Solvent viscosity effects on protein dynamics studied by ultrasonic absorption

Solvent viscosity is known to play an important role in the kinetics of biochemical reactions, and has been suggested to modulate the dynamic structure of proteins. The effect of viscous cosolvents, of various molecular sizes, on the apparent ultrasonic absorption of bovine serum albumin in solution, at 37 degrees, has been measured in attempt to investigate the following phenomena: 1) The predicted modulating effect of viscous cosolvents on the "internal friction" of proteins, and 2) Possible differences between the microscopic and macroscopic pictures of the solvent viscosity concerning the proposed effect. We have found that A) The absorption of ultrasound (3-17 MHz) by the protein increases with increasing the cosolvent concentration. B) That increase correlates with the solvent viscosity for small cosolvent molecules, but not with macromolecular cosolvents, and C) Dextran solutions with the same concentration by weight, reveal similar ultrasonic absorption, in spite of large differences in their viscosity. A possible explanation is discussed.     

Inhibitory effects of flavonoids on the reduction of progesterone to 20alpha-hydroxyprogesterone in rat liver

The first aim of this study is to characterize the reduction of progesterone in rat liver. Progesterone was mainly reduced to 20alpha-hydroxyprogesterone in the cytosolic fraction of rat liver. The amount of 20alpha-hydroxyprogesterone formed from progesterone in the cytosolic fraction was significantly larger in the males than in the females and this enzyme reaction proceeded not only in the presence of NADPH, but also in the presence of NADH. Furthermore, we attempted to evaluate the inhibitory effects of 15 flavonoids on the NADPH-dependent reduction of progesterone to 20alpha-hydroxyprogesterone in liver cytosol of male rats. The order of the inhibitory potencies was luteolin>apigenin>quercetin>myricetin=fisetin=kaempferol. Other flavonoids exhibited lower inhibitory potencies. Energy-minimized molecular models demonstrated that a planar benzopyrone ring (A and C rings) with a coplanar phenyl ring (B ring) is a structural characteristic determining the inhibitory effects of flavonoids other than isoflavones.     

Inhibitory action of gemfibrozil on cholesterol absorption in rat intestine

This study was designed to determine whether gemfibrozil inhibits intestinal lipid absorption. Male Sprague-Dawley rats received an oral dose of 30 mg gemfibrozil/kg body weight for 14 days. Mesenteric lymph cannulation was performed, and a lipid infusion containing 40 micromol/h (35.4 mg/h) of radiolabeled triolein and 2.74 micromol/h (1.06 mg/h) of radiolabeled cholesterol with the addition of 1 mg/h of gemfibrozil was infused intraduodenally at a rate of 3 ml/h for 8 h. The lymph was collected, and the radioactivity levels of the lumen and gut mucosa were measured after the infusion. Lymph cholesterol transport was depressed in gemfibrozil-treated rats, in terms of mass measurements as well as radioactivity in a lesser degree. More radioactive cholesterol remained in the proximal portion of the intestinal lumen and mucosa in the treated rats than in the control rats. More radioactive triglycerides also remained in the proximal intestinal lumen of treated rats, although no difference in lymphatic triglyceride transport was observed between the groups. A significant portion of the radioactive cholesterol remained in the lumen in the gemfibrozil-treated rats. Gemfibrozil increased biliary cholesterol excretion. Thus, this study shows that gemfibrozil inhibits cholesterol absorption in rat intestine.     

Inhibitory effects of surfactant protein A on surfactant phospholipid hydrolysis by secreted phospholipases A2

Hydrolysis of surfactant phospholipids by secreted phospholipases A(2) (sPLA(2)) contributes to surfactant dysfunction in acute respiratory distress syndrome. The present study demonstrates that sPLA(2)-IIA, sPLA(2)-V, and sPLA(2)-X efficiently hydrolyze surfactant phospholipids in vitro. In contrast, sPLA(2)-IIC, -IID, -IIE, and -IIF have no effect. Since purified surfactant protein A (SP-A) has been shown to inhibit sPLA(2)-IIA activity, we investigated the in vitro effect of SP-A on the other active sPLA(2) and the consequences of sPLA(2)-IIA inhibition by SP-A on surfactant phospholipid hydrolysis. SP-A inhibits sPLA(2)-X activity, but fails to interfere with that of sPLA(2)-V. Moreover, in vitro inhibition of sPLA(2)-IIA-induces surfactant phospholipid hydrolysis correlates with the concentration of SP-A in surfactant. Intratracheal administration of sPLA(2)-IIA to mice causes hydrolysis of surfactant phosphatidylglycerol. Interestingly, such hydrolysis is significantly higher for SP-A gene-targeted mice, showing the in vivo inhibitory effect of SP-A on sPLA(2)-IIA activity. Administration of sPLA(2)-IIA also induces respiratory distress, which is more pronounced in SP-A gene-targeted mice than in wild-type mice. We conclude that SP-A inhibits sPLA(2) activity, which may play a protective role by maintaining surfactant integrity during lung injury.     

Studies on mineral ion absorption by plants

Summary The effect of Al and P on the growth of lucerne (Medicago sativa) was studied in nutrient solutions in which aluminium phosphate did not precipitate. Al and P retained in the free space of the roots was washed out with 0.1N HCl/O₄ at 5°C. The inhibitory effect of Al on growth was much less at pH 5 than at pH 4.5, although 3 to 4 times as much Al was found in the roots and shoots of the pH 5 plants.It is suggested that the low toxicity of high contents of Al was due to a portion of the uptake at pH 5 being in the form of polymeric aluminophosphate complexes of low net charge density. The optimum pH for the formation and polymerization of such complexes is around 5, and their composition depends on the P/Al mole ratio of the initial solutions. Washing³²P-labelled roots in unlabelled P solutions containing Ca showed that 12–43 per cent more of the total label diffused out of the Al-treated roots at pH 5 than from control roots. This was consistent with estimates by solution analysis of 16–36 per cent (depending on the P/Al mole ratio) of the P present in the original uptake solutions being complexed with Al.     

Studies on mineral ion absorption by plants

Summary The rate of accumulation of phosphorus in the roots, and its transport to the shoots, of whole plants grown in very dilute nutrient solutions, did not conform to the kinetic models derived from studies with excised roots or tissue slices. The demand for phosphorus associated with the rate of plant growth, or the level of metabolic activity within the tissues, appeared to have a marked influence on the rate of phosphorus uptake at deficient to optimum (1 to 10 μM P) levels of supply. A hypothesis is presented whereby the rate of influx of orthophosphate into the root cortical cells is regulated by the turn-over rate of the pool of inorganic phosphate in the cytoplasm, and by the rate of transport of inorganic phosphate to the shoot. The turn-over rate of this labile pool depends on inherent factors, such as the relative growth rate of the species, and on environmental factors, including the supply of essential nutrients such as nitrogen. re]19720502     

Studies on mineral ion absorption by plants

Summary The relative yield response and phosphate uptake ofS. humilis, P. atropurpureus andD. intortum, grown on a very deficient soil, enriched with phosphate, were measured at four successive harvests during 42 days growth under controlled environmental conditions. Except at the first harvest (19–21 days), the relative yields ofS. humilis andP. atropurpureus at P₀ were identical, and slightly greater than that ofD. intortum; the relative response to applied phosphate was similar for the three species, up to the maximum yield attained. The higher relative yield ofD. intortum, at harvest 1, reflected the influence of a higher initial phosphate concentration in the small Desmodium seedling, compared toS. humilis andP. atropurpureus. The mean absorption rate for phosphate ofD. intortum, and to a lesser degreeS. humilis andP. atropurpureus, showed two distinct maxima: an initial peak at low soil activities (0.3 — 3μM P), and a second at higher soil activities (37–43μM P) when maximum yield had been attained, and luxury accumulation of phosphate appear to occur. The initial peak in was followed by a decline (significant (P=0.05) forD. intortum) at soil phosphate activities corresponding to maximum yield, suggesting that the rate of absorption by the roots was influenced by the demand for phosphate created within the growing plant. Mean absorption rates and relative growth rates , averaged over all phosphate levels, fell in the orderD. intortum >S. humilis >P. atropurpureus. Conversely, the efficiency of phosphate utilization by the plant, which may be expected to be greater in plants of low RGR, fell in the orderP. atropurpureus >S. humilis >D. intortum, and so counteracted the lower ofP. atropurpureus, and to a lesser extent,S. humilis. However,S. humilis had the advantage of a lower retention of phosphate in the root system, compared toP. atropurpureus, due to a relatively greater shortage of nitrogen in the tops when grown on symbiotically-fixed nitrogen.     

Inhibitory effects of some purinergic agents on ecto-ATPase activity and pattern of stepwise ATP hydrolysis in rat liver plasma membranes

Inhibitory effects of various purinergic compounds on the Mg(2+)-dependent enzymatic hydrolysis of [(3)H]ATP in rat liver plasma membranes were evaluated. Rat liver enzyme ecto-ATPase has a broad nucleotide-hydrolyzing activity, displays Michaelis-Menten kinetics with K(m) for ATP of 368+/-56 microM and is not sensitive to classical inhibitors of the ion-exchange and intracellular ATPases. P2-antagonists and diadenosine tetraphosphate (Ap(4)A) progressively and non-competitively inhibited ecto-ATPase activity with the following rank order of inhibitory potency: suramin (pIC(50), 4.570)>Reactive blue 2 (4.297)&z.Gt;Ap(4)A (3. 268)>pyridoxalphosphate-6-azophenyl-2',4'-disulfonic acid (PPADS) (2. 930). Slowly hydrolyzable P2 agonists ATPgammaS, ADPbetaS, alpha, beta-methylene ATP and beta,gamma-methylene ATP as well as the diadenosine polyphosphates Ap(3)A and Ap(5)A did not exert any inhibitory effects on the enzyme activity at concentration ranges of 10(-4)-10(-3) M. Thin-layer chromatography analysis of the formation of [(3)H]ATP metabolites indicated the presence of other enzyme activities on liver surface (ecto-ADPase and 5'-nucleotidase), participating in concert with ecto-ATPase in the nucleotide hydrolysis through the stepwise reactions ATP-->ADP-->AMP-->adenosine. A similar pattern of sequential [(3)H]ATP dephosphorylation still occurs in the presence of ecto-ATPase inhibitors suramin, Ap(4)A and PPADS, but the appearance of the ultimate reaction product, adenosine, was significantly delayed. In contrast, hydrolysis of [(3)H]ATP in the presence of Reactive blue 2 only followed the pattern ATP-->ADP, with formation of the subsequent metabolites AMP and adenosine being virtually eliminated. These data suggest that although nucleotide-binding sites of ecto-ATPase are distinct from those of P2 receptors, some purinergic agonists and antagonists can potentiate cellular responses to extracellular ATP through non-specific inhibition of the ensuing pathways of purine catabolism.     

Inhibitory effects of quinidine on rat heart muscarinic receptors

Quinidine inhibited binding of the labelled agonist [3H]oxotremorine M [( 3H]Oxo-M) and the labelled antagonist [3H]N-methylscopolamine [( 3H]NMS) to rat heart muscarinic receptors. Kinetic studies demonstrated that quinidine decreased the association rates (I50: 4 and 7.5 microM) and dissociation rates (I50: 100 and 68 microM) of [3H]Oxo-M and [3H]NMS, with different potencies. These cooperative effects explained the low Hill coefficients and apparent selectivity of quinidine competition curves.     

Substrate specificity of solvent viscosity effects in carboxypeptidase A catalyzed peptide hydrolysis

We have investigated the viscosity of carboxypeptidase A catalyzed Bz-Gly-Phe hydrolysis at pH 7.5 (Tris) and 0.5 mol.l-1 NaCl over the range 10-100 mp, varied by addition of glycerol or sucrose. In contrast to previous reports of strong viscosity effects on the corresponding Cbz-Ala-Ala-Ala hydrolysis, both the catalytic constant and the Michaelis constant are virtually independent of viscosity over the 10-fold range investigated. Furthermore, the CD spectra of carboxypeptidase A in the high-viscosity media point to no change in the alpha-helix and beta-sheet structure in these media. The data are compatible either with a compacter, more rigid enzyme-substrate structure or with a more prominent role of intramolecular nuclear reorganization compared to protein reorganization for Bz-Gly-Phe than for Cbz-Ala-Ala-Ala. These views can be given a preciser frame in terms of stochastic chemical rate theory.     

Inhibitory effects of psoralens on rat liver cyclic AMP phosphodiesterase

TMP (4,5',8-trimethylpsoralen) usually employed in PUVA therapy and TMA (4,6,4'-trimethylangelicin), phototherapeutic agent under clinical and biological investigation, show an inhibitory effect of competitive type on the low Km cyclic AMP phosphodiesterase present in rat liver. The two drugs exhibit Ki values of 135 and 320 microM, respectively.     

Fructooligosaccharides enhance mineral apparent absorption and counteract the deleterious effects of phytic acid on mineral homeostasis in rats

Phytic acid (PA) and fructooligosaccharides (FOS) such as inulin are two food components that are able to modify mineral absorption negatively or positively. The influence of PA and FOS on the cecal and apparent mineral absorption as well as on the mineral status (plasma, hepatic, and bone) were investigated in four groups of rats fed one of the experimental diets: a fiber-free (FF) diet, a FF diet containing 7 g/kg PA (FF + PA), a diet containing 100 g/kg inulin (FOS), or a FOS diet containing 7 g/kg PA (FOS + PA). The cecal enlargement together with the acidification of cecal pH in rats adapted to FOS diets led to an improved Ca and Mg cecal absorption. Mineral apparent absorption was significantly enhanced by FOS ingestion (Ca, +20%; Mg, +50%; Fe, +23%; Cu, +45%), whereas PA decreased this factor only for trace elements (Fe, -48%; Zn, -62%; Cu, -31%). These inhibitory effects of a FF + PA diet have repercussions on blood (Mg, -15%; Fe, -12%; transferrin saturation -31%), liver (Mg, -18%; Fe, -42%; Zn, -25%), and bone (Zn, -25%) variables. However, the introduction of FOS into a PA diet counteracted these observed deleterious effects by stimulating bacterial hydrolysis of PA (+60% in rats adapted to FOS + PA compared to those fed the FF + PA diet) and by improving cecal absorption of minerals.     

Inhibitory effects of ellagi- and gallotannins on rat intestinal alpha-glucosidase complexes

The clove ellagitannins and their related polygalloyl-glucoses inhibited maltase activity of rat intestinal alpha-glucosidases. The structure-activity relationship study of those galloylglucoses, varying the extent of galloylation on the glucose core, with the ellagitannins, indicated that an increasing number of galloyl units in the molecule lead to an increase in the inhibitory activity. Penta-O-galloyl-beta-D-glucose, with five galloyl groups showed the highest inhibitory activity. On the other hand, hexahydroxydiphenoyl units contained in the ellagitannins had little effect on the activity. After separation of maltase-glucoamylase and sucrase-isomaltase complexes from the crude mixture of the rat alpha-glucosidases, the inhibitory activities of the galloylglucose derivatives against each complex were examined. The inhibitory influence on the maltase-glucoamylase complex was more potent than on the sucrase-isomaltase complex.     

可乐定对背根神经节神经元GABA激活电流的抑制作用

本实验在新鲜分离大鼠背根神经节（ＤＲＧ）细胞上应用全细胞膜片的箝记录研究贤上腺素α２－受体激动剂可乐定（ｃｌｏｎｉｄｉｎｅ）对ＧＡＢＡ－激活电流的调制作用。发现缘大多数ＤＲＧ细胞对ＧＡＢＡ（１０＾－６￣１０＾－３ｍｏｌ／Ｌ）敏感（７２／７５）,产生浓度依赖性的内向电流;并且可被ｂｉｃｕｃｕｌｉｎｅ（１０＾－５￣１０＾－４ｍｏｌ／Ｌ）所阻断。在多数细胞中（５１／７２）预加可乐定（１０＾－８￣１０＾－     

Enzymatic hydrolysis of soluble cellulose derivatives as measured by changes in viscosity

Observation of changes in fluidity is presented as a method for following the enzymatic hydrolysis of soluble cellulose derivatives. The activity of different cell-free enzyme preparations may be compared by this method, providing certain precautions are observed. In general, results obtained by use of the fluidity method are similar to those obtained using the reducing sugar technique, indicating that the same enzyme system is measured by the two methods. Changes in the DP of the substratum may be followed within certain limits of molecular size. Results indicate that a random splitting of CMC occurs during enzymatic hydrolysis, with a concomitant decrease in intrinsic viscosity and an increase in reducing sugars. Certain inadequacies of the cellulose-cellobiose-glucose theory, together with more recent findings, have led to the postulation of an alternate explanation of the mechanism of cellulose hydrolysis.