Stereoselectivity and enantioselectivity of glutathione S-transferase toward stilbene oxide substrates

Isozyme 4-4 of rat liver glutathione S-transferase catalyzes the stereoselective addition of glutathione to the oxirane carbon of R-absolute configuration of cis-stilbene oxide, 2, to give 98 +/- 2% of the (1S,2S)-1,2-diphenyl-1-(S-glutathionyl)-2-hydroxyethane product with a turnover number (kc) of 0.22 s-1. The two enantiomers of trans-stilbene oxide, 3, are somewhat poorer substrates for the enzyme. Enantioselective addition of glutathione to 3 proceeds with turnover numbers of 0.12 s-1 and 0.023 s-1 for the (R,R,)- and (S,S)-antipodes, respectively.     

Ion-Exchange Chromatography Separates Activities Synthesizing and Degrading Fructose 2,6-Bisphosphate from C3 and C4 Leaves but Not from Rat Liver

Fructose-6-phosphate,2-kinase and fructose-2,6-bisphosphatase were separated on the basis of charge from leaves of C₃ (spinach, lettuce, and pea) and C₄ (sorghum and amaranthus) plants but not from rat liver—a tissue known to contain a bifunctional enzyme with both activities. [2-³²P]Fructose 2,6-bisphosphate binding experiments also suggest that the major forms of these activities reside on different proteins in leaves.     

Continuous high density expression of human beta 2-adrenergic receptors in a mouse cell line previously lacking beta-receptors

The PvuII fragment of human genomic clone LCV-517 which contains the entire coding region of a beta-adrenergic receptor gene was cloned into the SmaI site of the expression vector pMSG. The recombinant DNA was cotransfected with pRSVneo into mouse B-82 cells using the CaPO4 precipitation method. B-82 cells do not possess beta-adrenergic receptors but do contain prostaglandin E1 receptors that stimulate adenylate cyclase. Following transfection, several colonies expressing beta-adrenergic receptors were isolated. Analysis of ligand binding to expressed beta-receptors indicated that the protein encoded by the gene in clone LCV-517 was a beta 2-adrenergic subtype. Human beta 2-adrenergic receptors photoaffinity labeled with [125I]iodocyanopindolol diazirine migrated on sodium dodecyl sulfate-polyacrylamide gels consistent with a molecular mass of 68,000, demonstrating that the receptor is glycosylated to an extent of 25-30% by weight. Addition of isoproterenol to cultures of transfected cells resulted in a 3-4-fold stimulation of adenylate cyclase, an effect similar to that seen in control B-82 cells with prostaglandin E1. These data describe the production of stable murine clonal cell lines expressing human beta 2-adrenergic receptors and illustrate the utility of such lines in the biochemical and pharmacological characterization of receptor proteins.     

Molecular packing in type I collagen fibrils

Previous studies of the X-ray diffraction pattern of the crystalline regions of type I collagen fibrils yielded information on the unit cell parameters and also the orientation of the pseudo-hexagonally packed molecular segments in the overlap region. The absence of Bragg reflections at high angles attributable to the molecular segments in the gap region led to the suggestion that these segments were more mobile than those in the overlap region. We report a study of the low-angle Bragg reflections in a search for information about the nature of the orientation and packing of the molecular segments in the gap region. We conclude that the (m = 0, n = 0) helix layer plane of the molecular segments in the overlap region makes little or no contribution to the Bragg reflections at low angles, and identify three possible origins for the observed low-angle reflections in the electron density contrast associated with: (1) the "hole" created by the missing molecular segment in the gap region; (2) the telopeptides; or (3) the axial regularities in amino acid residues of a particular type, with periodicities of D/5 or D/6. Sufficient information is available to investigate the first two of these possibilities, and the results obtained suggest specific arrangements for the molecular segments in the overlap and gap regions, and specific connectivities between the molecular segments in successive overlap regions. In addition, we have examined the amino acid sequence and identified features related to the mobility of the molecular segments in the gap region and to the regions where it is thought that molecules are kinked.     

Crystallization and a preliminary X-ray diffraction study of isozyme 3-3 of glutathione S-transferase from rat liver

Crystals of the homodimeric isozyme 3-3 of glutathione S-transferase from rat liver have been obtained with the hanging drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of the enzyme required the presence of both the enzyme inhibitor (9R, 10R)-9, 10-dihydro-9-(S-glutathionyl)-10-hydroxyphenanthrene and the detergent beta-octylglucopyranoside. The crystals belong to the monoclinic space group C2, with cell dimensions of a = 88.24(8) A, b = 69.44(4) A, c = 81.28(5) A, beta = 106.01(6) degrees, and contain four dimeric enzyme molecules per unit cell. The crystals diffract to at least 2.2 A and are suitable for X-ray crystallographic structure determination at high resolution.     

Isolation of a major human placental substrate for the epidermal growth factor (urogastrone) receptor kinase: immunological cross-reactivity with transducin and sequence homology with lipocortin

Using as a starting material either a detergent extract or a protein fraction eluted from membranes with ethylene glycol bis (beta-aminoethyl ether)-N,N'-tetraacetic acid, we have isolated from human placental membranes a major substrate for the epidermal growth factor (urogastrone) receptor kinase (EGF kinase). The substrate was isolated both in an intact form, having a molecular mass of approximately 38-kDa (p38), and in a 35-kDa form (p35) representing a proteolytic cleavage product of p38. Both p38 and p35 cross-reacted with antibodies directed against bovine retinal transducin, but did not cross-react with antibodies directed against the 35-kDa beta subunit of human placental G-protein. Antisera directed against the placental EGF kinase substrate failed to react with either bovine or human placental src kinase substrate, p36. Conversely, antisera directed against p36 reacted only poorly with placental p38 or p35. Although p38 had a blocked amino terminus that precluded sequence analysis, p35 yielded an N-terminal sequence that was identical with residues 13-36 of human lipocortin. Our data clearly distinguish p38 from the previously described intestinal calcium binding protein calpactin I or p36 that is also a tyrosine kinase substrate, and our work points to a close relationship (if not identity) between p35 and a 35-kDa EGF receptor kinase substrate previously characterized in A431 cells. We conclude that p38 and p35, which very likely represent human placental lipocortin, may share only limited epitope homology with transducin alpha subunit; however, the possibility that p38, along with intestinal p36 and with a family of related calcium binding proteins, may, like transducin, play a role in receptor-mediated transmembrane signaling is discussed.     

Effects of hypoxia on lung mechanics in the newborn cat

The present study was designed to investigate the effects of hypoxia on lung mechanics in the newborn cat and to determine if vagal efferent innervation to the airways is involved in the response. We studied 11 animals, aged 2-7 days, anesthetized with a mixture of chloralose-urethane administered intraperitoneally. A tracheal cannula was inserted just below the larynx and following paralysis (pancuronium bromide), mechanical ventilation was initiated. A pneumothorax was created by a midline thoracotomy and an end-expiratory load was applied to maintain functional residual capacity. Animals were placed in a flow plethysmograph from which measurements of transpulmonary pressure, flow, and volume, mean inspiratory resistance, and dynamic compliance of the lung were calculated. The experimental protocol consisted of a series of 8-min trials, each preceded by a controlled volume history. The hypoxia challenge was composed of 1 min of ventilation with 40% O2, followed by 5 min exposure to 10% O2 and 2 min of recovery. In the majority of animals (7 out of 11), hypoxia had no effect on lung mechanics compared with control trials. Four animals responded to hypoxia with an increase in resistance and a decrease in compliance. Resistance remained elevated throughout the hypoxia with an average maximal increase of 47.2 +/- 22.2% (SD). Dynamic compliance was significantly decreased at the 2nd, 3rd, and 4th min only of hypoxia. Bilateral vagotomy abolished the response in the four animals and hypoxia had no effect on mechanics postvagotomy. Our data suggest that in most cases changes in lung mechanics do not play a causal role in the biphasic ventilatory response to hypoxia seen in the newborn.     

Activation of phosphoprotein phosphatases by growth hormone sequences with insulin-like activity

The N-terminal part sequences of pituitary growth hormone, N-acetyl-hGH 7–13 and hGH 6–13, promoted conversion of glycogen synthase b to glycogen synthase a in skeletal muscle and adipose tissue when injected intravenously. The peptides also caused conversion of phosphorylase a to phosphorylase b in liver and adipose tissue, but not in muscle, where the peptides antagonised activation of phosphorylase. Synthase phosphatase activity in muscle and phosphorylase phosphatase activity in liver increased after injection of peptide, with time courses of change similar to those seen for muscle synthase and liver phosphorylase activities. Injection of peptide also decreased both the cyclic AMP dependent and independent synthase kinase activities in muscle. These results show that the insulin-like activities of these peptides on glycogen synthase and phosphorylase involve both increases in protein phosphatase activities and inhibition of protein kinase activities. These results are discussed in relation to the insulin-like activities of growth hormone.