Molecular weights of subunits of acetyl CoA carboxylase in rat liver cytoplasm

Monomeric [14C] methyl avidin was shown to bind to sodium dodecyl sulfate-denatured biotinyl proteins and remain bound through polyacrylamide gel electrophoresis which allowed their detection by fluorography. This method was used to show that purified rat liver acetyl CoA carboxylase contained two high molecular weight forms of the enzyme (MR = 241,000 and 252,000) while rapidly prepared, crude rat liver cytoplasm contained two larger molecular weight (MR = 257,000 and 270,000) forms. Thus, the enzyme had undergone substantial proteolysis during purification. The crude enzyme preparation also contained a smaller biotinyl protein (MR = 141,000) which is likely a proteolytic product of the larger forms of acetyl CoA carboxylase.     

Insulin stimulates phosphorylation of a heat-stable protein in rat adipose tissue

Insulin in rat adipose tissue acts to increase the phosphorylation about 2.5-fold of a low molecular weight protein in the cytosol designated phosphoprotein m. Isoproterenol had no effect on the phosphorylation of phosphoprotein m. Some of the properties of phosphoprotein m are: soluble in 1% trichloro acetic acid, heat-stable and has a molecular weight of 23,000 on polyacrylamide gels in the presence of sodium dodecyl sulfate. Phosphoserine and phosphothreonine are the phosphorylated amino acid residues of phosphoprotein m. The physical and chemical properties of phosphoprotein m are similar to those of previously described inhibitor and modulator proteins.     

Semipreparative electrophoresis with prestained proteins.

A simple method for prestaining proteins with Coomassie blue G prior to acrylamidegel electrophoresis is described. Many, although not all, undenatured proteins can be stained. This prestaining technique can be conveniently coupled with a second electrophoresis step, using Sephadex as a supporting medium, to yield isolated protein in about 4 h.     

五种黄精属植物的蛋白水解酶谱研究

用蛋白水解酶复性电泳方法 (G- PAGE)分析了 5种黄精属植物根状茎和叶的蛋白水解酶的种类和活性。结果表明 :(1)它们的根状茎均含有 85k D和 55k D的蛋白水解酶 ;叶均含有 82 k D的蛋白水解酶 ;(2 )根状茎和叶的蛋白水解酶种类和活性有很大差异 ,叶的蛋白水解酶活性为根状茎的 10倍 ,它们的活性均受 p H影响 ,其最适 p H为 7;(3)每种植物都含有自己特有蛋白水解酶 ;(4 )蛋白水解酶在植物鉴定中有参考价值     

Covalent attachment of poly (U) template to 40S - mammalian ribosomal subunits

When 40S subunits are irradiated at 254nm in presence of [³H] poly (U), formation of a 40S subunit-poly (U) complex can be demonstrated either by filtration technique at low Mg⁺⁺ concentration or by polyacrylamide gel electrophoresis. No stable complex was detected using unirradiated samples under the same conditions. Electrophoresis of this complex in the presence of dodecyl sulfate showed that part of the poly (U) directly associates with 18S RNA. This association is not through proteins, since it is not disrupted by pronase treatment.     

Quantitation and characterization of the microtubule associated MAP2 in porcine tissues and its isolation from porcine (PK15) and human (HeLa) cell lines

A radioimmunoassay developed for the microtubule associated protein MAP₂ shows that this protein, or related polypeptides are present in all the porcine tissues studied. Nervous tissues (brain, 11 μg MAP₂/mg protein; cerebellum, 9.7 μg MAP₂/mg protein) contain much higher levels of MAP₂ than non-nervous tissues (kidney, 104 ng MAP₂/mg protein; lung 89 ng MAP₂/mg protein; spleen 66 ng MAP₂/mg protein; thyroid 21 ng MAP₂/mg protein; liver 9.7 ng MAP₂/mg protein). A heat resistant protein doublet of 300,000 with the ability to promote microtubule polymerization has been purified from pig kidney cells by affinity chromatography using MAP₂ antibodies. Using a similar purification method a protein of 200,000 daltons has been isolated from Hela cells.     

Association of two proteolipids of unknown function with ATP synthase from bovine heart mitochondria

ATP synthase, or F-ATPase, purified from bovine heart mitochondria in the absence of phospholipids is an assembly of 16 different subunits. In the presence of exogenous phospholipids, two additional hydrophobic proteins, a 6.8kDa proteolipid and diabetes associated protein in insulin sensitive tissue (DAPIT), were associated with the purified complex, with DAPIT at sub-stoichiometric levels. Both proteins are conserved in vertebrates and invertebrates, but not in fungi, and prokaryotic F-ATPases do not contain orthologues of either of them. Therefore, their roles are likely to be peripheral to the synthesis of ATP.     

Ankyrin is necessary for both drug-induced and ATP-induced shape change of human erythrocyte ghosts

Human erythrocyte membranes from ACD preseved blood were digested with trypsin (Protein: trypsin=100:1) and analyzed by SDS PAGE. After digestion for 20 sec at 0°C, only ankyrin disappeared but other bands including spectrin, band 4.1 and band 3 remained intact. In contrast to intact membranes, treatment with chlorpromazine or MgATP or HEPES did not induce shape change in these membranes. The number of transformable cells correlated closely with the amount of remaining ankyrin. We conclude that ankyrin is necessary for their shape change. This is the first direct evidence that ankyrin is involved in the maintenance of red cell shape. Three additional lines of indirect evidence are also presented.     

Role of a putative third subunit YhcB on the assembly and function of cytochrome bd-type ubiquinol oxidase from Escherichia coli

Recent proteome studies on the Escherichia coli membrane proteins suggested that YhcB is a putative third subunit of cytochrome bd-type ubiquinol oxidase (CydAB) (F. Stenberg, P. Chovanec, S.L. Maslen, C.V. Robinson, L.L. Ilag, G. von Heijne, D.O. Daley, Protein complexes of the Escherichia coli cell envelope. J. Biol. Chem. 280 (2005) 34409-34419). We isolated and characterized cytochrome bd from the ΔyhcB strain, and found that the formation of the CydAB heterodimer, the spectroscopic properties of bound hemes, and kinetic parameters for the ubiquinol-1 oxidation were identical to those of cytochrome bd from the wild-type strain. Anion-exchange chromatography and SDS-polyacrylamide gel electrophoresis showed that YhcB was not associated with the cytochrome bd complex. We concluded that YhcB is dispensable for the assembly and function of cytochrome bd. YhcB, which is distributed only in γ-proteobacteria, may be a part of another membrane protein complex or may form a homo multimeric complex.     

Additional evidence that the hemin-controlled translational repressor from rabbit reticulocytes is a protein kinase

Recent reports have suggested that the hemin-controlled translational repressor (HCR) which mediates the hemin control of protein synthesis in reticulocyte lysates, acts as a specific protein kinase, phosphorylating a subunit of the Met-tRNA_f binding factor (IF-1). We have found that crude and highly purified HCR can phosphorylate a 38,000 molecular weight component of IF-1, but that crude prorepressor (the precursor of HCR), which is not inhibitory, does not phosphorylate this component. Prolonged warming of the prorepressor induces the formation of the inhibitor and the protein kinase that phosphorylates the 38,000 molecular weight protein, and the formation of both is blocked by hemin. In addition, a brief incubation of the prorepressor with N-ethylmaleimide, which produces maximal inhibitory activity within 5 minutes, also induces formation of the protein kinase. These findings suggest that HCR and the protein kinase are the same protein and provide additional support for the concept that HCR controls protein synthesis by phosphorylating the Met-tRNA_f binding factor.     

Inhibition of photosynthetic oxygen evolution by calmodulin-type inhibitors and other calcium-antagonists

Several studies have recently implicated a role for Ca2+ in photosynthetic oxygen evolution (9-11). Our previous study indicated that Ca2+ was likely acting at the level of the Cl- cofactor requirement in photosystem II (9). We now demonstrate, through the use of calmodulin-type inhibitors ( calmidazolium and trifluoperazine) and metal Ca2+-antagonists (e.g., Tb+3 and La+3), the function of Ca2+ on the oxidizing side of photosystem II. In addition, the peroxide (H2O2) electron donation site was differentiated from the electron donation site of NH2OH, Mn2+ and diphenyl carbazide in the photosystem II complex.     

Glutathione S-transferase activities in rat and mouse sperm and human semen

Glutathione $\text{S}$-transferase activity was found in sperm of the rat and $\text{DBA}\text{2J}$ and C57 $\text{BL}\text{6J}$ mice. In rat sperm activities with benzo(a)pyrene 4,5-oxide, styrene 7,8-oxide, and 1-chloro-2,4-dinitrobenzene were 0.88, 1.07, and 26.1 nmoles/min/mg protein, respectively. Δ⁵-3-Ketosteroid isomerase activity of rat sperm was 4.9 nmoles/min/mg protein. These specific glutathione $\text{S}$-transferase and Δ⁵-3-ketosteroid isomerase activities in sperm represent 0.4–4.1% of rat liver cytosol values. Human semen also contained significant glutathione $\text{S}$-transferase activity. It is postulated that these enzymes could function in the metabolism and detoxification of certain electrophilic xenobiotics, if present in sperm.