Regulation of acetylcholine receptor synthesis at the level of translation in rat primary muscle cells.

Previous studies have shown that rat primary muscle cells do not respond to crude rat brain extract or one of its active components, ascorbic acid, with a significant increase in surface acetylcholine receptor (AChR) number. We report here that, although little or no response is seen on the cell surface, rat primary muscle cells do respond to both crude brain extract and to ascorbic acid with an approximately threefold increase in AChR alpha-subunit mRNA. The response of the mRNA is similar to that seen in the cloned L5 cells. However, while in L5 cells the increase in alpha-subunit mRNA is further translated into increased levels of alpha-subunit protein, there is no such increase in alpha-subunit synthesis in the primary cells. This study thus shows a regulation of surface AChR synthesis in rat primary cells at the level of alpha-subunit translation. This level of regulation is different from that involving subunit transcription or subunit assembly reported by others.

The crystallization of beef heart mitochondrial adenosine triphosphatase.

Conditions are described under which crystals are formed with the ATPase enzyme from beef heart mitochondria. Enzyme activity is retained during the crystallization process. Some unit cell parameters have been determined by electron microscopy of negatively stained crystals; comparison with the unit cell crystalline matris inclusions indicates that such inclusions could be ATPase crystals.

Evidence supporting the identity of beef heart mitochondrial chloroform-released adenosine triphosphatase (ATPase) with coupling factor I.

Highly purified mitochondrial chloroform-released beef heart ATPase had molecular weight 330 000, five bands (alpha, beta, gamma, delta, epsilon) in sodium dodecyl sulfate gel electrophoresis and could restore the oxidative-phosphorylation function of A particles. Maximal inhibition (90%) of the enzyme by N,N'-dicyclohexylcarbodiimide was achieved at a molar ratio of inhibitor to protein of 30 : 1. Chloroform introduced into an aqueous solution of beef heart coupling factor I protected it from cold inactivation.

Invited review: Bovine milk fat globule membrane as a potential nutraceutical.

For the last 15 yr, a great deal of knowledge has been accumulated on health beneficial factors, protein and nonprotein, of bovine milk fat globule membrane (MFGM). Among the health-beneficial components of the MFGM are cholesterolemia-lowering factor, inhibitors of cancer cell growth, vitamin binders, inhibitor of Helicobacter pylori, inhibitor of beta-glucuronidase of the intestinal Escherichia coli, xanthine oxidase as a bactericidal agent, butyrophilin as a possible suppressor of multiple sclerosis, and phospholipids as agents against colon cancer, gastrointestinal pathogens, Alzheimer's disease, depression, and stress. All of the above compel us to consider bovine MFGM as a potential nutraceutical.

A selective extraction of growth hormone from bovine pituitary gland and its further purification and crystallization.

A highly efficient method for the isolation of bovine growth hormone (GH) is described. The method is based on selective extraction of GH from 15,000 g subcellular sediment of anterior pituitary gland with 130-150 mM NH4HCO3, 1 mM EGTA, pH 7.2-7.4, at 2-6 degrees C for 60 min, purification of the extracted GH by ammonium sulfate fractionation, one-step ion-exchange column chromatography on DEAE-cellulose (or CM-cellulose), and gel filtration on Sephadex G-75. This 2-3 day procedure provides a highly pure hormone in high yield (up to 70-80 mg per 35-40 g of the whole pituitary gland), which can be crystallized by the batch method at a low ionic strength and isoelectric pH.

Quinone reductases of higher plants.

NAD(P)H: quinone oxidoreductase (DT-diaphorase) was detected in 100000 x g supernatant fractions of extracts of a wide variety of higher plants. Smaller amounts were also found in microsomes and chloroplast fractions. The enzyme was partially purified from soluble extracts of several plants and the quinone reductase from Catharanthus roseus was enriched 25-fold. Plant quinone reductases have molecular weights in the range of 38000-53000 as determined by gel filtration. The plant enzyme is far less sensitive to dicoumarol than its mammalian counterpart and it is inhibited by superoxide dismutase. Quinone reductase is capable of reducing simple p-benzoquinone and naphthoquinone including vitamins K3 and K1. These results indicate that, although the plant enzyme exhibits a similar substrate specificity, it is distinguishable from mammalian DT-diaphorase particularly with respect to its mechanism of reduction.

Solubilization and purification of xanthine oxidase from bovine milk fat globule membrane.

Bovine milk xanthine oxidase (XO) was isolated and purified from milk fat globule membrane (MFGM). The method included the following steps: solubilization of XO from MFGM in 200 mM dithiothreitol (DTT) at pH 8.0, fractionation of solubilized proteins with ammonium sulfate, chromatography on DEAE-Sepharose with gradient elution, and rechromatography of the XO fraction for final purification. The method is highly reproducible, is comparatively simple, and provides highly pure enzyme. Purified XO, analyzed by (8%) SDS-PAGE, had only one band of 140-150 kDa. XO showed a high specific activity of 2.5 units/mg of protein and an A280: A450 ratio of 4.8.

Association and coexpression of fatty-acid-binding protein and glycoprotein CD36 in the bovine mammary gland.

The involvement of glycoprotein CD36 and fatty-acid-binding protein (FABP) in cellular growth, differentiation, lipid transport and metabolism led us to examine the possible biochemical and physiological relationship(s) between these two proteins. We investigated three aspects of this relationship. We first attempted to identify any physical complex formed between CD36 and FABP in bovine milk fat globule membranes. These membranes are the product of mammary gland secretory epithelial cells. The second aspect studied was the effect of synthetic peptide analogs to the C-terminus (amino acid residues 121-131) of bovine mammary gland FABP on cell proliferation, as a result of the interaction of these peptides with the ectodomain of CD36. Finally, mammary gland CD36 and FABP coexpression was defined at different stages of lactation and during involution. Immunoprecipitation, Western immunoblotting with anti-FABP and anti-CD36, Northern-blot analysis and a mammary epithelial cell proliferation assay demonstrated that: (a) bovine milk fat globule membranes contain the complex of CD36 and FABP, and that this complex is, most likely, formed as a result of FABP binding to the cytoplasmic segments of CD36; (b) synthetic analog of the C-terminus of FABP with the sequence Val-Thr-Cys, identical to the sequence found in the CD36-binding domain of thrombospondin, was a more potent inhibitor of bovine mammary gland epithelial cell proliferation than a synthetic peptide with the Val-Cys-Thr sequence; (c) the expression of FABP and CD36 is related to the state of mammary cell differentiation, since it reaches its maximum during lactation and declines during the involutionary period.

Electron microscopy of beef heart F1-ATPase crystals.

The structure of thin crystalline plates of beef heart F1-ATPase has been investigated by a combination of electron microscopy and computer-based image processing. Both negatively stained thin crystals and thin sections of embedded crystals were used in the analysis. Some inherent twinning was observed in the thin crystals and two distinct orthorhombic crystal forms present in the microcrystal population were characterized. The form I crystals are space group P2(1)2(1)2 with unit cell parameters of a = 164 A, b = 324 A, and c = 118 A. The form I crystals have 1 molecule of beef heart coupling factor-ATPase/asymmetric unit and averaged reconstructions of projections of the (001) and (100) planes allowed the deduction of the packing of single F1-ATPase complexes in the crystals. The form II crystals have unit cell parameters of a = 156 A, c = 162 A, beta = 90 degrees and are either space group P2(1)2(1)2 or P222(1). Furthermore, based on the results presented in this report, it is clear that the monoclinic crystalline inclusions which have been observed in human mitochondria are not directly related to the form I or form II crystals of the F1-ATPase.

Characterization of a monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus (L.) G. Don.

Conditions have been established for the optimization of the specific activity of a membrane-bound monoterpene hydroxylase from cell suspension cultures of Catharanthus roseus. In time course studies, the hydroxylase and NADPH-cytochrome c reductase exhibited maximal activities 18-20 days after inoculation, i.e., during early stationary phase. By late stationary phase, enzyme activity had declined. In contrast an enzyme of primary metabolism achieved optimal specific activity by the 12th day and remained constant through day 26, synchronous with general growth. Effects of nutritional and hormonal factors on the specific activity of the hydroxylase and cell growth were evaluated. Inhibitors of hydroxylase activity were also assessed in vitro. A soluble form of the monoterpene hydroxylase has been detected in cultured cells possibly affording a useful source of this enzyme for further purification.

Kinetic Analysis of Corn Mitochondrial F(1)-ATPase.

The activation and catalytic mechanism of corn mitochondrial F(1) were examined for the two distinct forms of the enzyme which appear upon storage in ammonium sulfate or glycerol. Apparently irreversible differences in the stability of the two active forms were found. Nucleosidetriphosphate induced activation of the enzyme was found to produce lasting effects on subsequent catalysis. These effects varied with both the nucleotide used for activation, and the hydrolyzed species. The substrate and metal specificity were examined with the ATP activated enzyme. Mg(2+) and Ca(2+) were found to be the most effective at promoting ATP hydrolysis. The substrates were hydrolyzed in the order GTP > ITP > ATP regardless of which nucleotide was used for activation. While ATP and GTP hydrolysis exhibited kinetics typical of other ATPases, ITP showed a transition from negative to positive cooperativity at low substrate concentrations. Bicarbonate was found to affect primarily the kinetics of ATP hydrolysis. AMP-PNP proved to be a potent inhibitor with respect to ATP hydrolysis. The results are discussed in terms of possible catalytic mechanisms and the similarities of the corn mitochondrial F(1) to other ATPases.

Isolation and Antigenic Characterization of Corn Mitochondrial F(1)-ATPase.

Corn mitochondrial F(1)-ATPase was purified from submitochondrial particles by chloroform extraction. Enzyme stored in ammonium sulfate at 4 degrees C was substantially activated by ATP, while enzyme stored at -70 degrees C in 25% glycerol was not. Enzyme in glycerol remained fully active (8-9 micromoles P(i) released per minute per milligram), while the ammonium sulfate preparations steadily lost activity over a 2-month storage period. The enzyme was cold labile, and inactived by 4 minutes at 60 degrees C. Treatment with octylglucoside resulted in complete loss of activity, while vanadate had no effect on activity. The apparent subunit molecular weights of corn mitochondrial F(1)-ATPase were determined by SDS-polyacrylamide gel electrophoresis to be 58,000 (alpha), 55,000 (beta), 35,000 (gamma), 22,000 (delta), and 12,000 (epsilon). Monoclonal and polyclonal antibodies used in competitive binding assays demonstrated that corn mitochondrial F(1)-ATPase was antigenically distinct from the chloroplastic CF(1)-ATPases of corn and spinach. Monoclonal antibodies against antigenic sites on spinach CF(1)-ATPase beta and gamma subunits were used to demonstrate that those sites were either changed substantially or totally absent from the mitochondrial F(1)-ATPase.