Functional groups and quaternary structure of chicken liver xanthine dehydrogenase

Xanthine dehydrogenase from chicken liver is a dimeric enzyme, each hemimolecule containing one FAD and two Fe/S groups. Determination of sulfhydryl groups with 5,5-dithiobis(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuribenzoic acid (PMB) showed a variable number of sulfhydryl groups depending onpH, ionic strength, and nature of the reaction medium and buffer. The number of disulfide bonds was determined with DTNB and reducing conditions. Amino groups were determined with 2,4,6,-trinitrobencensulfonic acid (TNBS). At constant temperature andpH the reaction of DTNB and TNBS with native xanthine dehydrogenase showed an exponential dependence on time. From the obtained parameters the number of available sulfhydryl and amino groups at infinite concentration of enzyme and the rate constant of the equation were determined. The absorption spectrum of the enzyme changed with time when a chaotropic agent (1 M sodium nitrate) was added to the medium. This difference was detected by measuring the absorbance in the range 450–550 nm. The absorption spectrum (between 350 and 600 nm) also changed when a denaturating agent (sodium dodecyl sulfate) was added. This modification increased with time and depended on the medium.     

The Escherichia coli cytochrome b556 gene, cybA, is assignable as sdhC in the succinate dehydrogenase gene cluster

Abstract The cytochrome b556 -deficient mutant Escherichia coli K12 strain TK3D11 [7] could not grow with succinate as the sole carbon source, but could grow well on dl -lactate. This finding suggested that cytochrome b556 is primarily responsible for oxidative metabolism and utilization of succinate. 24 Amino acid residues at the amino-terminal of purified cytochrome b556 were determined. This sequence coincided completely with amino acid residues 4 to 27, predicted from the DNA sequence of the sdhC gene, one of the unassigned open reading frames of the sdh gene cluster recently reported by Wood et al. [16]. Based on these and other results, we concluded that cybA , the gene for cytochrome b556 , is assignable as sdhC .     

Relationship between carbon monoxide dehydrogenase and a small plasmid in Pseudomonas carboxydovorans

Abstract Isolation of plasmid DNA followed by plasmid curing was carried out to examine the relationship of plasmid to carbon monoxide dehydrogenase (CO-DH) production in carboxydobacteria. A small plasmid of almost identical size (1.52−1.76 × 10⁶) was present in Pseudomonas carboxydovorans, Azotobacter sp.1, and Azomonas sp.2. Azomonas sp.1 contained two kinds of plasmids (1.5 × 10⁶ and 2.47 × 10⁶). No plasmids were found in Pseudomonas carboxydohydrogena , JC1, and HY1. A plasmid-cured clone of P. carboxydovorans was obtained by growing the cells at 37°C. The cured cell was able to grow CO autotrophically on solid, but not in liquid, medium. CO-DH of the cured cell was active and consisted of three subunits similar to those found in the wild-type enzyme, with the exception that the β subunit of the enzyme was larger than that of the wild-type enzyme. These results suggest that the small plasmids do not carry genes encoding CO-DH but may have gene(s) for processing the β subunit of the enzyme.     

A photosystem other than PS370 also mediates the negative phototaxis of Halobacterium halobium

Abstract It was found that a photorepellent system other than photosystem 370 (PS370) also controls the behavior of Halobacterium halobium . Both the dependence of background illumination and wavelength where the response showed maximum action distinguished that photosystem from PS370 whose photoreceptor pigment is thought to be an intermediate of s-rhodopsin (sR). A mutant strain that has no detectable activity in PS370 and in photoattractant response was isolated. This mutant strain showed the repellent response due to the new photosystem.     

低温对不同耐寒力的黄瓜幼苗子叶的各细胞器中NAD~+-苹果酸脱氢酶的影响

NAD~+-MDH在黄瓜子叶中的定位是细胞溶质中占总活性的55～59％,线粒体为38～35％,叶绿体为7％。其同工酶谱亦为细胞溶质中带数最多,全青为5条,粤早3号为4条,线粒体和叶绿体均为1条,品种间无明显差异。黄瓜幼苗随低温胁迫的加剧,伤害逐步加重,子叶电解质渗出率明显增加,NAD~+-MDH活性亦不断下降,其中叶绿体的NAD~+-MDH对低温最敏感,1±1℃处理就能反映品种间耐寒力的差异。叶绿体和线粒体的NAD~+-MDH同工酶对低温的反应与活性变化一致,谱带数没有差异,只是活性降低。细胞溶质部分酶带较多,各条酶带对低温的反应不同。     

Distorted segregation and linkage of alcohol dehydrogenase genes in Camellia japonica L. (Theacease)

Alcohol dehydrogenase isozymes in Camellia japonica are encoded by two genes, Adh-1 and Adh-2. Both loci are expressed in seeds, and their products randomly associate into intragenic and intergenic dimers. Electrophoresis of leaf extracts reveals only the products of Adh-2. Formal genetic analysis indicated that the two Adh loci are tightly linked (combined estimate of r=0.004). Most segregations fit expected Mendelian ratios, but in some families distorted segregation was observed at Adh-1, Adh-2, or both loci. The deficient progeny class varied across families, and in two apparent backcrosses three rather than two phenotypic classes were recovered. The mechanism underlying these distortions is not known, but evidence is presented that suggests that the phenomenon is genic or segmental in nature. Plausible hypotheses include linkage of the Adh structural genes with a gametophytic self-incompatibility locus, translocation heterozygosity involving the segment bearing Adh-1 and Adh-2, or a combination of these two mechanisms.     

Structural evidence for ligand-induced sequential conformational changes in glyceraldehyde 3-phosphate dehydrogenase

Glyceraldehyde 3-phosphate dehydrogenase is a tetramer of four chemically identical subunits which requires the cofactor nicotinamide adenine dinucleotide (NAD) for activity. The structure of the holo-enzyme from Bacillus stearothermophilus has recently been refined using X-ray data to 2.4 A resolution. This has facilitated the structure determination of both the apo-enzyme and the enzyme with one molecule of NAD bound to the tetramer. These structures have been refined at 4 A resolution using the constrained-restrained parameter structure factor least-squares refinement program CORELS. When combined with individual atomic temperature factors from the holo-enzyme, these refined models give crystallographic R factors of 30.2% and 30.4%, respectively, for data to 3 A resolution. The apo-enzyme has 222 molecular symmetry, and the subunit structure is related to that of the holo-enzyme by an approximate rigid-body rotation of the coenzyme binding domain by 4.3 degrees with respect to the catalytic domains, which form the core of the tetramer. The effect of this rotation is to shield the coenzyme and active site from solvent in the holo-enzyme. In addition to the rigid-body rotation, there is a rearrangement of several residues involved in NAD binding. The structure of the 1 NAD enzyme is asymmetric. The subunit which contains the bound NAD adopts a conformation very similar to that of a holo-enzyme subunit, while the other three unliganded subunits are very similar to the apo-enzyme conformation. This result provides unambiguous evidence for ligand-induced sequential conformational changes in B. stearothermophilus glyceraldehyde 3-phosphate dehydrogenase.     

Distribution and properties of aldehyde dehydrogenase in regions of rat brain

Abstract: NAD-dependent aldehyde dehydrogenases (EC 1.2.1.3) were isolated from various subcellular organelles as well as from different regions of rat brain. The mitochondrial, microsomal, and cytosolic fractions were found to contain 40%, 28%, and 12%, respectively, of the total aldehyde dehydrogenase (5.28 ± 0.44 nmol NADH/min/g tissue) found in rat brain homogenate when assayed with 70 μ. M propionaldehyde at pH 7.5. The total activity increased to 17.3 ± 2.7 nmol NADH/min/g tissue when assayed with 5 m M propionaldehyde. Under these conditions the three organelles contained 49%, 23%, and 9%, respectively, of the activity. The enzyme isolated from cytosol possessed the lowest K _m. The molecular weight of the enzyme isolated from all three subcellular organelles was ∼100,000. Four activity bands were found by electrophoresis of crude homogenates, isolated mitochondria, or microsomes on cellulose acetate strips. Cytosol possessed just two of the forms. The total activity was essentially the same in homogenates obtained from cortex, subcortex, pons-medulla, or cerebellum. Further, the enzyme had the same molecular distribution and total activity in each of these four brain regions. Disulfiram was found to be an in vivo and in vitro inhibitor of the enzymes obtained from these brain regions. Mercaptoethanol, required for the stability of the enzyme, reversed the inhibition produced by disulfiram. The effect was greater for enzyme isolated from cytosol than from mitochondria. Calculations led to the prediction that aldehydes such as acetaldehyde are oxidized in cytosol.