Monooxygenase activity of human hemoglobin: role of quaternary structure in the preponderant activity of the beta subunits within the tetramer

Catalysis of para hydroxylation of aniline was measured for human ferrihemoglobin and various derivatives in a reconstituted system consisting of the appropriate hemoprotein (at 4 microM heme), reduced nicotinamide adenine dinucleotide phosphate (NADPH), cytochrome P-450 reductase, and aniline under atmospheric O2. The isolated subunits of hemoglobin (alpha 3+ and beta 3+4) were prepared by treatment with p-(hydroxymercuri)benzoate. Semihemoglobin (alpha heme2 beta 02) was prepared from ferrihemoglobin and apohemoglobin. Converse valency hybrids alpha 3+2(beta 2+-CO)2 and (alpha 2+-CO)2 beta 3+2 were prepared from appropriately ligated alpha and beta subunits. After chromatography, the hemoglobin derivatives were characterized by visible and 1H NMR spectroscopy and electrophoresis. At the same concentration of aniline, the alpha and beta subunits were much less active than the normal tetramer. alpha-Semihemoglobin and the alpha 3+2(beta 2+-CO)2 hybrid also displayed lower hydroxylase activity. The (alpha 2+-CO)2 beta 3+2 hybrid was about as active as normal alpha 3+2 beta 3+2. This result suggests that the activity of tetrameric hemoglobin primarily involves the beta subunits. Also transfer of the beta subunits from the beta 4 molecular environment to the alpha 2 beta 2 state enhances their monooxygenase activity approximately 15-fold. The hemoglobin derivatives were differently susceptible to substrate inhibition, the beta 4 species being most sensitive. Estimates of Vmax from the linear portions of the corresponding Lineweaver-Burk plots showed agreement within a factor of 2.5 for all of the hemoglobin derivatives, suggesting that the intrinsic O2-activating capacities of the derivatives are similar.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thermoregulation during prolonged exercise in heat: alterations with beta-adrenergic blockade

Thermoregulation and cardiovascular drift were studied under conditions of prolonged exercise in a warm environment (dry bulb temperature 31.7 +/- 0.3 degrees C, rh 44.7 +/- 4.7%) during beta-adrenergic blockade. Fourteen subjects performed 90-min rides on a cycle ergometer at a work rate equivalent to 40% of their control maximal O2 uptake under each of three treatments provided in a randomized double-blind manner: atenolol (100 mg/day), propranolol (160 mg/day), and a placebo. Exercise during the propranolol trial resulted in significantly higher forearm vascular resistance values and significantly lower forearm blood flows (FBF) compared with the placebo trial. However, the significantly lower FBF during propranolol did not significantly alter the rectal temperature (Tre) response to prolonged exercise. In addition, both beta-blockers produced lower FBF for any given Tre, suggesting that beta-adrenergic blockade affects FBF through nonthermal factors. The slight differences in Tre, despite the large differences in FBF between the various treatments, are apparently the result of an enhanced sweat loss and a lower mean skin temperature during exercise with beta-blockade. The uncoupling of FBF and sweat loss provides evidence of independent regulation. The reduction in FBF at any given Tre was concomitant to lower blood pressure values during beta-blockade and suggests that baroreflexes provide significant input to the control of skin blood flow when both pressure and temperature maintenance are simultaneously challenged.     

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).