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Biomedical subjects

M J Adams

Publications and source records attributed to M J Adams.

12 recordsLinked to original sources

Sheep 6-phosphogluconate dehydrogenase. Revised protein sequence based upon the sequences of cDNA clones obtained with the polymerase chain reaction.

Sheep liver 6-phosphogluconate dehydrogenase (6-PGDH) is an enzyme of the pentose phosphate pathway. Evidence has appeared which suggests that the 6-PGDH protein sequence determined previously by direct analysis of the protein isolated from ovine liver is incorrect. Determining the enzyme's DNA sequence was considered to be the best way of solving the problem. In the first instance, a degenerate forward and a degenerate reverse primer were designed on the basis of the known protein sequence, and a partial-length cDNA clone was isolated from total sheep liver cDNA using the polymerase chain reaction. The clone encoded the expected part of the protein sequence. The clone was unsuccessfully used as a prime-cut probe to screen a sheep liver library and a bovine heart library. As a result, the polymerase chain reaction was utilized again to successfully generate a family of overlapping cDNA clones encoding a mature protein of 482 amino acids. The mature protein sequence encoded by the cDNA differs significantly from the sequence derived by direct analysis of the protein, but on closer examination the fundamental difference is caused by the incorrect placement of three enzyme fragments obtained by cyanogen bromide cleavage during the direct sequence analysis of the protein. Placing the fragments in the correct order results in the two sequences being virtually identical except for some minor amino acid changes between the amide and acid forms, and a small number of deletions and insertions.

Amino Acid Sequence

Community-based projects for the prevention of developmental disabilities.

Community projects are a central feature of the Centers for Disease Control's (CDC's) Disabilities Prevention Program. Twenty-eight states now have such programs, and they use community projects to stimulate local involvement in disability prevention and as a setting to evaluate prevention needs and preventive interventions.

Child

The structure of 6-phosphogluconate dehydrogenase refined at 2.5 A resolution.

The three-dimensional structure of ovine 6-phosphogluconate dehydrogenase, refined at 2.5 A resolution with a residual for all data of 18.5%, is reported. This model, based on improved diffraction data and a corrected sequence, supersedes that reported earlier. Each subunit of the dimer has three domains: a beta-alpha-beta domain binds NADP; an all alpha domain provides much of the dimer interface; the C-terminal tail burrows into the second subunit.

Molecular Structure

Barley yellow mosaic virus (BaYMV) and BaYMV-M: two different viruses.

Serological and physical properties of isolated particles as well as reactions of barley cultivars after virus inoculation showed that the yellow disease of barley is caused by different viruses. Results summarized in this paper suggest that the European isolates BaYMV-So and BaYMV-NM are closely related to, or identical with, BaYMV-J first reported from Japan, whereas BaYMV-M differs in several properties. Therefore, it seems justified to describe BaYMV-M as a separate virus tentatively designated barley mild mosaic virus.

Edible Grain

Identification of haptoglobin alpha-2FF variants in mid-trimester maternal serum as potential markers for Down syndrome.

Exploratory protein analysis in mid-trimester maternal sera by 2-dimensional electrophoresis and image analysis was performed to determine the differences between mothers carrying fetuses with Down syndrome (DS) and control mothers. Nine haptoglobin alpha-1 and alpha-2 (Hp1 and Hp2) variants were detected. Three apparent isoforms of the Hp2FF protein having the same relative charge but different Mr's were detected, with the two smaller variants--Hp2FF (-0.7k) and Hp2FF (-1.3k)--believed to be first reported here. Ninety-two percent of the cases (n = 83) had the Hp2FF (-0.7k) protein, and 61% had Hp2FF (-1.3k). The geometric mean concentration of Hp2FF (Mr = 18.0 kd) was found to be significantly increased (P less than 0.01) in mid-trimester sera from cases (n = 27) compared to controls (n = 56). When the concentrations of the three Hp2FF isoforms were added, the cases had larger sums on average than controls (P less than 0.01).

Age Factors

Binding of coenzyme and substrate and coenzyme analogues to 6-phosphogluconate dehydrogenase from sheep liver. An X-ray study at 0.6 nm resolution.

The analogues of the coenzyme NADP+, nicotinamide--8-bromo-adenine dinucleotide phosphate (Nbr8ADP+) and 3-iodopyridine--adenine dinucleotide phosphate (io3PdADP+), were prepared. Nbr8ADP+ was found to be active in the hydrogen transfer adn io3PdADP+ is a coenzyme competitive inhibitor for 6-phosphogluconate dehydrogenase. The binding of NADP+, NADPH and NADPH together with 6-phosphogluconate as well as that of both analogues to crystals of the enzyme 6-phosphogluconate dehydrogenase has been investigated at 0.6-nm resolution using difference electron density maps. The molecules bind in a similar position in a cleft in the enzyme subunit distant from the dimer interface. The orientation of the coenzyme in the site has been determined from the io3PdADP+ -NADP+ difference density. The ternary complex difference density extends beyond that of the nicotinamide moiety of the coenzyme and tentatively indicates substrate binding. No clear identification of the bromine atom of Nbr8ADP+ can be made. However, the analogue is bound more deeply in the cleft than is NADP+. The NADPH density is the most clearly defined and has thus been used to fit a molecular model using an interactive graphics system, checking for preferred geometry. A possible conformation is presented which is significantly different from that of NAD+ in the lactate dehydrogenase ternary complex.

Animals

An examination of potential matrices for the affinity chromatography of NADP+-linked dehydrogenases with special reference to 6-phosphogluconate dehydrogenase.

1. 6-phosphogluconate dehydrogenase from sheep liver has been purified 350-fold by affinity chromatography with a final specific activity of 18 micronmol of NADP+/reduced min per mg of protein and an overall yield of greater than 40%. 2. A systematic investigation of potential ligands has been carried out: these included 6-phosphogluconate and NADP+, pyridoxal phosphate and several immobilized nucleotides. The results indicate that NADP+ is the most suitable ligand for the purification of 6-phosphogluconate dehydrogenase. 3. The effects of pH and alternative eluents have been examined in relation to the parameters known to affect the desorption phase of affinity chromatography; careful manipulation of the elution conditions permitted the separation of glucose 6-phosphate dehydrogenase, glutathione reductase and 6-phosphogluconate dehydrogenase from sheep liver on NADP+-Sepharose 4B. 4. A large-scale purification scheme for 6-phosphogluconate dehydrogenase is presented that uses the competitive inhibitors inorganic pyrophosphate and citrate as specific eluents.

Animals

A two-step purification procedure for sheep liver 6-phosphogluconate dehydrogenase.

A two-step procedure for the purification of 6-phosphogluconate dehydrogenase (EC 1.1.1.44; 6-PGDH) from sheep liver is described. The enzyme is directly bound to cellulose phosphate by batch extraction and eluted with a linear salt gradient. Purification is completed by affinity chromatography using NADP(+)-agarose. The result is 6-PGDH of high purity, greatly increased yield, and the highest specific activity yet achieved, with a significant reduction in the purification time.

Animals

Modification toward dominance of a recessive lethal in the mouse.

The effect of the recessive siren (srn) gene singly in the presence of one Danforth's short tail (Sd) gene was to modify the expression of srn towards dominance with 46 percent penetrance. All mice that could be classified as sirens from srn/+ x Sd/+ matings had the Sd type short tail, indicating that interaction between Sd and srn was the principal cause for modification towards dominance of srn. External expressivity was variable in Sd srn pups, with true sirens, semi-sirens and apposed leg pups produced, each traceable to pelvic abnormalities. Variation in the urogenital system was similar to that found in siren pups with the srn/srn genotype.

Animals