Purification of arogenate dehydrogenase from Phenylobacterium immobile

Phenylobacterium immobile, a bacterium which is able to degrade the herbicide chloridazon, utilizes for L-tyrosine synthesis arogenate as an obligatory intermediate which is converted in the final biosynthetic step by a dehydrogenase to tyrosine. This enzyme, the arogenate dehydrogenase, has been purified for the first time in a 5-step procedure to homogeneity as confirmed by electrophoresis. The Mr of the enzyme that consists of two identical subunits amounts to 69000 as established by gel electrophoresis after cross-linking the enzyme with dimethylsuberimidate. The Km values were 0.09 mM for arogenate and 0.02 mM for NAD+. The enzyme has a high specificity with respect to its substrate arogenate.     

Enzymatic function in crystals of delta 5-3-ketosteroid isomerase. Catalytic activity and binding of competitive inhibitors

Crystals of the steroid-metabolizing enzyme, delta 5-3-ketosteroid isomerase (EC 5.3.3.1) from Pseudomonas testosteroni, exhibit many enzymatic properties. Each enzyme subunit in the lattice binds a competitive inhibitor, progesterone, with the same stoichiometry (1:1) and affinity (KD = 6 X 10(-6) M) as the enzyme in solution. Another competitive inhibitor, 19-nortestosterone, competes with progesterone for the same binding sites in the crystal. The enzyme crystals catalyze the conversion of delta 5- to delta 4-ketosteroids, but because the enzyme is so efficient, and substrate diffusion into the crystal is so slow, substrate cannot penetrate deeply into the crystal before being converted to product. A general theoretical formulation is presented to account for the effects of substrate diffusion into enzyme crystals of different shapes and sizes. The dependence of apparent mean enzyme activity in steroid isomerase crystals as a function of crystal size is shown to be consistent with this theoretical formulation. These inhibitor binding and catalytic properties suggest that the enzyme is in an active conformation within these crystals.     

Prediction of the halothane (Hal) genotypes of pigs by deducing Hal, Phi, Po2, Pgd haplotypes of parents and offspring: results from a large-scale practice in Swedish breeds

Results from a large-scale study, comprising 75 different breeding herds, are reported on predicting the halothane ( Hal ) genotypes of individual pigs by making use of the known close linkage between Hal and three C blood marker loci ( Phi, Po2, Pgd ). The parents haplotypes (involving Hal and marker loci) were determined from the HAL phenotypes (halothane test results) and marker loci phenotypes of their offspring in the first one or two litters studied. In subsequent litters of the Hal -marker loci haplotyped parents, the offspring's expected Hal genotypes could be predicted on the basis of the marker loci haplotypes inherited by them. By comparing the expected and observed HAL phenotypes of offspring in subsequent litters, the predicted Hal genotype was found to be correct in 90–95 % of the 4000 offspring (from Nn × Nn and Nn × nn matings) of Swedish Landrace and Yorkshire breeds studied.
The order of the three marker loci was confirmed as Phi-Po2-Pgd but the position of Hal with regards to Phi could not be resolved. The recombination frequencies between the most distant loci in this region, viz. Hal-Pgd and Phi-Pgd , were estimated to be 3–4.5 % and 4–6 % , respectively. The easy and rapid electrophoretic techniques described in the study to phenotype PHI, PO2, PGD, also allowed phe-notyping of six other polymorphic protein systems on the same gels. Thus Hal genotyping and effective parentage control can be conducted simultaneously.     

Estimation of population parameters and their application to the development of fishery management models in two African rivers

The dynamics of fish populations in two African rivers are being investigated with a view to developing fishery management models for tropical non-floodplain rivers. Preliminary work was carried out in the Rufiji River, East Africa, to establish growth and mortality rates along with L _∞ and K for six species in a river where fishing mortality is zero. These parameters, derived from scale-reading and length frequency analysis, also allowed the development of phased management plans for the fishery resulting from impoundment.
A subsequent investigation has begun on a West African river, the Taia in Sierra Leone, to pursue observations over four seasons, two of which are now complete. A systematic daily sampling programme is being carried out of the whole community, to accumulate similar but more extensive data to those obtained previously. In the Taia, however, fishing mortality must also be estimated from catch surveys, and analysis is carried out using the recently developed ELEFAN programs which can be compared with results obtained by other methods such as scale-reading.     

Change in the ultraviolet spectrum of solubilized Ca2+-dependent ATPase from sarcoplasmic reticulum due to binding with Ca2+ ions.

Solubilized sarcoplasmic reticulum (SSR) was prepared by solubilizing fragmented sarcoplasmic reticulum (FSR) with a nonionic detergent (C12E8) then displacing the detergent with Tween 80, using a DEAE-cellulose column. The UV absorption of SSR decreased reversibly at about 286 and 292 nm on removal of free Ca2+ ions, while no change in the fluorescence spectrum was detectable. On the other hand, the fluorescence intensity of FSR decreased 3-4% on removal of free Ca2+ ions, as previously reported by Dupont [(1976) Biochem. Biophys. Res. Commun. 71, 544-550]. The UV absorption of FSR increased reversibly at about 270-280 nm on removal of free Ca2+ ions, but the rate of the change was very slow (k = about 0.1 min-1).     

Monitoring signal transduction in cancer: cDNA microarray for semiquantitative analysis.

This study targeted the development of a novel microarray tool to allow rapid determination of the expression levels of 58 different tyrosine kinase (tk) genes in small tumor samples. The goals were to define a reference probe for multi-sample comparison and to investigate the variability and reproducibility of the image acquisition and RT-PCR procedures. The small number of tk genes on our arrays enabled us to define a reference probe by artificially mixing all genes on the arrays. Such a probe provided contrast reference for comparative hybridization of control and sample DNA and enabled cross-comparison of more than two samples against one another. Comparison of signals generated from multiple scanning eliminated the concern of photo bleaching and scanner intrinsic noise. Tests performed with breast, thyroid, and prostate cancer samples yielded distinctive patterns and suggest the feasibility of our approach. Repeated experiments indicated reproducibility of such arrays. Up- or downregulated genes identified by this rapid screening are now being investigated with techniques such as in situ hybridization.