Regulation of the synthesis of human chorionic gonadotropin by strains of HeLa cells in culture

Summary Thirty-seven strains of HeLa cells were examined for their ability to synthesize human chorionic gonadotropin (hCG) and its alpha subunit (hCG-α) in culture. Synthesis of hCG-α and hCG also was investigated in the presence of sodium butyrate and 5-bromo-2′-deoxyuridine (BrdUrd). All HeLa strains synthesized hCG-α in culture. Sodium butyrate increased the synthesis of hCG-α in all HeLa cells; BrdUrd increased synthesis in 32 of the 37 strains examined. Although few HeLa strains synthesized hCG in the absence of inducers, hCG was detected in most strains in the presence of sodium butyrate. The synthesis of hCG and its alpha subunit is, therefore, a stable genetic characteristics of HeLa cells. Certain preparations of hCG and its subunits were generously provided through the Center for Population Research of the National Institute of Child Health and Human Development, NIH.     

Age-dependent changes in rat liver microsomal and mitochondrial NADPH-dependent lipid peroxidation.

Purified outer membrane proteins O-8 and O-9 were able to bind to the peptidoglycan sacculi in sodium dodecyl sulfate solution. Binding was stimulated by lipopolysaccharide, that of protein O-9 being stimulated more remarkably. Proteins which had been heated in sodium dodecyl sulfate solution did not bind to the peptidoglycan sacculi even in the presence of lipopolysaccharide, while heated lipopolysaccharide stimulated the binding of non-heated proteins. The removal by pronase of the lipoprotein covalently bound to the peptidoglycan sacculi did not change the protein binding ability of the sacculi.     

Translation of tyrosine aminotransferase mRNA in a modified reticulocyte system.

Tyrosine aminotransferase messenger RNA has been translated in a cell-free system derived from rabbit reticulocytes. Cytoplasmic poly(A)-containing RNA from rat liver was used as the source of the messenger RNA. The newly synthesized subunits of the enzyme were isolated by immunoprecipitation and identified and quantitated using polyacrylamide gel electrophoresis. These procedures were used to demonstrate that glucocorticoid induction is associated with increased cytoplasmic levels of functional tyrosine aminotransferase messenger RNA.     

Localization of Cu and heme a on cytochrome c oxidase polypeptides.

After mild dissociation of cytochrome $\text{c}$ oxidase protomers, and polyacrylamide gel electrophoresis, copper was found predominantly in polypeptides of Bands V (m.w. 12,100) and VII (m.w. 3,400), and heme a predominantly in polypeptides of Bands I (m.w. 35,300) and II (m.w. 21,000). Some copper was found in Band II – III, and heme a in Band V.     

Localization of retinoic acid-binding protein in nuclei

Retinoic acid-binding component has been detected in the nuclei of chick embryo skin. The physicochemical properties of this macromolecule are in agreement with the properties of the retinoic acid-binding protein isolated from tissue cytosol. Although no binding protein could be detected in normal colon or lung tissue, nuclei isolated from a transplantable colon tumor and Lewis lung carcinoma contained this protein.     

Purification and partial characterization of a procaryotic glycoprotein from the plasma membrane of Thermoplasma acidophilum

The obligate, thermophilic, acidophilic mycoplasma, Thermoplasma acidophilum, grows optimally at 56° C and pH 2.0. Its plasma membrane possessed 21–22 protein bands that were resolved by polyacrylamide gel electrophoresis. One major membrane protein, molecular weight 152 000, which stained for carbohydrate with periodic acid-Schiff reagent, accounted for 32% (w/w) of the total membrane proteins. It was isolated and further purified by concanavalin A affinity chromatography. The carbohydrate content amounted to less than 10% (w/w) compared to that of the entire glycoprotein. The carbohydrate moiety consisted mainly of mannose residues with branched α 1 → 2 linkages at the non-reducing ends of the glycopeptide as determined by permethylation followed by gas chromatography-mass spectrometry analysis. The reducing end was an N-glycosidic linkage between asparagine and N-acetylglucosamine. The amino acid composition of this glycoprotein showed 62 mol% hydrophobic residues, while the acidic amino acid content contributed 9 mol% more than that of the basic amino acids. The existence of membrane glycoproteins in the procaryotic, wall-less T. acidophilum may provide a protective coat for the plasma membrane. The stereochemistry and the conformation of the carbohydrate chains, in conjunction with water turgor, may contribute to the rigidity of the membrane and the cation binding.     

Spin-label studies of membrane-associated denatured hemoglobin in normal and sickle cells

A maleimide spin label (N-(1-oxyl-2,2,5,5-tetramethylpyrrolidinyl)-maleimide) was reacted with oxyhemoglobin-free cell stromata of normal and sickle cells. The EPR spectrum of spin-labeled red cell membranes showed that the spin labels are attached to at least two different binding sites. There was a major signal, A, which characterized a strongly immobilized environment and a minor signal, B, which characterized a weakly immobilized environment. Quantitative EPR measurements using equal amounts of Hb AA and Hb SS red blood cells demonstrated that Hb SS red cell membranes had an approximately four times higher EPR signal intensity than Hb AA red cell membranes ((7.98 ± 1.14) · 10⁵ and (2.2 ± 1.2) · 10⁵ spin labels/cell, respectively). Moreover, the ratio of signal intensities A and B are different in these cells. Comparative spectrophotometric studies of membrane-associated denatured hemoglobins of Hb AA and Hb SS red cell membranes suggested that the EPR signal A is derived from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membrane-associated denatured hemoglobin, while signal B is mainly from spin labels attached to membranes. The combination of EPR spectrum of Hb AA membranes pretreated with N-ethyl-maleimide and that of spin-labeled precipitated hemoglobin further strengthened this conclusion.     

Paramagnetic probes in NMR and EPR studies of membrane enzymes

The applications of paramagnetic probes to problems of structure and mechanism are discussed from the point of view of the membrane enzymologist. Problems unique to membrane systems are discussed, and a variety of nuclear and paramagnetic probes are evaluated. Three membrane ATPase (kidney (Na+ + K+)-ATPase, Ca2+-ATPase from sarcoplasmic reticulum and Mg2+-ATPase from kidney) are used to describe the types of experiments which can be done, the information which can be obtained and the limitations involved. Nuclear relaxation studies employing 1H, 7Li+, 31P and 205Tl+ nuclei are described. The advantages and disadvantages of Mn2+, Gd3+ and Cr3+ as paramagnetic probes are discussed in terms of the three ATPases. The theory and interpretation of Mn2+ and Gd3+ EPR spectra are evaluated in studies with the (Na+ + K+)-ATPase and Ca2+-ATPase, respectively.