Human N-acetylgalactosamine-4-sulphatase: protein maturation and isolation of genomic clones

N-Acetylgalactosamine-4-sulphatase (EC 3.1.6.1, G4S) is composed of a 57 kDa species in human liver that dissociates into 43 kDa and 8 kDa subunits under reducing conditions and, when deficient, causes the lysosomal storage disorder, mucopolysaccharidosis type VI. We isolated genomic clones containing the G4S first exon, including the leader peptide and the amino terminus of the 43 kDa polypeptide. Amino-terminal amino acid sequences of the 43 kDa and 8 kDa subunits indicated that the 8 kDa component is linked to the 43 kDa polypeptide by a single disulphide bond, does not contain the mannose-6-phosphate lysosomal targeting signal and is at the carboxyl terminus of G4S.     

Mutants of Rhodobacter sphaeroides lacking one or more pigment-protein complexes and complementation with reaction-centre, LH1, and LH2 genes

The photosynthetic apparatus of Rhodobacter sphaeroides is comprised of three types of pigment-protein complex: the photochemical reaction centre and its attendant LH1 and LH2 light-harvesting complexes. To augment existing deletion/insertion mutants in the genes coding for these complexes we have constructed two further mutants, one of which is a novel double mutant which is devoid of all three types of complex. We have also constructed vectors for the expression of either LH1, LH2 or reaction-centre genes. The resulting system allows each pigment-protein complex to be studied either as part of an intact photosystem or as the sole complex in the cell. In this way we have demonstrated that reaction centres can assemble independently of either light-harvesting complex in R. sphaeroides. In addition, the isolation of derivatives of the deletion/insertion mutants exhibiting spontaneous mutations in carotenoid biosynthesis provides an avenue for examining the role of carotenoids in the assembly of the photosynthetic apparatus. We show that the LH1 complex is assembled regardless of the carotenoid background, and that the type of carotenoid present modifies the absorbance of the LH1 bacteriochlorophylls.     

Positive identification of a lambda gt11 clone containing a region of fungal phytase gene by immunoprobe and sequence verification

Summary As the initial step in a project to provide a more cost-effective source of the phytase enzyme, this paper reports on the use of a polyclonal antibody raised to phytase purified from an isolate of Aspergillus niger (A. ficuum) to screen an A. niger lambda gt11 expression library and the use of amino acid sequencing to identify a clone containing part of the fungal phytase gene. The described use of amino acid sequence fragments to verify the cloning of a gene has potential applications in other cloning projects.Mention of a trademark or proprietary product does not constitute a guarantee or warranty by the U.S. Department of Agriculture and does not imply approval to the exclusion of other products that may also be suitable Offprint requests to: E. Mullaney     

Cytosolic free calcium concentrations in synaptosomes during histotoxic hypoxia

Altered cytosolic free calcium concentrations ([Ca²⁺]_i) accompany impaired brain metabolism and may mediate subsequent effects on brain function and cell death. The current experiments examined whether hypoxia-induced elevations in [Ca²⁺]_i are from external or internal sources. In the absence of external calcium, neither KCl depolarization, histotoxic hypoxia (KCN), nor the combination changed [Ca²⁺]_i. However, with external CaCl₂ concentrations as small as 13 M, KCl depolarization increased [Ca²⁺]_i instantaneously while hypoxia gradually raised [Ca²⁺]_i. The combination of KCN and KCl was additive. Increasing external calcium concentrations up to 2.6 mM exaggerated the effects of K⁺ and KCN on [Ca²⁺]_i, but raising medium calcium to 5.2 mM did not further augment the rise. Diminishing the sodium in the media, which alters the activity and perhaps the direction of the Na/Ca exchanger, reduced the increase in [Ca²⁺]_i due to hypoxia, but enhanced the KCl response. The changes in ATP following K⁺ depolarization, KCN or their combination in the presence of physiological calcium concentrations did not parallel alterations in [Ca²⁺]_i, which suggests that diminished activity of the calcium dependent ATPase does not underlie the elevation in [Ca²⁺]_i. Valinomycin, an ionophore which reduces the mitochondrial membrane potential, elevated [Ca²⁺]_i and the effects were additive with K⁺ depolariration in a calcium dependent manner that paralleled the effects of hypoxia. Together these results suggest that hypoxia-induced elevations of synaptosomal [Ca²]_i are due to an inability of the synaptosome to buffer entering calcium.     

Location and sequence of the todF gene encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1

The gene (todF) encoding 2-hydroxy-6-oxohepta-2,4-dienoate hydrolase in Pseudomonas putida F1 was shown to be located upstream of the todC1C2BADE genes. The latter form part of the tod operon and encode the enzymes responsible for the initial reactions in toluene degradation. The nucleotide (nt) sequence of todF was determined and the deduced amino acid (aa) sequence revealed that the hydrolase contains 276 aa with a Mr of 30,753. The deduced aa sequence was 63.5% homologous to that reported for 2-hydroxymuconic semialdehyde hydrolase which is involved in phenol degradation by Pseudomonas CF600.     

Bacterial oxidation of chemical carcinogens: formation of polycyclic aromatic acids from benz[a]anthracene.

A Beijerinckia strain designated strain B1 was shown to oxidize benz[a]anthracene after induction with biphenyl, m-xylene, and salicylate. Biotransformation experiments showed that after 14 h a maximum of 56% of the benz[a]anthracene was converted to an isomeric mixture of three o-hydroxypolyaromatic acids. Nuclear magnetic resonance and mass spectral analyses led to the identification of the major metabolite as 1-hydroxy-2-anthranoic acid. Two minor metabolites were also isolated and identified as 2-hydroxy-3-phenanthroic acid and 3-hydroxy-2-phenanthroic acid. Mineralization experiments with [12-14C]benz[a]anthracene led to the formation of 14CO2. These results show that the hydroxy acids can be further oxidized and that at least two rings of the benz[a]anthracene molecule can be degraded.     

The proton pore in the Escherichia coli F0F1-ATPase: substitution of glutamate by glutamine at position 219 of the alpha-subunit prevents F0-mediated proton permeability

Three mutations in the uncB gene encoding the a-subunit of the F0 portion of the F0F1-ATPase of Escherichia coli were produced by site-directed mutagenesis. These mutations directed the substitution of Glu-219 by Gln, or of Lys-203 by Ile, or of Glu-196 by Ala. Strains carrying either the Lys-203 or Glu-196 substitutions showed growth characteristics indistinguishable from the coupled control strain. Properties of membrane preparations from these strains were also similar to those from the coupled control strain. The substitution of Glu-219 by Gln resulted in a strain which was unable to utilise succinate as sole carbon source and had a growth-yield characteristic of an uncoupled strain. Membrane preparations of the Glu-219 mutant were proton impermeable and the F1-ATPase activity was inhibited by about 50% when membrane-bound. The results are discussed with reference to a previously proposed intramembranous proton pore involving subunits a and c.     

Testing the two-state model: anomalous effector binding to human hemoglobin

Three allosteric states are required to describe the relaxation of (carbon monoxy) hemoglobin following flash photolysis. Combined absorbance and fluorescence probes were used. The absorbance signals consist of a component corresponding to ligand recombination and a component for the R-T transition. The fluorescence of 8-hydroxy-1,3,6-pyrenetrisulfonate (HPT), an analogue of 2,3-diphosphoglycerate, shows rates and amplitudes correlated with the absorbance transients. Measurements were made at pII 6, 6.5, and 7.0 at CO partial pressures of 0.1 and 1 atm. The fractional photolysis was varied in each case to change the initial distribution of the R states. Data show an immediate absorbance change due to ligand dissociation, while the changes in the ligand isosbestic and the fluorescence signals occur with time constants of 80 microseconds (at pH 6.5). The signals then show a biphasic return to equilibrium, characteristic of the allosteric system. The measurements provide three independent probes of the kinetics of the substates of hemoglobin. Although the ligand binding data can be generally represented by a two-state model, the fluorescence data require T states with different affinities for HPT.     

In vitro metabolism of apolipoprotein E

Apolipoprotein E plays a major role in the uptake of chylomicrons and of very-low-density lipoprotein (VLDL) remnants by the liver. It has also been clearly demonstrated that apolipoprotein E rapidly and spontaneously exchanges between lipoproteins. To assess whether all lipoprotein-bound apolipoprotein E is available to participate in spontaneous transfer and/or exchange, the present study followed the fate of radiolabeled apolipoprotein E in an in vitro system. The results show that in vitro, apolipoprotein E can be considered as having both a spontaneously exchangeable pool and a nonexchangeable pool. Based upon specific radioactivity data, only a limited amount of apolipoprotein E originating in VLDL or in high-density lipoproteins (HDL) was capable of in vitro exchange with that in other lipoprotein fractions. Lipolysis of VLDL triacylglycerol by milk lipoprotein lipase, however, resulted in complete transfer of VLDL apolipoprotein E mass and radioactivity to HDL, supporting the potential for transformation of exchangeable apolipoprotein to a transferable pool in vivo. The results of these studies indicate that during the course of lipoprotein metabolism, conformational changes occur which alter the accessibility of apolipoprotein E. Such dynamic heterogeneity may have implications for the regulation of lipoprotein metabolism.