Antithrombin III binds to human platelets

The receptor site for antithrombin III (AT III) was investigated in normal human platelets. [¹²⁵I] iodinated AT III was utilized as tracer for the binding assay. Equilibrium of AT III binding was reached within 2 min. The binding capacity was pH-dependent with the optimum around pH 7.0. Binding specificity was demonstrated by inhibition of [¹²⁵I] AT III ligation using an excess amount of non-labeled AT III. The AT III·heparin complex did not supress [¹²⁵I] AT III binding. Analysis of binding data by Scatchard plot revealed a single class of binding sites with K_d of 3.2 × 10^?7 M and binding capacity of 3840 per platelet.     

Characterization of the filamentous hemagglutin from Bordetella pertussis by gel electrophoresis

Summary A highly purified preparation of filamentous hemagglutinin (FHA) from Bordetella pertussis was analyzed for its protein composition by gel electrophoretic methods. In this preparation of FHA the following native species could be detected by polyacrylamide gel electrophoresis (PAGE) at pH 3.2: S, and S₂ (inactive subunits or fragments); two monomers, a major form designated Ia (144K), and a minor form lb, differing only in net charge; and three oligomeric forms, designated II (213K), III (595K) and IV (1064K). Hemagglutinating activity was associated predominantly with component Ia. PAGE of FHA after derivatization with sodium dodecyl sulfate (SDS) showed there to be three major species, designated A, C and D. According to estimated molecular weight values, A, C and D are likely to correspond to S₂, Ia and II respectively. Isolated components II, III and IV yield all three SDS-species upon derivatization with SDS. Both moving boundary electrophoresis and gel electrofocusing showed hemagglutinating FHA to be a basic protein. Its apparent pI is 8.1.     

Effects of low temperature on wheat leaf proteins

Finnish wheat cultivars, winter wheat ‘Vakka’ and spring wheat ‘Apu’, grown for 9 days at 25° and then for 52 days at 2–4°, were called ‘hardened’. Proteins and esterases of young leaves were resolved by polyacrylamide gel electrophoresis (PAGE), porosity gradient PAGE (poroPAGE), isoelectric focusing (PAGIF) between pH 3 and 9, and by Na-dodecylsulfate (SDS)-PAGE. Hardened and unhardened leaves have different protein patterns after PAGE, PAGIF, poroPAGE and SDS-PAGE, respectively, as well as for esterases after PAGE and PAGIF. The PAGIF patterns of esterases show distinct changes of two bands especially for hardened leaves of winter wheat, one appearing, the other one disappearing.     

Arginine residues are critical for the heparin-cofactor activity of antithrombin III.

A dilution/quench technique was used to monitor the time course of chemical modification on the heparin-cofactor (a) and progressive thrombin-inhibitory (b) activities of human antithrombin III. Treatment of antithrombin III (AT III) with 2,4,6-trinitrobenzenesulphonate at pH 8.3 and 25 degrees C leads to the loss of (a) at 60-fold more rapid rate than the loss of (b). This is consistent with previous reports [Rosenberg & Damus (1973) J. Biol. Chem. 248, 6490-6505; Pecon & Blackburn (1984) J. Biol. Chem. 259, 935-938] that lysine residues are involved in the binding of heparin to AT III, but not in thrombin binding. Treatment of AT III with phenylglyoxal at pH 8.3 and 25 degrees C again leads to a more rapid loss of (a) than of (b), with the loss of the former proceeding at a 4-fold faster rate. The presence of heparin during modification with phenylglyoxal significantly decreases the rate of loss of (a). Full loss of (a) correlates with the modification of seven arginine residues per inhibitor molecule, whereas loss of (b) does not commence until approximately four arginine residues are modified and is complete upon the modification of approximately eleven arginine residues per inhibitor molecule. This suggests that (the) arginine residue(s) in AT III are involved in the binding of heparin in addition to the known role of Arg-393 at the thrombin-recognition site [Rosenberg & Damus (1973) J. Biol. Chem. 248, 6490-6505; Jörnvall, Fish & Björk (1979) FEBS Lett. 106, 358-362].     

Applications of β-gal-III isozyme from Bacillus coagulans RCS3, in lactose hydrolysis

Bacillus coagulans RCS3 isolated from hot water springs secreted five isozymes i.e. β-gal I-V of β-galactosidase. β-gal III isozyme was purified using DEAE cellulose and Sephadex G 100 column chromatography. Its molecular weight characterization showed a single band at 315 kD in Native PAGE, while two subunits of 50.1 and 53.7 kD in SDS PAGE. β-Gal III had pH optima in the range of 6-7 and temperature optima at 65 °C. It preferred nitro-aryl-β-d-galactoside as substrate having K_m of 4.16 mM with ONPG. More than 85% and 80% hydrolysis of lactose (1-5%, w/v) was recorded within 48 h of incubation at 55 °C and 50 °C respectively and pH range of 6-7. About 78-86% hydrolysis of lactose in various brands of standardized milk was recorded at incubation temperature of 50 °C. These results marked the applications of β-gal III in processing of milk/whey industry.     

Molecular Weight Determination of Gliadin Fractions in Gel Filtration by SDS-Polyacrylamide Gel Electrophoresis and Sedimentation Equilibrium

Gliadin was fractionated into three fractions; ω-gliadin, Fraction III (γ-gliadin) and Fraction IV (α- and β-gliadin). The determination of the molecular weights (MW) of the three fractions was performed by both SDS-polyacrylamide gel electrophoresis (SDS–PAGE) and sedimentation equilibrium. In SDS–PAGE, ω-gliadin gave three bands (MW 50,000, 54,000 and 64,000), Fraction III two bands (MW 38,000 and 46,000) and Fraction IV two bands (MW 33,000 and 38,000), The sedimentation analysis showed that each fraction was fairly homogeneous relative to molecular weight. The molecular weights obtained by sedimentation were 28,000 for Fraction III and 27,000 for both Fraction IV and ω-gliadin. The disagreement in molecular weight between sedimentation and gel electrophoresis was discussed.     

Structural requirements for signal transduction of the insulin receptor

Structural requirements for signal processing by human placental insulin receptors have been examined. Insulin binding has been found to change the physico-chemical properties of (alpha beta)2 receptors solubilized with Triton X-100, indicating a marked alteration of the form, i.e. size and shape, of the molecular complex. (a) The Stokes radius decreases from about 9.5 nm to 7.9 nm, as determined by PAGE with Triton X-100 in the buffer (Triton X-100/PAGE), and from 9.1 nm to 8.7 nm, as assessed by gel filtration. (b) The sedimentation coefficient s20,w rises from 10.1 S to 11.4 S. Upon dissociation of the receptor-hormone complex, the alterations are reversed. After autophosphorylation of hormone-bound (alpha beta)2-insulin receptors, phosphate incorporation was found for 7.9-nm receptor forms when receptor-insulin complexes were crosslinked with disuccinimide suberate prior to Triton X-100/PAGE. However, phosphate incorporation was demonstrated for the 9.5-nm receptor forms when receptor-insulin complexes were not prevented from dissociation. This strongly indicates that the (alpha beta)2 receptor is autophosphorylated after assuming its 7.9-nm form upon insulin binding. Moreover, the insulin-dependent structural alterations are not affected by autophosphorylation. In contrast to (alpha beta)2 receptors, the diffusion and the sedimentation behaviour of alpha beta receptors, which carry a dormant tyrosine kinase even in the hormone-laden state, has been found to be insensitive to insulin binding. Different molecular properties of alpha beta and (alpha beta)2 receptors have also been detected by hormone binding studies. Insulin binding to (alpha beta)2 and alpha beta receptors differs markedly with respect to pH, ionic strength, and temperature. This might indicate that the structure of the hormone binding domain of alpha beta receptor changes on association into the (alpha beta)2 species. Alternatively, distinct hormone-induced conformational alterations at the molecular level of alpha beta and (alpha beta)2 receptor species may lead to the different binding properties. Our data demonstrate that the (alpha beta)2-insulin receptor undergoes extended conformational alterations upon insulin binding. This capacity for structural changes coincides with the hormone-inducable enhancement of tyrosine autophosphorylation of the 7.9-nm insulin-bound receptor form. In contrast, alpha beta receptors appear to be locked in an inactive nonconvertable state. Thus, interaction between two alpha beta receptor units is required to allow extended conformational alterations, which are assumed to be the triggering event for augmented auto-phosphorylation.     

Effect of substrate loading rate of chemical wastewater on fermentative biohydrogen production in biofilm configured sequencing batch reactor

The influence of substrate loading rate on fermentative hydrogen (H2) production was studied in biofilm configured sequencing batch reactor using chemical wastewater as substrate. Reactor was operated with selectively enriched anaerobic mixed microflora at different organic loading rates (OLRs; 6.3, 7.1 and 7.9kg COD/m3 day) after adjusting the feed to a pH of 6.0 (acidophilic) to provide suitable environment for acidogenic bacterial function. Variation in H2 production rate was observed with change in OLR [specific hydrogen yield - 13.44molH2/kgCODRday (6.3kgCOD/m3day), 8.23molH2/kgCODRday (7.1kgCOD/m3 day) and 6.064molH2/kgCODR day (7.9kgCOD/m3 day)]. H2 yield showed reasonably good correlation with pH drop [6.3kgCOD/m3 day (R2 - 0.9796), 7.1kgCOD/m3 day (R2 - 0.9973), 7.9kgCOD/m3 day (R2 - 0.9908)]. Increase in OLR showed marked reduction in COD removal efficiency [22.6% - 6.3kgCOD/m3 day; 19.8% - 7.1kgCOD/m3 day and 17.2% - 7.9kgCOD/m3 day].     

Structure, stability, and thermodynamics of a short intermolecular purine-purine-pyrimidine triple helix

We have investigated the structure and physical chemistry of the d(C3T4C3).2[d(G3A4G3)] triple helix by polyacrylamide gel electrophoresis (PAGE), 1H NMR, and ultraviolet (UV) absorption spectroscopy. The triplex was stabilized with MgCl2 at neutral pH. PAGE studies verify the stoichiometry of the strands comprising the triplex and indicate that the orientation of the third strand in purine-purine-pyrimidine (pur-pur-pyr) triplexes is antiparallel with respect to the purine strand of the underlying duplex. Imino proton NMR spectra provide evidence for the existence of new purine-purine (pur.pur) hydrogen bonds, in addition to those of the Watson-Crick (W-C) base pairs, in the triplex structure. These new hydrogen bonds are likely to correspond to the interaction between third-strand guanine NH1 imino protons and the N7 atoms of guanine residues on the purine strand of the underlying duplex. Thermal denaturation of the triplex proceeds to single strands in one step, under the conditions used in this study. Binding of the third strand appears to enhance the thermal stability of the duplex by 1-3 degrees C, depending on the DNA concentration. The free energy of triplex formation (-26.0 +/- 0.5 kcal/mol) is approximately twice that of duplex formation (-12.6 +/- 0.7 kcal/mol), suggesting that the overall stability of the pur.pur base pairs is similar to that of the W-C base pairs.(ABSTRACT TRUNCATED AT 250 WORDS)     

Coagulation inhibitors and glycaemic control in non-insulin-dependent diabetes mellitus

The relationship between long-term glycaemic control and the activity of coagulation inhibitors was investigated in 60 non-insulin-dependent diabetes mellitus (NIDDM) patients not on insulin therapy. Overall, the activities of antithrombin III (AT III) (median 96%, range 65–133%), protein C (127%, 24–190%) and protein S (130%, 54–163%) were not reduced. Patients in poor long-term glycaemic control as verified by increased glycated haemoglobin (HbA1c) demonstrated significantly decreased median AT III activity in comparison with patients in good glycaemic control (92% vs 101%,P=0.016). However, individual values for AT III activity were not below the critical limit of 60%. An inverse correlation between AT III activity and long-term glycaemic control (HbA1c) was calculated (r=–0.378,P=0.0029). As AT III concentrations were found to be normal, we propose that non-enzymatic glycation leads to reduced activity of AT III without affecting its concentration.     

Changes of antithrombin III (AT III) in AT III deficient patients during long-term anticoagulant treatment

Antithrombin III deficient patients with manifest thromboembolic diseases need long term coumarin treatment. There are contradictory data on the change of AT III during this therapy. The authors observed 5 patients with severe AT III decrease type I, 3 with functional abnormality and 2 with a pathological heparin binding. AT III function was determined by the Gerendás-Rák method and with chromogenic substrate. AT III antigen was measured with Behring M-Partigen and Laurell rocket electrophoresis. Crossed immunoelectrophoresis was carried out in all patients. In patients with type I AT III decrease, AT III hasn't changed even in a long period of more than 10 years. In the other types AT III became normal. The pathological heparin binding wasn't changed.     

Isolation and partial characterization of phenol oxidases from Mangifera indica L. sap (latex)

Mango sap (latex), a by-product in mango industry, was separated into upper non-aqueous phase and lower aqueous phase. Aqueous phase contains very low protein (4.3 mg/ml) but contains high specific activities for peroxidase and polyphenol oxidase. The aqueous phase of sap was subjected to ion-exchange chromatography on DEAE-Sephacel. The bound protein was separated into three enzyme peaks: peak I showed peroxidase activity, peak II showed polyphenol oxidase activity and peak III showed activities against substrates of peroxidase as well as polyphenol oxidase. On native PAGE and SDS-PAGE, each peak showed a single band. Based on the substrate specificity and inhibitor studies peak III was identified as laccase. Although they showed variations in their mobility on native PAGE, these enzymes showed similar molecular weight of 100,000 ± 5000. These enzymes exhibited maximum activity at pH 6 however, polyphenol oxidase showed good activity even in basic pH. Peroxidase and polyphenol oxidase showed stability up to 70 °C while laccase was found to be stable up to 60 °C. Syringaldazine was the best substrate for laccase while catechol was the best for polyphenol oxidase. Thus, mango sap a by-product in mango industry is a good source of these phenol oxidases.     

菜心和水稻绿叶中不同等电点的乙醇酸氧化酶

The proteins with glycolate oxidase activity from B.parachinensis Bailey and rice( Oryza sativa )green leaves were prepared respectively.From the second protein peak on DEAE\|Cellulose column,two glycolate oxidases,expressed as B.parachinensis Bailey GO Ⅲ(specific activity natove 13 2 U·mg -1 ·min -1 )and rice GOⅢ(specific activity 8 8 U·mg -1 ·min -1 ),could not migrate anywhere in 4%～20% native\|PAGE under a pH8.3 buffer system.GOⅢ's p I was about pH8.3. The protein containing B.parachinensis Bailey GOⅢ showed 67±2,43±2,and 38±2 kD in SDS PAGE,band 43±2 kD was the subunit of B.parachinensis Bailey GOⅢ.From the two proteins above,another group of glycolate oxidases,expressed as B.parachinensis Beiley GOⅠ(specific activity 5 U·mg -1 ·min -1 )and rice GOⅠ(specific activity 1 2 U·mg -1 ·min -1 ),showed only one 43±2 kD band in SDS\|PAGE,and was purified on the Sepharose\|6B column which migrated towards anode in the same native\|PAGE showing the M r about 420 kD,or 460 kD and 260 kD respectively.GOⅠ's p I was smaller than pH8.3.Antibody against B.parachinensis Bailey GOⅠ was prepared and its efficacy was about 1/1600 in ELISA.By native\|PAGE,Western blot and rocket immunoelectrophoresis,the third group of glycolate oxidases,expressed as B.parachinensis Bailey GOⅡ and rice G0Ⅱ,were confirmed in crude protein of green leaves and migrated towards cathode under the same native\|PAGE,so GOⅡ's p I was higher than pH8.3.The M r of B.parachinensis Bailey GOⅡ was about 669 kD determined by native\|PAGE Western blot.Rice GOⅠ,rice GOⅢ and rice GOⅡ showed different quantitation under different physiological conditions.Rice GOⅡ could be induced by glycolate.     

赤豆子叶β-半乳糖苷酶的纯化及其性质的研究

β-半乳糖苷酶 ( EC3.2 .1 .2 )广泛存在于动植物的组织中 ,如在杏仁、桃子、大豆、咖啡豆等植物 ,蜗牛 ,哺乳动物的肠道中都有 β-半乳糖苷酶 .同样 ,微生物也能产生β-半乳糖苷酶 ,俗称乳糖酶 .乳糖操纵子学说的提出就是建立在对微生物β-半乳糖苷酶研究基础之上的 .在过去的研究中 ,关于微生物、动物来源的乳糖酶报道较多[1] ,而对于植物来源的β-半乳糖苷酶研究报道却相对较少[2 ] .它可能降解多糖中 β-构型半乳糖苷键 ,为种子生长发育提供必要的能量来源 .但目前对β-半乳糖苷酶在植物中确切的生理生化功能尚不清楚 .为了进一步阐明…     

Recognition of a guanine-cytosine base pair by 8-oxoadenine.

Two oligodeoxyribonucleotides, d-CTTCTTTTTTATTTT, I(A), and d-ATTATTTTTTATTTT, II(A), where C is 5-methylcytosine and A is 8-oxoadenine, were prepared and their interactions with the duplex d-GAAGAAAAAAYAAAA/d-TTTTZTTTTTTCTTC, III.IV(Y.Z), were studied. Oligomers I(A) and II(A) each form triplexes with III.IV(G.C) at temperatures below 20 degrees C as shown by continuous variation experiments, melting experiments, and circular dichroism (CD) spectroscopy. The CD spectra of these triplexes are almost identical to those formed by I(C) and II(C), oligomers which contain cytosine in place of 8-oxoadenine. This suggests that the 8-oxoadenine-containing triplexes have conformations which are very similar to those of the cytosine-containing triplexes. The melting temperature (Tm) for dissociation of the third strand of triplex II.III.IV(A.G.C) is 22 degrees C at pH 7.0 and 8.0, whereas the Tm of the corresponding transition in triplex II.III.IV(C.G.C) decreases from 28 degrees C at pH 7.0 to 17 degrees C at pH 8.0. The pH dependence of the Tm in the latter triplex reflects the necessity of protonating the N-3 of cytosine in order for it to form two hydrogen bonds with G of the G.C base pair. It appears that the keto form of 8-oxoadenine can potentially form two hydrogen bonds with the N-7 and O-6 atoms of G of the G.C base pair, when the 8-oxoadenine is in the syn conformation and in contrast to cytosine does not require protonation of the base. Oligomer I(A) does not form triplexes with III.IV(Y.Z) when Y.Z is A.T or T.A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Endopeptidases from Plasmodium knowlesi

Extracts of rhesus monkey erythrocytes infected with Plasmodium knowlesi were fractionated by polyacrylamide gel electrophoresis (PAGE) and several zones of endopeptidase activity were demonstrated by an imprint-digest method. The enzymes were active only under acid conditions; activity was detected at pH 3.2 but not between pH 6.4 and 8.9 using haemoglobin, albumin or erythrocyte lysate as the substrate. Optimized PAGE conditions separated highly active parasite enzymes with Rf values of 73, 63 and 53 (+/- 7%), as well as a red cell endopeptidase, Rf44. Of two other minor bands of activity, one was associated with platelets.     

The effect of tetrazole and its amino derivatives on the kinetics of peroxidase oxidation of chromogenic substrates

The peroxidase-catalyzed oxidation of 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (PDA), and 3,3',5,5'-tetramethylbenzidine (TMB) was found to be activated by tetrazole and 5-aminotetrazole (AT) and weakly inhibited by 1,5-diaminotetrazole. The activating action of tetrazole and AT on the PDA and TMB oxidation was clearly discompetitive and that on ABTS was non-competitive. The coefficients (degrees) of activation alpha were determined for three substrates and two activators; they depended on the substrate type and the buffer nature and increased along with the pH growth from 6.4 to 7.2. For AT and tetrazole, the maximal alpha values were 4140 and 800 M(-1), respectively, upon the PDA oxidation and 3570 and 540 M(-1), respectively, upon the TMB oxidation. Lower alpha values (145 and 58 M(-1) for tetrazole and AT, respectively) were characteristic of the peroxidase oxidation of ABTS. The activation of peroxidase oxidation of the substrates by tetrazole and AT at pH > or = 5.4 was explained by the nucleophilic nature of the activators interacting with the amino acid residues in the peroxidase active site according to the mechanism of acid-base catalysis. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2004, vol. 30, no. 3; see also http://www.maik.ru.     

Antithrombin III stimulates prostacyclin production by cultured aortic endothelial cells

Prostacyclin (PGI2) is a potent vasodilator and an inhibitor of platelet aggregation. We found that antithrombin III (AT III), an anticoagulant present in circulating blood, stimulated PGI2 production by cultured bovine aortic endothelial cells in a dose- and time-dependent manner. The stimulation of PGI2 production by AT III was observed at physiological concentrations and was inhibited by the addition of anti-AT III antiserum and heparin. These results suggest that AT III may stimulate PGI2 production by binding to heparin-like molecules on the endothelial cell membrane.