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Query: UMLS:C0027960 (mole)
 21,279 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Treatment of bovine thyroid with the non-ionic detergent Triton X-100 extracts most of the cell protein and leaves insoluble residue. This Triton-insoluble cytoskeleton consists of five major polypeptides on sodium dodecyl sulfate polyacrylamide gels. One of these polypeptides is actin. Based on DNase inhibition assay, 30% of the total actin is associated with the cytoskeleton as the filamentous form. Thyroid actin from the cytoskeleton has been solubilized by dialysis against a low ionic strength buffer at pH 8.0 and purified to homogeneity by a polymerizing-depolymerizing cycle. The overall purification was about 144-fold with a yield of 10%. Bovine thyroid actin is very similar to actins from other tissues on the basis of: (1) comigration with rabbit skeletal muscle actin during gel electrophoresis in sodium dodecyl sulfate, (2) its amino acid composition, which includes about 1 mole of 3-methylhistidine per 42,000 g, (3) its ability to bind and inhibit pancreatic deoxyribonuclease I, and (4) its ability to form 7-8 nm microfilaments which is similar to that of skeletal filamentous actin. Thyroid actin contains beta- and gamma-isoactins, with isoelectric points more alkaline than the alpha-actin of rabbit skeletal muscle.
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PMID:Actin in Triton-insoluble cytoskeleton of thyroid. 628 13

Neurotoxins that act at specific receptor sites on voltage-sensitive sodium channels have been used as molecular probes to identify and purify protein components of sodium channels from mammalian brain. Photoreactive derivatives of scorpion toxin have been prepared and used to covalently label sodium channels in intact synaptosomes. Two polypeptides, alpha with Mr approximately 270,000 and beta with Mr approximately 38,000, are specifically labeled indicating that they are components of the scorpion toxin receptor site on the sodium channel. The sodium channel can be solubilized with retention of specific binding of [3H] saxitoxin using nonionic detergents such as Triton X-100. The solubilized saxitoxin receptor has molecular weight of 316,000 +/- 63,000 and binds 0.9 g of Triton X-100 and phospholipid per g of protein. The solubilized receptor can be purified 750-fold by ion exchange chromatography, wheat germ lectin/Sepharose chromatography and sucrose gradient sedimentation to a final specific activity of 1488 pmol/mg. Analysis of the polypeptide chain composition of the most highly purified fractions indicates that alpha and beta comprise 65% of the protein of these fractions and are only the polypeptides whose presence correlates with saxitoxin binding activity. These studies lead to a working hypothesis of sodium channel structure in which the intact channel is comprised of a complex with Mr of approximately 316,000 containing one mole of alpha (Mr approximately 270,000) and one to three moles of beta (Mr approximately 38,000).
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PMID:Molecular Properties of neurotoxin receptors sites associated with sodium channels from mammalian brain. 628 40

In cultured melanotic melanoma, a marked decrease of pigmentation has been found to be induced by the addition of tunicamycin [Y. Mishima and G. Imokawa (1983) J. Invest. Dermatol. 81, 106-114]. Since it appears that this impaired pigmentation arises from the loss of asparagine-linked sugar chains serving as a signal for transport of tyrosinase from GERL (Golgi-associated endoplasmic reticulum of lysosomes) to premelanosomes, tyrosinases from the membrane fraction of Greene's hamster melanoma have been purified, and the structures of their sugar chains have been analyzed. Two kinds of tyrosinases were purified by Triton X-100 solubilization; DEAE-cellulose, Sephadex G-200, and DEAE-Sephadex column chromatography; and preparative polyacrylamide gel electrophoresis. The two tyrosinases were separated by polyacrylamide gel electrophoresis, and both corresponded to Mr 69,000. Their asparagine-linked sugar chains were released by hydrazinolysis and analyzed. The sugar chains of the two tyrosinases were identical except for the sialic acid contents. One mole of each tyrosinase contained 1 mol of high-mannose-type sugar chains and 3 mol of complex-type sugar chains. The former chain has Man3 approximately 5 X GlcNAc2 and the latter has Man3 X GlcNAc beta 1----4(+/- Fuc alpha 1----6)GlcNAc as their core structures. The complex-type sugar chains are composed of mono-, bi-, tri-, and tetraantennary sugar chains, with +/- Sia alpha 2----3Gal beta 1----4GlcNAc beta 1----as their outer chains.
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PMID:Purification of hamster melanoma tyrosinases and structural studies of their asparagine-linked sugar chains. 643 39

The effect of apolipoprotein C-II (apoC-II) on the bovine milk lipoprotein lipase (LpL)-catalyzed hydrolysis of a homologous series of saturated phosphatidylcholines was examined with respect to the fatty acyl chain length of the substrates. Dilauryl-, dimyristoyl-, dipalmitoyl-, and distearoylphosphatidylcholine solubilized by Triton X-100 and sonicated vesicles of dimyristoylphosphatidylcholine were used as substrates. The maximal rate of the LpL-catalyzed hydrolysis of each of these lipids was determined in the absence and presence of apoC-II. The activation factor (the ratio of enzyme activity with apoC-II to that without the activator protein) increased with increasing mol ratios of apoC-II to LpL and was maximal at a ratio of approximately 50. At all apoC-II/LpL mole ratios tested, the activation factor increased as a function of fatty acyl chain length. A quantitative relationship between fatty acyl chain length and the extent of maximal activation of LpL by apoC-II was observed: the logarithm of the activation factor is a linear function of the number of carbon atoms of a single fatty acyl chain of the substrates.
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PMID:Chain length dependence of phosphatidylcholine hydrolysis catalyzed by lipoprotein lipase. Effect of apolipoprotein C-II. 664 74

A kinetic scheme is proposed for the action of cobra venom phospholipase A2 on mixed micelles of phospholipid and the nonionic detergent Triton X-100, based on the "dual phospholipid model." (formula; see text) The water-soluble enzyme binds initially to a phospholipid molecule in the micelle interface. This is followed by binding to additional phospholipid in the interface and then catalytic hydrolysis. A kinetic equation was derived for this process and tested under three experimental conditions: (i) the mole fraction of substrate held constant and the bulk substrate concentration varied; (ii) the bulk substrate concentration held constant and the Triton X-100 concentration varied (surface concentration of substrate varied); and (iii) the Triton X-100 concentration held constant and the bulk substrate concentration varied. The substrates used were chiral dithiol ester analogs of phosphatidylcholine (thio-PC) and phosphatidylethanolamine (thio-PE), and the reactions were followed by reaction of the liberated thiol with a colorimetric thiol reagent. The initial binding (Ks = k1/k-1) was apparently similar for thio-PC and thio-PE (between 0.1 and 0.2 mM) as were the apparent Michaelis constants (Km = (k-2 + k3)/k2) (about 0.1 mol fraction). The Vmax values for thio-PC and thio-PE were 440 and 89 mumol min-1 mg-1, respectively. The preference of cobra venom phospholipase A2 for PC over PE in Triton X-100 mixed micelles appears to be an effect on k3 (catalytic rate) rather than an effect on the apparent binding of phospholipid in either step of the reaction.
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PMID:Kinetic analysis of the dual phospholipid model for phospholipase A2 action. 671 70

Bilirubin diglucuronide, the major pigment in human bile is formed in two steps. Bilirubin is converted to bilirubin monoglucuronide by transfer of the glucuronosyl moiety of uridine diphosphoglucuronic acid catalyzed by the microsomal enzyme, uridine diphosphoglucuronate glucuraonosyl transferase (UDP glucuronyl transferase, EC 2.4.1.17). Bilirubin monoglucuaronide is converted to bilirubin diglucuronide in vitro by two enzymatic mechanisms: (a) UDP glucuronyl transferase-mediated transfer of a second mole of glucuronic acid form UDP-glucuronic acid to bilirubin monoglucuronide; (b) dismutation of 2 moles of bilirubin monoglucuronide to 1 mole of bilirubin diglucuronide and 1 mole of unconjugated bilirubin, catalyzed by bilirubin monoglucuronide dismutase (bilirubin glucuronoside glucuronosyl transferase EC 2.4.1.95). Assay methods for the three enzymatic mechanisms in human liver homogenate by high-pressure liquid chromatography analysis of underivatized bilirubin tetrapyrroles have been developed. UDP glucuronyl transferase was activated in five human liver homogenates with digitonin, Triton X-100, or UDP-N-acetylglucosamine. Greatest activation was observed with Triton X-100. The pH optimum for conversion of bilirubin to bilirubin monoglucuronide was 7.4, and UDP glucuronyl transferase activity was 625 +/- 51 nmoles per 20 min per gm liver. At high initial bilirubin concentrations (342 microM), the product of UDP glucuronyl transferase assay with bilirubin as substrate was predominantly bilirubin monoglucuronide. At lower initial bilirubin concentrations (6.5 to 34 microM), up to 15% bilirubin diglucuronide was formed. Glucuronyl transferase-mediated UDP glucuronic acid-dependent conversion of bilirubin monoglucuronide to diglucuronide was assayed using UDP-14-C-glucuronic acid. The pH optimum was 7.4, and the rate was 21 +/- 7 nmoles per gm liver per 20 min. The rate of bilirubin diglucuronide formation by enzymatic dismutation of bilirubin monoglucuronide was 470 +/- 112 nmoles per gm liver per min. The pH optimum was 6.6. The products of enzymatic dismutation were of the IX alpha configuration.
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PMID:Bilirubin mono- and diglucuronide formation by human liver in vitro: assay by high-pressure liquid chromatography. 679 86

Band 3 protein extracted from human erythrocyte membranes by Triton X-100 was recombined with the major classes of phospholipid occurring in the erythrocyte membrane. The resulting vesicle systems were characterized with respect to recoveries, phospholipid composition, protein content and vesicle size as well as capacity and activation energy of sulfate transport. Transport was classified into band-3-specific fluxes and unspecific permeability by inhibitors. Transport number (sulfate ions per band 3 per minute) served as a measure of functional therapy after reconstitution. The transport properties of band 3 proved to be insensitive to replacement of phosphatidylcholine by phosphatidylethanolamine, while sphingomyelin and phosphatidylserine gradually inactivated band-3-specific anion transport when present at mole fractions exceeding 30 mol%. The activation energy of transport remained unaltered in spite of the decrease in transport numbers. The results, which are discussed in terms of requirements of band 3 protein function with respect to the fluidity and surface charge of its lipid environment, provide a new piece of evidence that the transport function of band 3 protein depends on the properties of its lipid environment just as the catalytic properties of some other membrane enzymes. The well-established species differences in anion transport (Gruber, W. and Deuticke, B. (1973) J. Membrane Biol. 13, 19-36) may to some extent reflect this lipid dependence.
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PMID:Phospholipid dependence of the anion transport system of the human erythrocyte membrane. Studies on reconstituted band 3/lipid vesicles. 683 Jul 94

Phosphatidylserine decarboxylase from Escherichia coli, an intrinsic membrane protein, catalyzes the conversion of phosphatidylserine to phosphatidylethanolamine. The physical and kinetic properties of the purified enzyme were studied in several detergents under assay conditions. The active form of the enzyme is an oligomer, probably a trimer, and the enzyme activity was unaffected by the concentration of the nonionic poly(oxyethylene) ether detergent present in the assay medium, so long as the detergent micelle/substrate mole ratio was less than one. When this ratio was greater than one, the detergent acted as an inhibitor by competing with enzyme-containing micelles for substrate. The zwitterionic and bile salt detergents that were tested inactivated the enzyme by dissociating the oligomer. The native, Triton X-100 solubilized, enzyme was modified with a cross-linking reagent. Activity of the cross-linked enzyme was retained after the Triton X-100 was replaced by a zwitterionic sulfobetaine detergent and conformed to the same kinetic model as with the poly(oxyethylene) ether detergents. The cross-linked enzyme was also active when solubilized by the bile salt detergents although the activity did not conform to any simple kinetic model. These data indicate that the oligomer is the active form of the enzyme under assay conditions and that certain nondenaturing detergents can inactivate this enzyme by dissociating the enzyme complex.
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PMID:Kinetics and protein subunit interactions of Escherichia coli phosphatidylserine decarboxylase in detergent solution. 701 77

The structure of the major protein constituent of photosynthetic membranes in higher plants, the chlorophyll a/b-light harvesting complex (LHC), was studied by x-ray diffraction and electron microscopy. The LHC was purified from Triton X-100 solubilized thylakoid membranes of the pea, and contained 6 mol of chlorophylls a and b per mole of a polypeptide of 27,000 molecular weight. X-ray diffraction showed that in the presence of 10 mM MgCl2, purified LHC forms planar aggregates that stack with a period of 51 A. Within each layer, LHC molecules pack with a center-to-center distance of 85 A but without long-range order. However, when LHC is incorporated into single-walled vesicles of plant lecithin, the addition of NaCl above 10 mM, or MgCl2 above 2 mM, led to the formation of plaques of hexagonal lattices, with a lattice constant of 125 A. The large domain size and high degree of order in the plane of the membrane are evident from the sharp lattice lines observed to 7 A resolution on the equator of the x-ray pattern. Freeze-fracture electron micrographs demonstrated an aligned stacking of the lattices in adjacent membranes, resulting in crystallinity in the third dimension over short distances. Micrographs of negatively stained membranes revealed a hexagonal lattice of the same lattice constant, formed by surface-exposed parts of the LHC molecules which are probably responsible for the ordered stacking of lattices. In both the LHC aggregates and in the reconstituted membrane lattices the diffracted x-ray intensities at 10-A spacing on the equator indicate that the LHC molecule contains paralled alpha-helices or beta-sheets that are oriented perpendicular to the planar arrays.
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PMID:Formation of crystalline arrays of chlorophyll a/b - light-harvesting protein by membrane reconstitution. 703 99

After treatment of red cell ghosts with chymotrypsin, the predominant intrinsic peptides remaining in the membrane fraction are 15,000 and 9,000 daltons mol wt. After partial extraction with Triton X-100, the residual membrane vesicles have almost no other stained peptides and such vesicles are reported to carry out anion transport activities sensitive to specific inhibitors. In vesicles derived from cells treated with DIDS(4,4'-diisothiocyano-2,2'-stilbene disulfonic acid), an irreversible inhibitor of anion transport that is highly localized in an abundant intrinsic protein known as band 3, the probe is largely recovered in the 15,000 dalton peptide. The part of band 3 from which it is derived is a previously reported 17,000 transmembrane segment (Steck, T.L., Ramos, R., Strapazon, E., 1976, Biochemistry 15:1154). The 9,000-dalton peptide is present in the vesicles in a one-to-one mole ratio with the 15,000-dalton peptide, suggesting that both are derived from the same protein. This conclusion is supported by the finding that the 35,000-dalton C-terminal end of band 3, derived by chymotrypsin treatment of cells, is further proteolysed if the cells are converted to ghosts and its disappearance coincides with the appearance of the 9,000-dalton fragment. Evidence is presented that the 9,000-dalton fragment crosses the bilayer and that it is closely associated with the 15,000-dalton peptide.
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PMID:Intrinsic segments of band 3 that are associated with anion transport across red blood cell membranes. 720 45


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