Absence Of PP Research Articles

Degradation of the Rubber Network during Dynamic Vulcanization of EPDM/PP Blends Using Phenolic Resol

Abstract Physical blends and thermoplastic vulcanizates (TPVs) based on EPDM and PP were prepared in a batch mixer. Dynamic vulcanization of TPVs using resol/SnCl2 was studied as a function of time. Static and dynamic vulcanization of EPDM in the absence of PP were also studied. Crosslinking of EPDM in the absence of PP is more efficient via static than dynamic vulcanization. For EPDM/PP-based TPVs the extent of crosslinking reaction versus time decreases as the amount of EPDM increases. Degradation of the EPDM network occurs during dynamic vulcanization, due to the combined action of shearing and high temperature, and increases as EPDM becomes more and more the continuous phase.

Structure and properties of dynamically cured EPDM/PP blends

AbstractThe structure and properties of polyolefin blends of ethylene–propylene–diene terpolymer (EPDM) and polypropylene were studied. Blends were prepared in a laboratory internal mixer where EPDM was cured with PP under shear with dicumyl peroxide (DCP) at different shear conditions (blend–cure). Blends were also prepared for comparison from EPDM which were dynamically cured in the absence of PP and blended later (cure–blend). The effect of DCP concentration, intensity of the shear mixing, and rubber/plastic composition were studied. In blend–cure, the melt viscosity increased with increasing DCP concentration in blends of 75% EPDM and 25% PP, but it decreased with increasing DCP concentration in blends of 75% PP and 25% EPDM. In cure–blend, however, the melt viscosity increased with increasing DCP concentration for all compositions. The melt viscosity decreased with increasing intensity of the shear mixing presumably due to the formation of the smaller segregated microdomain of the crosslinked EPDM gels in both blend–cure and cure–blend materials. The crystallization rate was higher in EPDM/PP blends than in PP homopolymer. The crystallization rates for various blending conditions were also compared.

Guanasone 5'-(alpha,beta-methylene)triphosphate enhances specifically microtubule nucleation and stops the treadmill of tubulin protomers.

Substitution of pp(CH2)pT for GTP in the polymerization of microtubular protein results in a marked enhancement of both the rate and the extent of microtubule nucleation. Comparison of the kinetics of microtubule polymerization and pp(CH2)pG hydrolysis reveals that massive microtubule nucleation occurs in the absence of pp(CH2)pG hydrolysis. The shortest microtubule nuclei formed in the presence of pp(CH2)pG are curled ribbons on three protofilaments 0.15 to 0.2 micrometer long. No specific effect of pp(CH2)pG on microtubule propagation is observed. Nucleotide chase experiments suggest that the rings of microtubular protein present at 4 degrees C are not incorporated directly into the microtubule. Microtubules polymerized by pp(CH2)pG do not show the treadmill of tubulin protomers characteristic of the microtubules polymerized by GTP. Nucleotide analysis of microtubules polymerized by pp(CH2)pG and GTP reveals that 95% of the exchangeable nucleotide contained in the microtubules is p(CH2)pG and GDP, respectively. pp(CH2)pG blocks the treadmill of tubulin protomers from microtubules assembled by GTP by suppressing depolymerization at the depolymerization end of the microtubule. On the other hand, GTP promotes the treadmill of tubulin from microtubules assembled by pp(CH2)pG by reactivating the depolymerization end of the microtubule.

Purification of kinetic properties of human erythrocyte Mg 2+-dependent inorganic pyrophosphatase

Inorganic pyrophosphatase (pyrophosphate phosphohydrolase, EC 3.6.1.1) from human erythrocyte henolysates has been purified up to 10 000-fold. The purified enzyme is homogenous and has a specific activity of 79.75 μmol PP i hydrolysed · min −1 · mg −1 at pH 8 ad 37°C. It was confirmed that it is a dimer with a molecular weight of 42 000, composed of two identical protomers. From kinetic studies, it is proposed that human erythrocyte inorganic pyrophosphatase activity depends on free Mg 2+ concentration in different ways. This ion constitutes part of the substrate (the Mg · PP i complex; K m = 1.4 · 10 −4 M) and probably acts as an allosteric activator (kinetic activation constant: K a Mg 2+ = 7.5 · 10 −4 M). Equilibrium binding studies performed in the absence of PP i showed 4 binding sites for Mg 2+, all having the same high affinity (dissociation constant: K d Mg 2+ = 4 · 10 t-6 M). Since the concentration of free Mg 2+ in red blood cells is very low and may vary with the oxygenation state, it is likely that in vivo erythrocyte pyrophosphatase activity is regulated.

Adenosine triphosphate sulfurylase from Penicillium chrysogenum: Equilibrium binding, substrate hydrolysis, and isotope exchange studies

ATP sulfurylase from Penicillium chrysogenum was purified to homogeneity. The enzyme binds 8 mol of free ATP ( K s = 0.53 mM) or AMP ( K s = 0.50 mM) per 440,000 g. The results are consistent with our earlier report that the enzyme is composed of eight identical subunits of M r 55,000 ( J. W. Tweedie and I. H. Segel, 1971, Prep. Biochem. 1, 91–117; J. Biol. Chem. 246, 2438–2446 ). In the absence of cosubstrates, the purified enzyme catalyzes the hydrolysis of MgATP (to AMP and MgPP i) and adenosine 5′-phosphosulfate (APS) (to AMP and SO 4 2−). MgATP hydrolysis is inhibited by nonreactive sulfate analogs such as nitrate, chlorate, and formate (uncompetitive with MgATP). In spite of the hydrolytic reactions it is possible to observe the binding of MgATP and APS to the enzyme in a qualitative (nonequilibrium) manner. Neither inorganic sulfate (the cosubstrate of the forward reaction) nor formate or inorganic phosphate (inhibitors competitive with sulfate) will bind to the free enzyme in detectable amounts in the absence or in the presence of Mg 2+, Ca 2+, free ATP, or a nonreactive analog of MgATP such as Mg-α,β-methylene-ATP. Similarly, inorganic pyrophosphate (the cosubstrate of the reverse reaction) will not bind in the absence or in the presence of Mg 2+ or Ca 2+. The induced binding of 32P i (presumably to the sulfate site) can be observed in the presence of MgATP. The results are consistent with the obligately ordered binding sequence deduced from the steady-state kinetics ( J. Farley et al., 1976, J. Biol. Chem. 251, 4389–4397 ) and suggest that the subsites for SO 2− 4 or MgPP i appear only after nucleotide cleavage to form E~AMP · MgPP i or E~AMP · SO 4 complexes. The suggestion is supported by the relative values of K ia (ca. 1 m m for MgATP) and K iq (ca. 1 α m for APS) and by the inconsistent value of k −1 calculated from V f K i a K m A (The value is considerably less than V r) Purified ATP sulfurylase will also catalyze a Mg 32PP i-MgATP exchange in the absence of SO 4 2−. A 35SO 4 2−-APS exchange could not be demonstrated in the absence or presence of MgPP i. This result was not unexpected: The rate of APS hydrolysis (or conversion to MgATP) is extremely rapid compared to the expected exchange rate. Also, the pool of APS at equilibrium is extremely small compared to the sulfate pool. The V values for molybdolysis, APS hydrolysis (in the absence of PP i), ATP synthesis (from APS + MgPP i), and Mg 32PP i-MgATP exchange at saturating sulfate are all about equal (12–19 μmol × min −1 × mg of enzyme −1). The rates of Mg 32PP i-MgATP exchange in the absence of sulfate, APS synthesis (from MgATP + sulfate), and MgATP hydrolysis (in the absence of sulfate) are considerably slower (0.10 – 0.35 μmol × min −1 × mg of enzyme −1). These results and the fact that k 4 calculated from V r K i q K m Q is considerably larger than V f suggest that the rate-limiting step in the overall forward reaction is the isomerization reaction E~AMP-SO 2− 4 → E APS. In the reverse direction the rate-limiting step may be SO 2− 4 release or isomerization of the E~AMP · MgPP i · SO 4 2− complex. (The reaction appears to be rapid equilibrium ordered.) Reactions involving the synthesis or cleavage of APS are specific for Mg 2+. Reactions involving the synthesis or cleavage of ATP will proceed with Mg 2+, with Mn 2+, and, at a lower rate, with Co 2+. The results suggest that the enzyme possesses a Mg 2+-preferring divalent cation (activator) binding site that is involved in APS synthesis and cleavage and is distinct from the MeATP or MePP i site. The equilibrium binding of about one atom of 45Ca 2+ per subunit (possibly to the activator site) could be demonstrated ( K s = 1.4 mM).

Guanosine monophosphate synthetase from ehrlich ascites cells. Multiple inhibition by pyrophosphate and nucleosides

GMP synthetase (xanthosine-5′-phosphate: ammonia ligase (AMP-forming), EC 6.3.4.1) from Ehrlich ascites cells was found to be subject to multiple inhibition by its reaction product, PP i, and some analogs of adenosine. PP i and the nucleoside (N) inhibitors were also capable of individually inhibiting this enzyme. Under no conditions did the inhibition appear to be irreversible or “pseudoinactivating” in nature. The individual inhibition by PP i was competitive with respect to ATP ( K 1 = 0.42 mM ). Conversely, in the absence of PP i, the binding of N was noncompetitive with ATP, but shifted to a competitive pattern when PP i was present. Furthermore, with the inhibitors in concert, there was an apparent lowering of the K I values for both inhibitors. This data was consistent with either PP i functioning to tighten the binding of N at a noncatalytic site (positive cooperativity) or with PP i actually opening a second binding site for N in addition to the non-catalytic site. Although this study did not distinguish which of these events was occurring, it did reveal that the intensity of the effect of PP i appeared to be constant. That is, for various N inhibitors with a range of independently determined K I values from 26 to 1650 μM, the ratio of their K I values determined in the absence of PP i to the values determined in the presence of PP i was always 38 ± 1.

F-actin-heavy meromyosin complex studied by optical homodyne and heterodyne methods

Quasi-elastic scattering of laser light is now becoming a powerful tool for the study of dynamic properties of both biological and non-biological macromolecules. In solutions of biological macromolecules, aggregation-disaggregation of molecules under study usually depends on solvent conditions. Therefore, special attention must be paid in light-beating spectroscopy. By use of both homodyne and heterodyne methods, the spectral densities of solutions of F-actin and of a complex of F-actin and heavy meromyosin were measured. The half-width of the heterodyne spectrum was much wider than that of the homodyne spectrum. This was probably due to polydispersity of F-actin. The results showed that laser light scattering gave information about the dynamics of free filaments, although low frequency rheometry has suggested the rubber-like elasticity of the solution of F-actin and heavy meromyosin. The interaction between F-actin and heavy meromyosin was studied in the presence of pyrophosphate (PP i). At PP i concentrations between 10 and 100 μM, the spontaneous bending motion of F-actin occurred as if heavy meromyosin were not present, although turbidity of the solution indicated that heavy meromyosin was, in fact, bound to F-actin. This meant that there were two types of binding state of the F-actin-heavy meromyosin complex. A plausible model is that, in the absence of PP i, the two heads of one heavy meromyosin molecule simultaneously interact with two neighbouring monomers in F-actin, whereas a single head binding occurs in the presence of PP i. Above 100 μM PP i, heavy meromyosin seemed to dissociate from F-actin. However, it was not clear whether or not this was due to a direct effect of PP i on heavy meromyosin alone, because the dynamic properties of F-actin also changed. Assuming that actin filaments are cross-linked by myosin, a simple model is proposed to explain qualitatively the rubber-like elasticity of the solution of an F-actin-heavy meromyosin complex.

Purification and properties of the enzyme ATP-sulfurylase and its relation to vitamin A

1. 1. The enzyme ATP-sulfurylase which catalyzes the formation of adenosine 5′-phosphosulfate (APS) was purified about 1000-fold over supernatant fraction by ammonium sulfate and acid precipitation and by elution from Sephadex G-200, agarose and hydroxyapatite columns. 2. 2. The molecular weight of the enzyme was between 800 000 and 900 000; its optimum pH, at 8.0; its K m with respect to ATP, 1.6 · 10 −3M; K m (sulfate), 1.0 · 10 −4M; K m (APS), 2.5 · 10 −4 M; K m (PP 1), 3.7 · 10 −5M. There was inhibition at high PP 1 and APS concentrations and at low concentrations of ADP. Cu 2+ and Co 2+ were strongly inhibitory. p-Chloromercuribenzoate (PCMB) and EDTA also inhibited: the former inhibition was completely relieved by glutatione; the latter, by Mg 2+. The enzyme was irreversibly inactivated by deoxycholate. It was unstable in Tris-HCl buffer and decayed to about 15% activity in 24 h whether measured by ATP generation or molybdolysis. It was completely stabilized by phosphate but not by barbiturate or sulfate ions. 3. 3. The mechanism of the reaction was explored by a study of the reaction kinetics and the following was observed: (a) the rate of exchange of 32PP i into ATP was greater in presence than in absence of sulfate and greater (even in absence of SO 4 2− than the overall reaction of APS synthesis; (b) there was no exchange of 35SO 4 2− into APS in presence or absence of PP 1; and (c) the rate of reaction of APS with P i at low APS concentrations was independent and at high concentrations was dependent on PP i concentration. We conclude that the reaction proceeds through intermediate formation of an AMP-enzyme complex. 4. 4. 14C-Labeled vitamin A was administered to vitamin A-deficient rats, and ATP-sulfurylase was isolated after 3 days. Although radioactivity remained associated with the enzyme over some of the purification steps, highly active enzyme which had lost all radioactivity was obtained by fractionation on agarose columns. Similar results were obtained when the labeled vitamin was homogenized with rat embryo livers or injected into newborn rats. We conclude that neither vitamin A nor a metabolite of the vitamin is an integral or necessary part of the enzyme and suggest that since the enzyme is unstable, particularly under conditions of low phosphate concentration, vitamin A may help to stabilize the enzyme.

Physicochemical studies on denaturation of myosin-adenosinetriphosphatase

Denaturation of myosin A- and myosin B-ATPase was investigated in 0.5–0.6 M KC1 and at 30 °C. and pH 7.0. The denaturation of myosin A proceeded in accordance with the first-order law until ATPase disappeared, while that of myosin B proceeded according to the first-order law to a constant value of ATPase. From the results on denaturation of the three parts of myosin B separated by ultracentrifugation and of synthetic actomyosin, it was shown that the rapid denaturation of the first-order myosin B was due to myosin A contaminant in the preparation and/or released from the “heavy” components of myosin B. Under present conditions, the denaturation of myosin A was not affected by the addition of cysteine and by passing nitrogen or oxygen through the solution. By salting-out analysis, it was found that the main peak of myosin A moved from 38.5 to 32% saturation of ammonium sulfate with denaturation, and that a subunit, occupying about 10% of the total, was revealed in the precipitation range of 50–60%. This subunit was water soluble and considerably heat stable and traveled in the ultra-centrifuge as one peak, its s 20 w being 2.1–2.8. The reduced viscosity of myosin A and the weight-average molecular weight of myosin A increased with decrease of ATPase. When PP was added and ATPase was assayed using Ca ++ as an activator of ATPase, a lag phase appeared in the denaturation of ATPase. After the lag phase, the denaturation proceeded with a smaller velocity than in the absence of PP, while, when Mg ++ was used as a modifier of ATPase, the effect of PP could not be observed. From these results a reaction scheme was proposed which satisfactorily explained the course of the denaturation under various conditions.