spectra are observed in the pH range 5.5 to 8.5 when synthetic 8 alpha-imidazolyl-FMN is bound to the apoflavodoxins from Azotobacter vinelandii and from Clostridium pasterianum. The observed spectral perturbations of these two flavodoxin complexes follow a single proton ionization dependence with respective pKa values of 6.7 and 6.8. No pH-induced spectral perturbations were observed when 8 alpha-(N-CH3)-imidazolium FMN was bound to either flavodoxin. Similar approaches are described to determine the 8 alpha-imidazolyl pKa values of the 8 alpha-histidyl-FAD coenzyme of the cholesterol oxidases from Schizophyllum commune and from Gleocystidium chrysocreas. Previous work has shown the former enzyme contains an 8 alpha-N1-histidyl-FAD (W. C. Kenney et al. (1979) J. Biol. Chem. 254, 4689-4690) while experiments reported here show the latter enzyme also contains one 8 alpha-N1-histidyl-FAD per mole of enzyme. The pKa value for the 8 alpha-imidazole substituent on the flavin of S. commune cholesterol oxidase is 5.4 while that determined for the G. chrysocreas enzyme is 6.2. These results demonstrate that the pKa of the 8 alpha-imidazole substituent can be determined in enzymes containing an 8 alpha-histidylflavin, provided that the enzyme is stable in the pH range required to observe ionization. Furthermore it is shown this the pKa value can differ even on comparison of enzymes from different sources that catalyze the same reaction.     

Trehalose synthase of Mycobacterium smegmatis: purification, cloning, expression, and properties of the enzyme.

Trehalose synthase (TreS) catalyzes the reversible interconversion of trehalose (glucosyl-alpha,alpha-1,1-glucose) and maltose (glucosyl-alpha1-4-glucose). TreS was purified from the cytosol of Mycobacterium smegmatis to give a single protein band on SDS gels with a molecular mass of approximately 68 kDa. However, active enzyme exhibited a molecular mass of approximately 390 kDa by gel filtration suggesting that TreS is a hexamer of six identical subunits. Based on amino acid compositions of several peptides, the treS gene was identified in the M. smegmatis genome sequence, and was cloned and expressed in active form in Escherichia coli. The recombinant protein was synthesized with a (His)(6) tag at the amino terminus. The interconversion of trehalose and maltose by the purified TreS was studied at various concentrations of maltose or trehalose. At a maltose concentration of 0.5 mm, an equilibrium mixture containing equal amounts of trehalose and maltose (42-45% of each) was reached during an incubation of about 6 h, whereas at 2 mm maltose, it took about 22 h to reach the same equilibrium. However, when trehalose was the substrate at either 0.5 or 2 mm, only about 30% of the trehalose was converted to maltose in >or= 12 h, indicating that maltose is the preferred substrate. These incubations also produced up to 8-10% free glucose. The K(m) for maltose was approximately 10 mm, whereas for trehalose it was approximately 90 mm. While beta,beta-trehalose, isomaltose (alpha1,6-glucose disaccharide), kojibiose (alpha1,2) or cellobiose (beta1,4) were not substrates for TreS, nigerose (alpha1,3-glucose disaccharide) and alpha,beta-trehalose were utilized at 20 and 15%, respectively, as compared to maltose. The enzyme has a pH optimum of about 7 and is inhibited in a competitive manner by Tris buffer. [(3)H]Trehalose is converted to [(3)H]maltose even in the presence of a 100-fold or more excess of unlabeled maltose, and [(14)C]maltose produces [(14)C]trehalose in excess unlabeled trehalose, suggesting the possibility of separate binding sites for maltose and trehalose. The catalytic mechanism may involve scission of the incoming disaccharide and transfer of a glucose to an enzyme-bound glucose, as [(3)H]glucose incubated with TreS and either unlabeled maltose or trehalose results in formation of [(3)H]disaccharide. TreS also catalyzes production of a glucosamine disaccharide from maltose and glucosamine, suggesting that this enzyme may be valuable in carbohydrate synthetic chemistry.