Similar MW Research Articles

Evidence for the specific binding of growth hormone to a receptor-like protein in rabbit serum

The binding in vitro of 125I-human or bovine growth hormone (GH) to normal female rabbit serum has been studied using gel filtration to separate bound and free hormone. On Ultrogel AcA34 columns, a substantial peak of specific 125I-GH binding was observed at a MW-120000. This peak was not precipitable by 12.5% polyethylene glycol, a method used widely for solubilized hormone receptors. Assuming a 1:1 binding stoichiometry between GH (MW 21000) and the binding protein, the MW of the binding protein would be ∼ 100000. Gel filtration of serum alone, followed by assessment of 125I-hGH binding in column fractions, indicated the binding protein had a similar MW (83000–107000). Specific binding of 125I-hGH was dependent on incubation time (equilibrium being reached in 2 h at 21°C), Ca 2+ concentration (0.5–2.0 mM) and serum concentration (a 1: 5 dilution of serum giving 45.2 ± 1.7% specific binding; mean ± SE, n = 10). Binding was completely reversible ( t 1 2 ∼ 1.5 h ) and specific for somatotrophic but not lactogenic hormones. Scatchard analysis revealed linear plots with a K a 1.59 ± 0.11 × 10 9 M −1 and capacity 3700 fmol/ml serum. The presence in rabbit serum of a high affinity, GH-specific, binding protein raises important questions regarding its identity and possible physiological role in modulating the delivery and/or activity of GH in vivo.

Characterization of the prolactin receptor in cell fractions from rat liver

[ 125I]Prolactin (PRL) was covalently cross-linked to its binding sites in subcellular fractions of female rat livers using NHSAB and UV irradiation. Analysis by non-reducing SDS-PAGE showed that all fractions with specifically bound radioactive hormone contained a major autoradiographic band (eliminated with unlabeled PRL) of similar electrophoretic mobility consistent with a MW of 36K for the receptor. In addition, microsomal membranes were treated with a zwitterionic detergent (CHAPS), solubilizing 30–60% of the specifically bound radioactivity. SDS-PAGE analysis of [ 125I]PRL cross-linked to CHAPS-soluble and CHAPS-insoluble material showed an autoradiographic band with a similar MW. These results suggest that prolactin receptors in different hepatocyte membranes are similar in MW and do not appear to be linked by disulfide bonds to other membrane proteins. Some of the binding sites appear to interact with membrane constituents in ways that affect their solubility by CHAPS.

Coconut α- d-galactosidase isoenzymes: isolation, purification and characterization

α- d-Galactosidases (α- d-galactoside galactohydrolase, EC 3.2.1.22) from normal coconut endosperm were isolated and partially purified by a combination of ammonium sulfate fractionation, SP-Sephadex C50–120 ion-exchange chromatography and Sephadex G-200 and G-100 gel filtration. Two molecular forms of the enzyme, designated as A and B, were eluted after SP-Sephadex C50–120 ion-exchange chromatography. α- d-Galactosidase A, which is the major isoenzyme, was partially purified 43-fold on Sephadex G-200 and has a MW of about 23 000 whereas α- d-galactosidase B was partially purified 23-fold on Sephadex G-100 and has a similar MW of about 26 600. Both isoenzymes exhibited optimum activity at pH 7.5. The apparent K m and V max of α- d-galactosidase A were obtained at 3.46 × 10 −4M and 1.38 × 10 −3 M p-nitrophenyl α-< d-galactoside, respectively. A distinct substrate inhibition was noted. The enzyme was inhibited strongly by d-galactose and to a lesser extent by myo-inositol, d-glucose-6-phosphate, l-arabinose, melibiose and iodoacetic acid. Similarly, makapuno α- d-galactosidase was localized in the 40–70 % (NH 4) 2SO 4 cut but its optimum activity at pH 7.5 was considerably lower as compared to the normal. Its K m was obtained at 6.75 × 10 −4 M p-nitrophenyl α- d-galactoside while the V max was noted at 5.28 × 10 −3 M p-nitrophenyl α- d-galactoside. Based on the above kinetic data, the possible cause(s) of the deficiency of α- d-galactosidase activity in makapuno is discussed.

Gibberellin-controlled pectinic acid and protein secretion in growing cells

Gibberellic acid (GA) promoted cell expansion in a suspension culture of spinach. Preceding this response, weakly acidic pectinic acid was released into the medium. GA did not alter the concentration of sorbitol (0.7 M) needed to prevent growth, and sorbitol at this concentration did not block the action of GA upon the release of pectinic acid. GA suppressed the release into the medium of a peroxidase of MW ca 40 000 and favoured the intracellular accumulation of a polypeptide of similar MW. All the responses to GA were antagonized by 2,4-dichlorophenoxyacetic acid but not by kinetin. A hypothesis is presented in which the promotion of growth by GA is mediated by peroxidase, its phenolic substrates and the cell-wall pectic polysaccharides.

Regulation of homoserine dehydrogenase in developing organs of soybean seedlings

The regulation of homoserine dehydrogenase (HSD) activity (EC 1.1.1.3) by L-threonine, L-cysteine and K + was examined using extracts of organs of soybean seedlings harvested 3, 6, 11, and 19 days after germination. K + stimulated HSD activity from each source at least 2-fold. HSD activity was completely inhibited by 10 mM L-cysteine while 10 mM D-cysteine was not inhibitory. A progressive decrease in sensitivity of NAD-dependent HSD to inhibition by 10 mM L-threonine occurred in all organs except the leaf during the sampling period. This progressive decrease in sensitivity of the HSD to threonine inhibition was detected only when K + was present in the assay mixtures. Four major molecular forms, including one rapidly migrating form (form I) and three more slowly migrating forms (forms II, III, IV) of HSD, were identified in extracts of soybean organs by polyacrylamide electrophoresis. Chromatographic and electrophoretic data indicate that form I, which was not inhibited by threonine or stimulated by K +, was of lower MW than forms II, III and IV which were of similar MW. These latter 3 forms were inhibited by threonine and stimulated by K +. During soybean seedling development form II increased in amount and forms I and IV decreased in amount. This alteration in the amounts of the forms of HSD occurred during the same period as the decrease in the amount of threonine inhibition. Since K + stimulation of HSD decreased during soybean organ development and K + enhanced threonine inhibition, this might account for the observed decrease in threonine inhibition.

Genetic variability and improvement of seed proteins in wheat

Albumins, globulins, gliadins and glutenins presumably comprising 100 percent of the wheat seed proteins were sequentially extracted and electrophoresed on SDS-polyacrylamide gels. The SDS-electrophoretic patterns within each of the four fractions from T. boeotiaum, T. urartu, T. turgidum, T. timopheevii, T. aestivum, Ae. speltoides and Ae. squawosa were similar. They differed from one species to another only in a few minor components or density of certain components. Similarity in MW's of components, as indicated by the SDS-electrophoretic patterns, suggests that the wheats and Aegilops exhibit no variability for structural genes coding seed proteins. A minimum of 60 to 70 and a maximum of 360 to 420 structural genes with major or minor effects control the total seed protein in T. aestivum. Presumably, only one or the other homoeoallele was expressed in the polyploids. Different components of albumins and globulins presumably had distinct MW's and amino acid composition, while the components of gliadins and glutenins could be classified into a few groups each containing one or more components with the same MW and nearly identical amino acid composition. The genes for components with similar MW's and amino acid composition arose through multiplication of a single original gene and perhaps share the same regulatory mechanism. Seed protein content and quality in wheat might be improved through the incorporation of structural genes, coding for polypeptides with distinct MW's, from distantly related species, rather than by manipulation of the structural genes within the Triticum-Aegilops group. Regulatory mutants similar to opaque-2 of corn could be used to alter the proportion of gliadins in relation to albumins and globulins, to improve amino acid composition of wheat proteins.