Shang Y Chen

Chaotropic anions and the surface potential of bilayer membranes

The chaotropic anions perchlorate and thiocyanate adsorb to artificial phospholipid membranes. The negative electrostatic potential they produce at the surface of the membranes was measured by two independent techniques. The conductance produced by neutral carriers of cations and anions was measured to estimate changes in the surface potentials of planar black lipid films and the electrophoretic mobility of phospholipid vesicles was used to monitor changes in the zeta potentials of spherical bilayer membranes. Qualitatively similar results were obtained with the two techniques. The results, moreover, agreed with the change in surface potential produced by these anions at an air water interface, as measured directly with an ionizing electrode (Randles, J. E. B. (1957) Discuss. Faraday Soc. 24, 194-199). The results obtained with artificial bilayers may explain the observation (Wieth, J. O. (1970) J. Physiol. 207, 581-609) that thiocyanate increases the sodium or potassium and decreases the sulfate permeability of erythrocyte membranes.

Growth hormone and liver mitochondria: Effects on cytochromes and some enzymes

Abstract The effects of hypophysectomy and subsequent growth hormone treatment on liver mitochondrial levels of cytochromes, flavoprotein, cytochrome oxidase and NADH oxidase were studied in rats. Hypophysectomy lowered cytochrome a , a 3 , b , c , and c 1 , concentrations and cytochrome oxidase activity. On the other hand, flavoprotein concentration and NADH oxidase activity were increased. Administration of either human or bovine growth hormone restored the cytochrome levels. Cytochrome oxidase activity was also increased but did not reach its original level. Only the bovine hormone exerted a lowering effect on flavoprotein and NADH oxidase activities. These and other results suggest that growth hormone influences mitochondrial electron transport.

Distribution of 3H-acetyl human growth hormone in subcellular fractions of rat liver

Abstract 3 H-Acetyl human growth hormone ( 3 H-HGH) (∼60 μg) was injected intravenously into hypophysectomized rats. Radioactivity appeared in all the subcellular fractions of liver as early as 2 minutes after the injection. Until 10 minutes postinjection virtually all of the tissue radioactivity was precipitable with trichloroacetic acid, whereas at 20 and 40 minutes after injection all of the radioactivity was soluble in trichloroacetic acid. Liver uptake (cpm/mg protein) peaked at about 20 minutes postinjection. The appearance of radioactivity in the crude nuclear fraction closely followed that of the whole homogenate. Radioactivity in the microsomal and mitochlondrial fractions was highest at the early time periods (up to 10 minutes) and then declined progressively. The radioactivity in the final supernatant or cytoplasmic fraction increased slowly, peaked at 20 minutes, and remained the highest. Pretreatment with unlabeled or 14 C-labeled HGH significantly reduced the mitochondrial and microsomal uptake of 3 H-HGH. In contrast with these results, when 3 H-HGH was added in vitro just before homogenization and fractionation of liver, most of the radioactivity (77%) appeared in the cytoplasmic fraction. These results are interpreted to mean that the appearance of 3 H-HGH in the particulate fractions at these early time periods may have functional significance, and that important initial binding sites of growth hormone in the liver cell may be mitochondria and microsomes.

Fluorometry of Selenium in Human Hair, Urine and Blood

This communication introduces a special lyophilization process for selenium determination by fluorometric methods. It permits a small sample volume, with several modifications including a single test tube process. Samples and standard are lyophilized first, then digested with nitric-perchloric acid mixture in a heated sand bath. Selenium is complexed with 2,3-diaminonaphthalene and extracted by n-hexane in the same test tube. The n-hexane layer is transferred to a cuvette and measured fluorometrically. Selenium concentration in healthy children from Long Island (aged 5-18 years) was: hair 0.765 +/- 0.114 microgram/g (n = 52), urine 28.65 +/- 8.27 micrograms/g creatinine (n = 66), and serum 95.4 +/- 14.4 ng/ml (n = 44). The current literature reflects an increase in the role of selenium in human nutrition. Thus, a simple but reliable method for determination of selenium in biological materials is needed in the clinical and research laboratory.

Fluorescence studies of human growth hormone

Abstract The emission maximum of human growth hormone, excited at 280 nm, in aqueous solution (pH 7.5) and in the presence of 6 M guanidine is at 348 nm. Rates of increase of tryptophanyl fluorescence of human growth hormone with ethanol or dioxane (up to 30%, by vol.) are identical to those shown by N- acetyl- l - tryptophanamide . Perturbation of tyrosyl fluorescence by ethanol is identical to that of N- acetyl- l - tyrosinamide whereas by dioxane it is only 39%. Tryptophanyl fluorescence of human growth hormone is quenched by about 35% between pH 9 and II in the aqueous solution but not in the presence of 6 M guanidine. These results suggest that the single tryptophanyl residue of human growth hormone is accessible to solvent molecules and may be situated close to tyrosyl residue(s).

Activation of microsomal glucose-6-phosphatase and associated pyrophosphatase and pyrophosphate-glucose phosphotransferase activities of human liver by aluminum oxide

Abstract Glucose-6-phosphatase activity and associated inorganic pyrophosphatase and pyrophosphate-glucose phosphotransferase activities of human liver microsomes was increased more than two-fold when the pH of the microsomal suspension was adjusted to 9.5 by Al2O3 powder and stirred overnight. Nearly 68% of glucose-6-phosphatase activity did not sediment at 105,000 × g after this procedure. The lipid content, pH optimum, Km, and antigenic characteristics of Al2O3 treated and intact microsomes were similar, but the enzymatic activity of treated fractions was more stable to heat (30°) and the RNA content was reduced by 50%. Electron microscopy indicated that the treated microsomes were essentially free of ribosomes and fragmented. The treatment appears to have dissociated the microsomal membrane by removing ribosomes, thus exposing more enzymatic active sites.

Molecular weight of microsomal glucose-6-phosphatase of rat and human liver

Abstract Salt-washed and unwashed and aluminum oxide treated microsomes of human liver, and washed microsomes of liver of normally fed, 1 day fasted and alloxan diabetic rats were subjected to polyacrylamide gel electrophoresis in presence of sodium dodecylsulfate and urea. Unwashed and aluminum oxide treated microsomes showed several protein bands, but salt-washed microsomes gave only one major protein band. This major protein band which appeared in all the preparations showed glucose-6-P activity on incubation of the electrophoresed gel with the substrate and subsequent staining with ammonium sulfide. Molecular weight of this protein was estimated to be 63,000 ± 6,800. Glucose-6-P and pyrophosphatase activities were measured after subjecting lyophilized microsomes, aluminum oxide treated supernatant and residue fractions of human and rat liver to ionizing radiation in vacuo at different doses (Mrads). There was no significant difference between either the surviving fraction of glucose-6-P and pyrophosphatase activities or among the different preparations of human and rat liver at a given radiation dose. Computer analysis of the pooled data gave a value of 9.16 Mrads for 37% survival of the activity which corresponds to a molecular weight of 70,000.

Kinetics of uptake and subcellular distribution of human growth hormone in liver slices

Abstract Uptake of 3 H-acetyl human growth hormone ( 3 H-HGH) and 3 H-acetyl bovine growth hormone ( 3 H-BGH) into slices of hypophysectomized rat liver and human liver (obtained from autopsy) has been measured both as a function of time and concentration of the labeled hormones in the incubation medium. Uptake kinetics as a function of concentration of 3 H-HGH or 3 H-BGH in rat liver slices followed a sigmoid curve. Similar sigmoid curve was obtained when increasing concentrations of 3 H-HGH were incubated with human liver slices. In contrast, uptake kinetics of 3 H-BGH in human liver slices followed a hyperbolic curve. Addition of unlabeled HGH or BGH into the incubation medium reduced the uptake of 3 H-HGH in rat liver slices but in human liver slices only human growth hormone competed. This binding-site difference along with the reported molecular differences between human and bovine growth hormones may well explain the different biological specificities of the two hormones.

450 ZINC (Zn) AND BIRTH DEFECTS (BD)

Zn deficiency has been reported in infants with BD and in children born with achondroplasia. 304 families were studied comparing parental age, Zn nutritional status and infant weight at the time of birth.There is a statistically significant correlation (p < .01) between infant hair Zn and maternal age at time of birth. The decrease in infant hair Zn seen with teenage (14-20) and elder mothers (31-40) may be related to the incidence of increased BD which occurs in these groups. Infant hair Zn was lowest in the heaviest infants and in a group with BD made up of porencephaly, anencephaly and microcephaly as the presenting sign (N=4; Zn=134±10). The heavier children may represent the infants of potential diabetic mothers (who have more BD). Placental Zn was 18-19 ug/g and did not correlate well with maternal or infant hair Zn. Finally, diet did not explain parental Zn status since paired deficiencies did not occur. Therefore, an association between Zn levels, high-risk age groups and those children with BD is apparent.

Hepatic microsomal membrane: Activation of glucose-6-phosphatase

Liver glucose-6-phosphatase is a constitutive enzyme firmly bound to endoplasmic reticulum [ 1 ] . Membrane localization and orientation of the enzyme may be visualized to play a role in the vectorial release of glucose from liver and regulation of blood glucose. Formation and distribution of the enzyme in developing hepatocytes has been used to study biogenesis and differentiation of the membrane [2]. Like other membrane-bound enzymes the activity is increased, but becomes very labile when solubilized with detergents [ 1 ] . Similar increase in the activity also occurs when the pH is increased by 1 M NH,OH [3] or Al203 [4] and the enzyme becomes more stable. We have shown [5] that pyridoxal-5’-phosphate (PLP) inactivates the enzyme, by competing with the substrate and by reaction at the active site. This has offered us an opportunity to investigate the mechanism of activation by NH,OH which seems to be the method of choice as recently shown by Arion et al. [6]. The rationale of the present experimental approach are: [l] glucose-6-phosphatase activity expressed in the absence of NH,OH is due to exposed enzyme sites which are accessible to the substrate or its competitor, and [2] the activity increment which results after NH,OH treatment is due to enzyme sites which are not accessible to substrate or its competitor in the absence of any dispersing agent. It follows from such a model that if one partially inactivates the enzyme by reaction with PLP and then treats with NH,OH (raising the pH), percent activation