In vitro excystation of Paragonimus heterotremus metacercariae.

In vitro excystation studies were carried out on the metacercariae cysts of Paragonimus heterotremus obtained from naturally infected crabs Potamon spp. The effects of elastase, trypsin, trypsin-dog bile, trypsin-bile salt, and dithiothreitol (DTT) were examined. The trypsin-dog bile medium stimulated maximum excystation. Of the media that contained 1 mM DTT, the optimum conditions for the excystation were shown to be pH 9, temperature of 39-40 C, and osmolarity of 250-350 mOsm. The DTT acceleration was antagonized by all of the following 6 protease inhibitors: leupeptin (0.5-4 microg/ml), L-trans-epoxysuccinyl leucylamido (4-guanidine) butane (1-8 microM), N-tosyl-L-phenylalanine chloromethyl ketone (0.1-0.4 mM), N alpha-p-tosyl-L-lysine chloromethyl ketone (25-200 microg/ml), iodoacetic acid (0.5-4 mM), and phenylmethylsulfonyl fluoride (1-4 mM). These results suggest that a number of extrinsic and intrinsic factors may modulate excystation.     

Pertussis toxin catalyzed ADP-ribosylation of a 41 kDa G-protein impairs insulin-stimulated glucose metabolism in BC3H-1 myocytes

In this study, we examined the effects of pertussis toxin (PT) on the ADP-ribosylation of guanine nucleotide binding proteins (G-proteins) and various insulin-stimulated processes in cultured BC3H-1 myocytes. Treatment of intact myocytes with 0.1 microgram/ml PT for 24 hours resulted in the complete ribosylation of a 41 kDa protein. The 41 kDa PT substrate was immunoprecipitated with antibodies directed against a synthetic peptide corresponding to a unique sequence in the alpha subunit of Gi-proteins. PT treatment of intact cells had no effect on insulin receptor binding or internalization. However, PT inhibited insulin-stimulated glucose transport at all insulin-concentrations tested (1-100 ng/ml). Maximally stimulated glucose transport was reduced by 50% +/- 15%. Insulin-stimulated glucose oxidation was also decreased by 31% +/- 8%. The toxin had no significant effect on the basal rates of glucose transport and glucose oxidation. The time course of PT-induced inhibition on glucose transport correlated with the time course of the "in vivo" ADP-ribosylation of the 41 kDa protein. The results suggest that a 41 kDa PT-sensitive G-protein, identical or very similar to Gi, is involved in the regulation of glucose metabolism by insulin in BC3H-1 cells.     

Protease treatment of pertussis toxin identifies the preferential cleavage of the S1 subunit.

Trypsin digestion of pertussis toxin (PT) preferentially cleaved the S1 subunit at Arg-218 without detectable degradation of the B oligomer. The fragment produced, termed the tryptic S1 fragment, appears to remain associated with the B oligomer. Chymotrypsin digestion of PT also preferentially cleaved the S1 subunit without detectable degradation of the B oligomer. The chymotryptic S1 fragment possessed a slightly lower apparent molecular weight than the tryptic S1 fragment and was more accessible to the respective protease. Trypsin- and chymotrypsin-treated PT and PT required the presence of dithiothreitol and ATP for optimal enzymatic activity. Trypsin-treated PT showed approximately a 2-4-fold higher level of expression of ADP-ribosyltransferase and NAD-glycohydrolase activities than PT. Chymotrypsin-treated PT also exhibited approximately a 2-fold greater level of ADP-ribosyltransferase activity than PT. The observed increase in activity of protease-treated PT was due primarily to a shorter time for activation in PT mediated ADP-ribosylation of transducin. In addition, trypsin-digested PT possessed the same cytotoxic potential for Chinese hamster ovary cell clustering as PT. One possible role for the generation of a proteolytic fragment of the S1 subunit of PT would be to produce a catalytic fragment with increased efficiency for ADP-ribosylation of G proteins in vivo.     

Fluorescence in situ hybridization with Y chromosome-specific probe in decondensed bovine spermatozoa

This study was carried out to demonstrate bovine Y chromosome-bearing spermatozoa by rapid fluorescence in situ hybridization (FISH), using a digoxigenin (Dig)-labeled DNA probe specific to bovine Y chromosome. Before the FISH procedure, sperm heads were treated for decondensation with dithiothreitol (DTT) and glutathione (GSH) with or without heparin supplementation. Concentrations of either above 2 mM DTT or above 100 mM GSH induced swelling of the sperm head, which resulted in sufficient detection of the Y chromosome signal in sperm nuclei by rapid FISH (49.8 to 53.4%). When FISH was used with 2 mM DTT or 100 mM GSH on specimens from 7 sires, the rate of detection of the Y chromosome signal varied among sires (5.4 to 49.6%), especially that of the GSH treatment. Supplementation of GSH with heparin (100 U/mL), however, could induce reliable, repeatable detection of the Y chromosome signal in sperm nuclei of all the 7 sires (48.4 to 50.3%). These results show that in bovine spermatozoa decondensed with GSH and heparin, rapid FISH can detect Y chromosome-bearing spermatozoa.     

Effects of oxidizing and reducing agents on ovine pulmonary artery responses to nitric oxide donors, sodium nitroprusside and 3-morpholino-sydnonimine

Nitrovasodilators-sodium nitroprusside (SNP; 10(-9)-10(-4) M) and 3-morpholino-sydnonimine (SIN-1; 10(-9)-10(-4) M) produced concentration-dependent relaxation of the fourth generation sheep pulmonary artery, preconstricted with 5-hydroxytryptamine (1 microM). Oxidizing agents [oxidized glutathione (GSSG, 1 mM) and CuSO4 (5 and 20 microM)] and reducing agents [dithiothreitol (DTT, 0.1 mM), ascorbic acid (1 mM) and reduced glutathione (GSH, 1 mM)] caused opposite effects on nitric oxide (NO)-induced vasodilation in the artery. Ascorbic acid and GSH potentiated the NO responses, while GSSG and CuSO4 inhibited relaxation caused by the nitrovasodilators. DTT, however, reduced the relaxant potency and efficacy of SNP and SIN-1. Pretreatment of the pulmonary artery strips with DTT (0.1 mM) inhibited SNP (10 microM)-induced Na(+)-K(+)-ATPase activity, while ascorbic acid (1 mM) and GSH (1 mM) had no effect either on basal or SNP (10 microM)-stimulated 86Rb uptake, an index of Na(+)-K(+)-ATPase activity, in ovine pulmonary artery. The results suggest that reducing agents like ascorbic acid may have beneficial effect in improving the vascular function under oxidative stress.     

Stabilization and protection of an enzymatic system, participating in the transformation of beta-carotene into retinal, during its isolation]

The authors studied the effect of dithiothreitol (DTT), carotenoids and protease inhibitors on stabilization and protection of the enzyme catalysing the conversion of beta-carotene into retinal during the enzyme isolation from the rabbit small intestine. The addition of 1 mM DTT into the homogenization mixture increased the activity of the enzyme 5 times compared with control. The additional introduction of 0.7 mg/ml soybean trypsin inhibitor or 2.10(-4) M phenylmethylsulfonyl fluoride increased the enzyme activity 2.1 and 1.2 times, respectively. Lutein, beta-carotene and lycopene at a concentration of 10 mg/ml increased the enzyme activity 2.1, 1.9 and 1.6 times respectively. The effects of DTT, lutein and the protease inhibitor depended on their concentrations and was of an independent additive character. The maximum activity of the isolated enzyme exceeded the control without DTT 15 times.     

Possible coupling of prostaglandin E2 receptor with pertussis toxin-sensitive guanine nucleotide-binding protein in osteoblast-like cells.

In cloned osteoblast-like cells, MC3T3-E1, prostaglandin E2 (PGE2) stimulated the formation of inositol phosphates in a dose-dependent manner in the range between 10 nM and 10 microM. Pertussis toxin inhibited the effect of PGE2 dose-dependently in the range between 1 ng/ml and 1 micrograms/ml. In the cell membranes, pertussis toxin catalyzed ADP-ribosylation of a protein with an Mr of about 40,000. Pretreatment of membranes with 10 microM PGE2 in the presence of 2.5 mM MgCl2 and 100 microM GTP markedly attenuated this pertussis toxin-catalyzed ADP-ribosylation of the protein in a time-dependent manner. G12 was detected in these cells by immunoblotting with purified anti-G12 alpha antibodies. The results indicate the possible coupling of PGE2 signalling with pertussis toxin-sensitive GTP-binding protein, which is probably G12, in osteoblast-like cells.     

NMR measurements of the diffusional permeability of water in cultured colonic epithelial cancer cells

The water residence time and diffusional water permeability in colonic epithelial T84 cancer cells was measured using (1)H NMR spectroscopy; the values estimated were 35.2+/-2.8 ms and (7.4+/-0.6)x10(-3)cms(-1), respectively. Water permeability was inhibited to approximately 10% of its original value by the mercurial diuretic, p-chloromercuribenzenesulfonate (PCMBS; 1mM), and fully restored by dithiothreitol (DTT; 1mM). The permeability was also inhibited reversibly to approximately 55%, by extracellular glibenclamide (1mM), an inhibitor of some ATP-binding cassette (ABC) transporters, including the cystic fibrosis transmembrane conductance regulator (CFTR). Addition of the phosphodiesterase inhibitor, 3-isobutyl-1-methylxanthine (IMBX; 0.1-1mM) and the adenylate cyclase activator, forskolin (0.1-1mM) did not alter water permeability. It is concluded that in T84 cells water diffuses through the membrane lipid bilayer and via channels that are inhibited by PCMBS, including the channels that are known to be inhibited by glibenclamide.     

Redox modification of sodium-calcium exchange activity in cardiac sarcolemmal vesicles

Na-Ca exchange activity in bovine cardiac sarcolemmal vesicles was stimulated up to 10-fold by preincubating the vesicles with 1 microM FeSO4 plus 1 mM dithiothreitol (DTT) in a NaCl medium. The increase in activity was not reversed upon removing the Fe and DTT. Stimulation of exchange activity under these conditions was completely blocked by 0.1 mM EDTA or o-phenanthroline; this suggests that the production of reduced oxygen species (H2O2, O2-.,.OH) during Fecatalyzed DTT oxidation might be involved in stimulating exchange activity. In agreement with this hypothesis, the increase in exchange activity in the presence of Fe-DTT was inhibited 80% by anaerobiosis and 60% by catalase. H2O2 (0.1 mM) potentiated the stimulation of Na-Ca exchange by Fe-DTT under both aerobic and anaerobic conditions; H2O2 also produced an increase in activity in the presence of either FeSO4 (1 microM) or DTT (1 mM), but it had no effect on activity by itself. Superoxide dismutase did not block the effects of Fe-DTT on exchange activity; however, the generation of O2-. by xanthine oxidase in the presence of an oxidizable substrate stimulated activity more than 2-fold. Hydroxyl radical scavenging agents (mannitol, sodium formate, sodium benzoate) did not attenuate the stimulation of activity observed with Fe-H2O2. Exchange activity was also stimulated by the simultaneous presence of glutathione (GSH; 1-2 mM) and glutathione disulfide (GSSG; 1-2 mM). Neither GSH nor GSSG was effective by itself and either 0.1 mM EDTA or o-phenanthroline blocked the effects on transport activity of the combination of GSH + GSSG. Treatment of the GSH and GSSG solutions with Chelex ion-exchange resin to remove contaminating transition metal ions reduced (by 40%) the degree of stimulation observed with GSH + GSSG. Full stimulating activity was restored to the Chelex-treated GSH and GSSG solutions by the addition of 1 microM Fe2+; Cu2+ was less effective than Fe2+ whereas Co2+ and Mn2+ were without effect. In the presence of 1 microM Fe2+, GSH alone produced a slight increase in transport activity, but this was markedly enhanced by the addition of Chelex-treated GSSG. The results indicate that stimulation of exchange activity requires the presence of both a reducing agent (DTT, GSH, O-.2, or Fe2+) and an oxidizing agent (H2O2, GSSG, and perhaps O2) and that the effects of these agents are mediated by metal ions (e.g. Fe2+).(ABSTRACT TRUNCATED AT 400 WORDS)     

Muscarinic cholinergic stimulation of inositol phosphate production in cultured embryonic chick atrial cells. Evidence for a role of two guanine-nucleotide-binding proteins.

These studies demonstrate a novel mechanism for the coupling of the muscarinic receptor to phospholipase C activity in embryonic chick atrial cells. In monolayer cultures of atrial cells from hearts of embryonic chicks at 14 days in ovo, carbamylcholine stimulated the sequential appearance of InsP3, InsP2 and InsP1 with an EC50 (concn. causing 50% of maximal stimulation) of 30 microM. In the presence of 15 mM-Li, a 5 min exposure to carbamylcholine (0.1 mM) increased InsP3 levels to a maximum of 47 +/- 12% over basal, InsP2 to 108 +/- 13% over basal and InsP1 to 42 +/- 5% over basal. This effect was blocked by 5 microM-atropine. Incubation of these cells with pertussis toxin (15 h; 0.5 ng/ml) inhibited carbamylcholine-stimulated InsP3, InsP2 and InsP1 formation by 42 +/- 7%, 30 +/- 3% and 48 +/- 7% respectively. The IC50 (concn. causing 50% inhibition) for pertussis toxin inhibition of all three inositol phosphates was 0.01 ng/ml, with a half-time of 6 h at 0.5 ng/ml. This partial sensitivity to pertussis toxin was not due to incomplete ADP-ribosylation of the guanine-nucleotide-binding protein (G-protein), since autoradiography of polyacrylamide gels of cell homogenates incubated with [32P]NAD+ in the presence of pertussis toxin demonstrated that incubation of cells with 0.5 ng of pertussis toxin/ml for 15 h resulted in complete ADP-ribosylation of pertussis toxin substrates by endogenous NAD+. In cells permeabilized with saponin (10 micrograms/ml), 0.1 mM-GTP[S] (guanosine 5'-[gamma-thio]triphosphate) stimulated InsP1 by 102 +/- 15% (mean +/- S.E.M., n = 4), InsP2 by 421 +/- 67% and InsP3 by 124 +/- 33% above basal. Incubation of cells for 15 h with 0.5 ng of pertussis toxin/ml decreased GTP[S]-stimulated InsP1 production in saponin-treated cells by 30 +/- 10% (n = 3), InsP2 production by 45 +/- 7% (n = 4) and InsP3 production by 49 +/- 6% (n = 4). These data demonstrate that in embryonic chick atrial cells at least two independent G-proteins, a pertussis toxin-sensitive G-protein and a pertussis toxin-insensitive G-protein, play a role in coupling muscarinic agonist binding to phospholipase C activation and to inositol phosphate production.     

Effects of DTT and thioredoxins on rat argininosuccinate synthetase activity in vitro

Argininosuccinate synthetase (ASS, EC 6.3.4.5), the third enzyme of urea-cycle, was studied in desactivated extracts of rat liver. The enzyme is activated, in vitro, by Mg2+ ions (5 mM) and dithiothreitol (DTT: 10 mM). After reduction by DTT, thioredoxins isolated from rat liver were able to activate ASS by 370%.     

Coimmobilization of lactate dehydrogenase and low molecular weight thiols on sepharose

Lactate dehydrogenase (EC 1.1.1.27) and dithiothreitol (DTT) were coimmobilized on Sepharose activated with cyanogen bromide. It was demonstrated that addition of 10 mM DTT (but not 2-mercaptoethanol) during immobilization increased the enzyme specific activity 1.5-5-fold, depending on the initial extent of Sepharose activation by cyanogen bromide. The total activity increased two- to threefold. The lactate dehydrogenase preparations were rich in matrix-immobilized sulfhydryl groups (1.8-13.0 nmol per ml gel). The presence of DTT increased the stability of immobilized lactate dehydrogenase.     

Coimmobilization of Lactate Dehydrogenase and Low-Molecular-Weight Thiols on Sepharose

Lactate dehydrogenase (EC 1.1.1.27) and dithiothreitol (DTT) were coimmobilized on Sepharose activated with cyanogen bromide. It was demonstrated that the addition of 10 mM DTT (but not 2-mercaptoethanol) during immobilization increased the enzyme specific activity 1.5–5-fold depending on the initial extent of Sepharose activation by cyanogen bromide. The total activity increased two- to threefold. The lactate dehydrogenase preparations were rich in matrix-immobilized sulfhydryl groups (1.8–13.0 nmol per ml gel). The presence of DTT increased the stability of immobilized lactate dehydrogenase.     

Solubilization of proteins from human lymph node tissue and two-dimensional gel storage

In the present study, we compared six different solubilization buffers and optimized two-dimensional electrophoresis (2-DE) conditions for human lymph node proteins. In addition, we developed a simple protocol for 2-D gel storage. Efficient solubilization was obtained with lysis buffers containing (a) 8 M urea, 4% CHAPS (3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate), 40 mM Tris base, 65 mM DTT (dithiothreitol) and 0.2% carrier ampholytes; (b) 5 M urea, 2 M thiourea, 2% CHAPS, 2% SB 3-10 (N-decyl-N,N-dimethyl-3-ammonio-1-propanesulfonate), 40 mM Tris base, 65 mM DTT and 0.2% carrier ampholytes or (c) 7 M urea, 2 M thiourea, 4% CHAPS, 65 mM DTT and 0.2% carrier ampholytes. The optimal protocol for isoelectric focusing (IEF) was accumulated voltage of 16,500 Vh and 0.6% DTT in the rehydration solution. In the experiments conducted for the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), best results were obtained with a doubled concentration (50 mM Tris, 384 mM glycine, 0.2% SDS) of the SDS electrophoresis buffer in the cathodic reservoir as compared to the concentration in the anodic reservoir (25 mM Tris, 192 mM glycine, 0.1% SDS). Among the five protocols tested for gel storing, success was attained when the gels were stored in plastic bags with 50% glycerol. This is the first report describing the successful solubilization and 2D-electrophoresis of proteins from human lymph node tissue and a 2-D gel storage protocol for easy gel handling before mass spectrometry (MS) analysis.     

A versatile transition metal salt reaction for a wide range of common biochemical reagents: an instantaneous and quantifiable color test

A rapid and sensitive spot test amenable to visual or spectrophotometric quantitation has been developed for a wide variety of biochemical reagents by utilizing the transition metal salt cupric chloride and its large number of related colored compounds. This assay is potentially a widely applicable multipurpose test for rapidly detecting the presence of unknown substances. Combination of the test sample with the working reagent results in the immediate formation of a distinctive colored product that may be precipitable. Some compounds require the further addition of sodium hydroxide in order to generate the distinctively colored product. Distinctive reactions occur with the following reagents, and their limit of visual detection is indicated in parentheses: ammonium bicarbonate (12.5 mM), ammonium acetate (25 mM), ammonium hydroxide (0.1%), ammonium sulfate (2%), ammonium persulfate (0.02 mM), L-(+)-cysteine (0.07 mM), dithiothreitol (DTT) (1.25 mM), EDTA (0.6 mM), ethylene glycol bis(beta-aminoethyl ether) N,N'-tetraacetic acid (5 mM), D-glucose (6 mM), glycerol (0.3%), imidazol (12.5 mM), DL-methionine (100 mM), mercaptoethanol (0.05%), sodium azide (19 mM, 0.1%), sodium dithionite (0.25%), sodium metabisulfite (25 mM), sodium nitrite (6.2 mM), sodium periodate (3.1 mM), sodium sulfite (12.5 mM), sodium thiosulfite (12.5 mM), sucrose (6 mM), and N,N,N',N'-tetramethylethylenediamine (0.05%). A distinctive exothermic reaction occurs with hydrogen peroxide, but without color change. Compounds reacting insignificantly include 50 mM Tris buffer, urea, N,N'-methylene bisacrylamide, sodium dodecyl sulfate, isopropyl alcohol, sodium fluoride, trichloroacetic acid, phenol, mannose, K2HPO4, guanidine HCl, chloramine-T, magnesium chloride, and boric acid, where the solids were tested at approximately 10 mg/ml. Spectrophotometric standard curves were developed for DTT and sodium azide utilizing the clear supernatants resulting from these reactions. Combinations of at least four reagents could be discriminated, as demonstrated with mixtures of glucose, sodium azide, EDTA, and DTT. In addition ammonium sulfate could be detected to a limit of 4% in the presence of protein, DTT, and EDTA in a 50 mM Tris buffer. Spot tests were developed which utilized reagent-impregnated filter paper and gave distinctive colored products on addition of 5 microliter of test sample.     

Toxicity of the sulfhydryl-containing radioprotector dithiothreitol

The toxicity of the sulfhydryl-containing radioprotective agent dithiothreitol (DTT) has been studied using Chinese hamster V79 cells growing in monolayer in minimal essential medium containing 10% fetal calf serum. DTT at low concentrations (between 0.4 and 1.0 mM) caused cell killing, but higher concentrations (above 2 mM) or lower concentrations (0.1 mM) did not. This DTT-induced toxicity was prevented by catalase, glutathione, the use of serum-free medium, or lowering incubation temperature; was slightly decreased by dimethyl sulfoxide; and was enhanced by some metal chelators but prevented by desferal, an iron chelator. Experiments involving simultaneous exposure of cells to DTT and H2O2 showed that low concentrations of DTT enhanced H2O2-induced toxicity, but high concentrations of DTT prevented the H2O2 toxicity. These results are consistent with the proposal that toxicity results from autoxidation of DTT to produce H2O2, which in turn reacts via the metal-catalyzed Fenton reaction to produce the ultimate toxin, .OH radicals, although chemical studies show that rates of autoxidation of various sulfhydryl compounds do not correlate with the observed toxicity.     

Thionamides and arsenite inhibit specific T3 binding to the hepatic nuclear receptor

Methimazole (MMI) and propylthiouracil (PTU) are widely used for the treatment of Graves' disease. However, no studies have been reported on the action of these drugs on binding of L-triiodothyronine (T3) to the nuclear receptor. T3 receptors of rat liver nuclei, prepared by differential centrifugation, were extracted with 0.4 M KCl and 5 mM dithiothreitol (DTT). In the assessment of T3 binding to the DTT-reduced receptor, the hepatic nuclear extract was chromatographed on Superose 6 to remove DTT and isolate proteins of relative mass approximately 50,000 (chromatographed nuclear receptors (CNRs)), prior to the addition of [125I]T3 of high specific activity (3300 microCi/micrograms; 1 Ci = 37 GBq). MMI or PTU at 2 mM reduced specific T3 binding to CNR by 84% and 85%, respectively. The inhibitory effects of these reagents and 2 mM sodium arsenite (which complexes dithiols) were additive. Scatchard analyses indicated that neither MMI nor PTU (at 2 mM) significantly altered the affinity constant (Ka) (from 2.41 x 10(9) to 1.74 x 10(9) M-1 for PTU and 1.79 x 10(9) M-1 for MMI), while they both decreased (p less than 0.02) maximal binding capacity (from 0.36 +/- 0.02 to 0.19 +/- 0.02 pmol/mg protein for MMI and 0.17 +/- 0.02 pmol/mg protein for PTU). Dose-response curves showed that 50% inhibition was attained at 0.6 mM PTU or 1.0 mM MMI with approximately 25% inhibition by both at 0.1 mM. Artefactual binding effects by MMI and PTU on [125I]T3 were excluded by chromatography experiments. Similar results were obtained using nuclear receptors prepared from livers of hyperthyroid rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Oxidation and reduction of pig skeletal muscle ryanodine receptors

Time-dependent effects of cysteine modification were compared in skeletal ryanodine receptors (RyRs) from normal pigs and RyR(MH) (Arg(615) to Cys(615)) from pigs susceptible to malignant hyperthermia, using the oxidizing reagents 4,4'-dithiodipyridine (4, 4'-DTDP) and 5,5'-dithio-bis(2-nitrobenzoic acid) (DTNB) or the reducing agent dithiothreitol (DTT). Normal and RyR(MH) channels responded similarly to all reagents. DTNB (1 mM), either cytoplasmic (cis) or luminal (trans), or 1 mM 4,4'-DTDP (cis) activated RyRs, introducing an additional long open time constant. 4,4'-DTDP (cis), but not DTNB, inhibited channels after >5 min. Activation and inhibition were relieved by DTT (1-10 mM). DTT (10 mM, cytoplasmic or luminal), without oxidants, activated RyRs, and activation reversed with 1 mM DTNB. Control RyR activity was maintained with 1 mM DTNB and 10 mM DTT present on the same or opposite sides of the bilayer. We suggest that 1) 4,4'-DTDP and DTNB covalently modify RyRs by oxidizing activating or inhibiting thiol groups; 2) a modified thiol depresses mammalian skeletal RyR activity under control conditions; 3) both the activating thiols and the modified thiols, accessible from either cytoplasm or lumen, reside in the transmembrane region; 4) some cardiac sulfhydryls are unavailable in skeletal RyRs; and 5) Cys(615) in RyR(MH) is functionally unimportant in redox cycling.     

Phosphatidylcholine stimulates the activity of UDP-Gal beta 1-4 galactosyltransferase in normal human kidney proximal tumour cells

Effects of various lipid components of low density lipoproteins (LDL) and serine on the regulation of UDP-Gal-beta 1-4-galactosyltransferase (GalT-2) activity have been investigated in normal proximal tubular (PT) cells. Addition of exogenous serine (0.1-0.75 mM), cholesterol (0-200 micrograms/ml medium), linoleic acid and oleic acid (0.1-0.75 mM) for 4 hr at 37 degrees C did not suppress the activity of GalT-2 in PT cells. Similarly, incubation of cells with glucosylceramide and lactosylceramide (25-50 micrograms/ml medium) did not alter GalT-2 activity in cells as compared to control. In contrast, palmitic acid (0-0.75 mM), phosphatidylethanolamine and sphingomyelin (0-200 micrograms/ml) stimulated GalT-2 activity by 20-36% as compared to control. Incubation of PT cells with D-alpha-dipalmitoyl phosphatidylcholine (0-200 micrograms/ml medium) also stimulated the activity of GalT-2, maximum stimulation (200%) occurring with 25 micrograms phosphatidylcholine/ml medium. However, at a higher concentration (200 micrograms/ml), the stimulation of the activity of GalT-2 was in the order of 27% compared to control. Dioleylphosphatidylcholine did not alter GalT-2 activity in PT cells. Thus, it is concluded that (i) various lipid components, sphingosine and serine present in LDL are not involved in the LDL-mediated suppression of GalT-2 activity in normal PT cells, and (ii) stringent structural requirements in the phosphatidylcholine molecule are necessary to exert a time and concentration dependent stimulation of GalT-2 activity.     

Electron microscopy of human factor VIII/Von Willebrand glycoprotein: effect of reducing reagents on structure and function

The structure of native and progressively reduced human factor VIII/von Willebrand factor (FVIII/vWF) was examined by electron microscopy and SDS gel electrophoresis and then correlated with its biological activities. Highly resolved electron micrographs of well-spaced, rotary- shadowed FVIII/vWF molecules showed their structure to consist of a very flexible filament that contains irregularly spaced small nodules. Filaments ranged from 50 to 1,150 nm with a mean length of 478 nm and lacked fixed, large globular domains as seen in fibrinogen and IgM. A population of multimeric FVIII/vWF species ranging in molecular weight from 1 to 5 million daltons and differing in size alternately by one and two subunits was observed on SDS-2% polyacrylamide-0.5% agarose gel electrophoresis. With progressive reduction of disulfide bonds by dithiothreitol (DTT), the electron microscopic size of FVIII/vWF decreased in parallel with increased electrophoretic mobility on SDS- agarose gels; between 0.1 and 0.5 mM DTT its structure changed from predominantly fibrillar species to large nodular forms. A 50% loss of vWF specific activity and FVIII procoagulant activity occurred at 0.4 mM DTT and 1 mM DTT, respectively, corresponding to the reduction of 4 and 12 disulfide bonds of the 62 disulfides per 200,000-dalton subunit. We conclude that reduction of a few critical disulfide bonds results in a major structural change by electron microscopy and a concomitant loss of approximately 50% of the vWF function.