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)     

Biochemical studies of taste sensation. XI. Isolation, characterization and taste ligand binding activity of plasma membranes from catfish taste tissue

Variants of creatine kinase-MM (variant of ATP:creatine N-phosphotransferase, EC 2.7.3.2), present in human heart and skeletal muscle, have been purified to homogeneity using DEAE-Sepharose column chromatography and column chromatofocusing techniques. Creatine kinase-MM I-IV were present in both heart and skeletal muscle, while MM-V was found only in heart. The number, ratio and elution profile of the variants during chromatofocusing remained identical even when they were purified in the presence of proteinase inhibitors. MM-I-V, on chromatofocusing, were eluted at pH 8.3, 7.9, 7.6, 7.2 and 6.8, respectively. Isoelectric focusing revealed the pI of MM-I-V to be 7.2, 6.9, 6.7, 6.4 and 6.2. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis showed a doublet pattern for creatine kinase-MM variants III-V. However, polyacrylamide gel electrophoresis without SDS indicated homogeneity because each variant showed a single band. The doublet pattern observed in the presence of SDS may reflect the presence of two subunits of slightly different mass.     

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.     

Hypophysiotrophic thyrotropin releasing hormone (TRH) synthesizing neurons. Ultrastructure, adrenergic innervation and putative transmitter action

The neuropeptide thyrotropin releasing hormone (TRH) is capable of influencing both neuronal mechanisms in the brain and the activity of the pituitary-thyroid endocrine axis. By the use of immunocytochemical techniques, first the ultrastructural features of TRH-immunoreactive (IR) perikarya and neuronal processes were studied, and then the relationship between TRH-IR neuronal elements and dopamine-beta-hydroxylase (DBH) or phenylethanolamine-N-methyltransferase (PNMT)-IR catecholaminergic axons was analyzed in the parvocellular subnuclei of the hypothalamic paraventricular nucleus (PVN). In control animals, only TRH-IR axons were detected and some of them seemed to follow the contour of immunonegative neurons. Colchicine treatment resulted in the appearance of TRH-IR material in parvocellular neurons of the PVN. At the ultrastructural level, immunolabel was associated with rough endoplasmic reticulum, free ribosomes and neurosecretory granules. Non-labelled axons formed synaptic specializations with both dendrites and perikarya of the TRH-synthesizing neurons. TRH-IR axons located in the parvocellular units of the PVN exhibited numerous intensely labelled dense-core and fewer small electron lucent vesicles. These axons were frequently observed to terminate on parvocellular neurons, forming both bouton- and en passant-type connections. The simultaneous light microscopic localization of DBH or PNMT-IR axons and TRH-synthesizing neurons demonstrated that catecholaminergic fibers established contacts with the dendrites and cell bodies of TRH-IR neurons. Ultrastructural analysis revealed the formation of asymmetric axo-somatic and axo-dendritic synaptic specializations between PNMT-immunopositive, adrenergic axons and TRH-IR neurons in the periventricular and medial parvocellular subnuclei of the PVN. These morphological data indicate that the hypophysiotrophic, thyrotropin releasing hormone synthesizing neurons of the PVN are directly influenced by the central epinephrine system and that TRH may act as a neurotransmitter or neuromodulator upon other paraventricular neurons.     

Heat exchanges in wet suits

Flow of water under foam neoprene wet suits could halve insulation that the suits provided, even at rest in cold water. On the trunk conductance of this flow was approximately 6.6 at rest and 11.4 W . m-2 . C-1 exercising; on the limbs, it was only 3.4 at rest and 5.8 W . m-2 . degrees C-1 exercising; but during vasoconstriction in the cold, skin temperatures on distal parts of limbs were lower than were those of the trunk, allowing adequate metabolic responses. In warm water, minor postural changes and movement made flow under suits much higher, approximately 60 on trunk and 30 W . m-2 . degrees C-1 on limbs, both at rest and at work. These changes in flow allowed for a wide range of water temperatures at which people could stabilize body temperature in any given suit, neither overheating when exercising nor cooling below 35 degrees C when still. Even thin people with 4- or 7- mm suits covering the whole body could stabilize their body temperatures in water near 10 degrees C in spite of cold vasodilatation. Equations to predict limits of water temperature for stability with various suits and fat thicknesses are given.