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1.
Commercial [5-14C]mevalonate is shown to contain several radioactive impurities, which give artifactually high amounts of Hyamine bound, volatile acidic radioactivity when incubated with killed or living rat renal cortex slices, as compared with [5-14C]mevalonate purified either by liquid-liquid partition chromatography or through the enzymically generated by ion-exchange chromatography. The artifactual results were not diluted by incubation with increasing amounts of unlabelled mevalonate, whereas the and []cholesterol produced by rat renal cortex slices incubated with purified [5-14C]mevalonate were both diluted to the same extent by unlabelled mevalonate. It is concluded that is genuinely oxidized to , and that purification of substrate before its use is necessary. Production of and various []lipids from purified [5-14C]mevalonate, as a function of time and substrate concentration, by renal cortex and liver slices, is described. 相似文献
2.
Zoltán Spolarics Jozsef Mandl Raymund Machovich Patrick Lambin Tamás Garzó Ferenc Antoni István Horváth 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,845(3):389-395
125I-labelled complexed with thrombin or plasmin bound to hepatocytes in a concentration-and time-dependent manner. The apparent values calculated from displacement experiments were 7.9 · 10?8 M for and 8.5 · 10?8 M for . Association of these complexes was only partially reversible; after a 180 min incubation period, 50–60% of the bound radioactivity was internalized by the cells. itself bound also to hepatocytes, but the affinity of the complexes was higher than that of the inhibitor alone, and was not internalized, either. 125I-labelled thrombin or plasmin bound to hepatocytes as well. These bindings were also concentration-dependent and could be decreased with an excess of unlabelled ligands. Binding rates and amounts of the bound proteinases were higher than those of their complexes. The complex competed with the complex in binding to hepatocytes, whereas there was no competition between these complexes and the antithrombin III-thrombin complex. These results suggest that the binding sites of hepatocytes for and antithrombin III-proteinase complexes are different. 相似文献
3.
Yukihiro Shoyama Yasuo Kishimoto 《Biochemical and biophysical research communications》1976,70(4):1035-1041
A doubly labeled 3-ketoceramide, [1-14C] lignoceroyl [1-3H2] 3-ketosphingosine ( ratio, 3.61) was injected into the left ventricle of rat heart. The ceramide isolated from the livers of the animals after 1 hr incubation contained an equal ratio of 3.60. This finding strongly supports the existence for direct conversion of 3-ketoceramide to ceramide in rat liver. 相似文献
4.
The uptake of d-glucose, 2-aminoisobutyric acid and glycine was studied with intestinal brush border membrane vesicles of a marine herbivorous fish: Boops salpa. The uptake of these three substances is stimulated by an Na+ electrochemical gradient (). For glucose, an increase of the electrical membrane potential generated by a concentration gradient of the liposoluble anion, SCN?, increases the Na+-dependent transport. This responsiveness to the membrane potential was confirmed by valinomycin. Differently from glucose, uptake of glycine and 2-aminoisobutyric acid requires, besides the Na+ gradient, the presence of Cl? on the external side of the vesicles. In the absence of Cl?, amino acid uptake is not stimulated by the Na+ gradient and is not influenced by an electrical membrane potential generated by SCN? gradient () or by a K+ diffusion potential (). This Cl? requirement differs from the Na+ requirement, since a Cl? gradient () does not result in an accumulation of glycine or 2-aminoisobutyric acid similar to that produced by an Na+ gradient. 相似文献
5.
Shirley Rainier Mahendra K. Jain Fausto Ramirez Panayiotis V. Ioannou James F. Marecek Roger Wagner 《生物化学与生物物理学报:生物膜》1979,558(2):187-198
Synthesis and phase transition characteristics of aqueous dispersions of the homologous (12 : 0, 14 : 0, 16 : 0) diphosphatidylglycerols (cardiolipins) and phosphatidyldiacylglycerols are reported. Electron microscopy of the negatively stained aqueous dispersions reveals a characteristic lamellar structure suggesting that these phospholipid molecules are organized as bilayers in the aqueous dispersions. The phase transition temperature () and the enthalpy of transition () increase monotonically with chain length in the cardiolipin and phosphatidyldiacylglycerol series; for phosphatidyldiacylglycerol is higher than that for cardiolipin of the same chain-length. The transition temperatures for the enantiomeric and for the diastereomeric, are approximately the same. The molar enthalpy for the transition of cardiolipin-NH4+ bilayers is approximately twice the value for the phosphatidylcholines of the same chain length, i.e., the molar enthalpy per acyl chain is approximately the same in the two systems. The transition temperatures for metal ion salts of C1 6-cardiolipin exhibit a biphasic dependence upon the unhydrated ionic radii, i.e. the highest is observed for Ca2+- cardiolipin and decreases for the salts of ions with smaller and larger ionic radii than that of Ca2+. The lowest is observed for Rb+-cardiolipin. Monovalent metal salts of cardiolipin exhibit two phase transitions. This effect may result from different conformational packing of the four acyl chains due to differences in metal-phosphate binding. 相似文献
6.
The cell cycle time of Calliphora vicina prohaemocytes was examined using the labelled mitoses method after the administration of a pulse of H3-thymidine. The total cycle time occupied 9.1 hr, while , S and occupied 1.6 hr, 2.7 hr and 4.8 hr respectively. 相似文献
7.
A-Side (4-R)-(4-2H)-reduced nicotinamide adenine dinucleotide (NADD) was prepared by a stepwise oxidation of ethanol-d6 to acetate in the presence of NAD, alcohol dehydrogenase, and aldehyde dehydrogenase. The B-side (4-S) isomer of NADD was prepared using the glucose dehydrogenase activity of glucose-6-phosphate dehydrogenase to oxidize to oxidize glucose-1-d in 40% dimethyl aulfoxide. Subsequent purifieation of the reduced nucleotides was achieved using a column of strongly basic polystyrene macroporous resin (AG MP-1) eluted with 0.2 m LiCl, pH 10, and applying the pooled NADD peak to a polyacrylamide gel (Bio-Gel P-2) column. The final ratio obtained for these preparations was below 2.3. Preparation of the deuterated reduced nucleotides in this manner allows production of specifieally deuterated substrates by coupled enzymatic synthesis. L-Malate-2-d was prepared by coupled synthesis of A-side NADD to the reduction of oxaloacetate by the A-side enzyme malate dehydrogenase. 相似文献
8.
Qβ plus strands with a 70 S ribosome bound to the coat cistron initiation site were used as template for Qβ replicase. Minus strand synthesis proceeded until the replicase reached the ribosome. The ribosome was removed and elongation was continued in a substrate-controlled, stepwise fashion. The nucleotide analog N4-hydroxyCMP was introduced into the positions complementary to the third and fourth nucleotides of the coat cistron. The minus strands were elongated to completion, purified and used as template for Qβ replicase. The final plus strand preparation consisted of four species, with the sequences -A-U-G-G- (wild type), -A-U-A-G- (mutant C3), -A-U-G-A- (mutant C4) and -A-U-A-A- (mutant ) at the coat initiation site. The ribosome binding capacity of the mutant RNAs relative to wild type was <0.1 (C3), 3.2 (C4) and 0.3 (). The finding that mutant C3 no longer formed an initiation complex suggests that the interaction of the ribosome binding site with fMet-tRNA plays an essential role in the formation of the 70 S initiation complex. The fact that mutant C4 RNA bound more efficiently than wild type, and that mutant RNA showed substantial ribosome binding capacity whereas the single mutant C3 did not, can be explained by assuming that an A residue following the A-U-G triplet interacts with a complementary U residue in the anticodon loop sequence. In the case of this additional base-pair may offset the reduced codon-anticodon interaction resulting from the modification of the A-U-G codon. 相似文献
9.
Jose C. Gan 《International journal of biological macromolecules》1980,2(2):97-104
An material has been purified to homogeneity from the soluble fraction of normal human liver by procedures adapted from those employed for plasma . The liver material, in contrast to a previous report1 has the same molecular weight as the corresponding normal plasma . The subunit structure, immunoelectrophoretic and immunological properties of the liver glycoprotein are identical to those of normal plasma . Amino acid and carbohydrate compositions of the liver material are similar to those of obtained from the plasma. The material has also been isolated and purified from the microsomal fraction of liver It has the same molecular weight and immunological properties as glycoprotein obtained from the cytosol. Although inhibitors of lysosmal proteases were added during the homogenization of the liver, the purified glycoprotein is devoid of trypsin-inhibitory capacity. The loss of inhibitory activity could be due to extensive cellular autolysis before autopsy. 相似文献
10.
M.Alfonsina Desiderio Angela Sessa Antonio Perin 《Biochimica et Biophysica Acta (BBA)/Molecular Cell Research》1985,845(3):463-468
The administration of preferential adrenergic receptor antagonists to uninephrectomized rats revealed the mediation in diamine oxidase activity increase that occurs in the remaining kidney undergoing compensatory hypertrophy. In fact, or , but not , , or this enzyme enhancement. Further studies with adrenoceptor agonists, such as epinephrine (, , , ), isoproterenol (, ) or terbutaline () showed that also in normal rat kidney diamine oxidase activity is under the control of through a mechanism that involves new synthesis of mRNA and protein. Theophylline, an inhibitor of phosphodiesterase, or forskolin, an activator of adenyl cyclase, increased diamine oxidase activity as does epinephrine or nephrectomy. Thus, catecholamine-triggered coupled to adenyl cyclase are involved in the regulation of diamine oxidase activity in normal and hypertrophic rat kidney. 相似文献
11.
The temperature-dependent relationship between K+ active influx, Mg2+-ATPase activity, transmembrane potential (ΔΨ) and the membrane lipid composition has been investigated in mycoplasma PG3. Native organisms were grown in a medium containing 10 μg/ml cholesterol and either oleic plus palmitic (chol (+), O + P) or elaidic (chol (+), E) acids. Adapted cells were grown in a medium free of exogenous cholesterol and supplemented with elaidic acid (chol (?), E).Arrhenius plots of 42K+ active influx gave a linear relationship for (chol (+), O + P) cells (). On the other hand, when oleic plus palmitic acids are replaced by elaidic acid, an upward discontinuity appears between 28 and 30°C, which is associated with a large increase in the apparent activation energy of the process ().Finally, a biphasic response with a break at approx. 23°C (; ) is observed for (chol (?), E) organisms. From the lack of correspondence between these effects on the K+ influx and the temperature dependence of both the Mg2+-ATPase activity and ΔΨ, it is suggested that changes in the membrane lipid composition affect the K+ transport at the level of the K+ carrier itself.Differential scanning calorimetry, steady-state fluorescence polarization of diphenylhexatriene and freeze-fracture electron microscopy experiments further suggest that the effect is largely due to modifications of the membrane microviscosity and that the K+ carrier is associated with the most fluid lipid species present in the membrane. 相似文献
12.
Adult female Dipetalonema viteae worms obtained from hamsters were introduced beneath the dorsal skin of , , and mice. The microfilaraemia from the transplanted worms in mice was higher and persisted longer than in , and in mice was intermediate between these two strains. The transplanted adult worms were killed earlier in compared to mice; adult worms were killed before the microfilariae were cleared from the circulation. D. viteae infective larvae did not reach maturity in mice but when female worms were implanted into mice which had been infected with third-stage infective larvae 6 or 19 days previously, microfilarial production was inhibited. Outbred as well as inbred nude mice infected with 10 or 20 infective larvae died by Day 15, whereas the normal littermate control mice that received the same number of infective larvae remained alive and healthy. There was no difference in the duration and level of microfilaraemia from implanted female worms in outbred nudes and their heterozygous littermate control mice. In contrast, microfilaraemia in inbred Nu+ was similar to that of inbred only until Day 45; thereafter the microfilaraemia declined to zero around Day 160 in the Nu+, at which time it was still high and persisted longer in the . Transplanted adults were viable in inbred for a longer time than in Nu+. When infected, amicrofilaraemic nudes, littermate controls, and three strains of mice were challenged, a very low level short-lasting microfilaraemia resulted. 相似文献
13.
[14C]Guanidine was observed in the urine after subcutaneous administration to rats of l-[guanidino-14C]arginine or l-[guanidino-14C]canavanine. []Hydroxyguanidine was additionally detected in the urine after injection of dl-[guanidino-14C]canavanine. These metabolites were characterized by high-voltage electrophoresis and paper chromatography, by enzymatic conversion of [14C]hydroxyguanidine to [14C]guanidine, and by repeated recrystallization of isolated urinary []guanidine as the picrate salt with no significant loss of specific activity. These experiments demonstrate that both l-arginine and l-canavanine can serve as precursors of guanidine in the rat. 相似文献
14.
Soluble consisting predominantly of αβ-units with below 170 000 was prepared by incubating pure membrane-bound (35–48 μmol Pi/min per mg protein) from the outer renal medulla with the non-ionic detergent dodecyloctaethyleneglycol monoether (C12E8). and potassium phosphatase remained fully active in the detergent solution at C12E8/protein ratios of 2.5–3, at which 50–70% of the membrane protein was solubilized. The soluble protomeric was reconstituted to Na+, K+ pumps in phospholipid vesicles by the freeze-thaw sonication procedure. Protein solubilization was complete at C12E8/protein ratios of 5–6, at the expense of partial inactivation, but and potassium phosphatase could be reactivated after binding of C12E8 to Bio-Beads SM2. At C12E8/protein ratios higher than 6 the activities were irreversibly lost. Inactivation could be explained by delipidation. It was not due to subunit dissociation since only small changes in sedimentation velocities were seen when the C12E8/protein ratio was increased from 2.9 to 46. As determined immediately after solubilization, was 7.4 S for the fully active , 7.3 S for the partially active particle, and 6.5 S for the inactive particle at high C12E8/protein ratios. The maximum molecular masses determined by analytical ultracentrifugation were 141 000–170 000 dalton for these protein particles. Secondary aggregation occurred during column chromatography, with formation of enzymatically active or with S and apparent molecular masses in the range 273 000–386 000 daltons. This may reflect non-specific time-dependent aggregation of the detergent micelles. 相似文献
15.
Christine De Paillerets Jacques Gallay Michel Vincent Monique Rogard Annette Alfsen 《生物化学与生物物理学报:生物膜》1981,644(1):134-142
The lipid dynamics of the adrenocortical microsomal membranes was studied by monitoring the fluorescence anisotropy and excited state lifetime of a set of anthroyloxy fatty acid probes (2-, 7-, 9- and 12-(9-anthroyloxy)-stearic acid (AP) and 16-(9-anthroyloxy)palmitic acid (AS). It was found that a decreasing polarity gradient from the aqueous membrane interface to the membrane interior, was present. This gradient was not modified by the proteins, as evidenced by comparison of complete membranes and derived liposomes, suggesting that the anthroyloxy probes were not in close contact with the proteins. An important change of the value of the mean rotational relaxation time as a function of the position of the anthroyl ring along the acyl chain was evidenced. In the complete membranes, a relatively more fluid medium was evidenced in the C16 as compared to the C2 region, while the rotational motion appeared to be the most hindered at the C7–C9 level. In the derived liposomes, a similar trend was observed but the mobility was higher at all levels. The decrease of the mean rotational relaxation time was more important for 12-AS and 16-AP. Temperature dependence of the mean rotational relaxation time of 2-AS, 12-AS and 16-AP in the complete membranes revealed the existence of a lipid reorganization occurring around 27°C and concerning mainly the C16 region. The extent to which the acyl chain reacted to this perturbation at the C12 level depended on pH. The presence of proteins increased the apparent magnitude of this reorganization and also modified the critical temperature from approx. 23°C in the derived liposomes to approx. 27°C in the complete membranes. Thermal dependence of the maximum velocity of the , the second enzyme in the enzymatic sequence, responsible for the biosynthesis of the in the adrenal cortex microsomes, was studied. The activation energy of the catalyzed reaction was found to be low and constant () in the temperature range 16–40°C at pH 7.5, 8.5 and 9, corresponding to the minimum, intermediate and maximum rate, respectively. A drastic increase of the activation energy () was observed at temperature below 16°C at pH 7.5. A correlated change of the as function of temperature was detected; at 36°C while at 13°C the value shifted to 8.7. The pH range of the group ionization was narrower at 13°C. In contrast with the behaviour of the , the was apparently unaffected by the lipid reorganization at 27°C. It is suggested that this enzyme possesses a different and more fluid lipid environment than the bulk lipids. 相似文献
16.
《Insect Biochemistry》1987,17(3):433-437
We have studied the metabolism of the hyperglycemic/cardioacceleratory peptide periplanetin CC-2 (pGlu-Leu-Thr-Phe-Thr-Pro-Asn-Trp-NH2), in Periplaneta americana using both in vitro and in vivo methods. We focused on metabolism in hemolymph, and homogenates of fat body (a presumed target organ) and Malpighian tubules (previously suggested to degrade the nonpurified cardioacceleratory factors). Despite using low concentrations (6 nM) of [4-3H-Phe]CC-2, enzymatic degradation in homogenates of fat body and Malpighian tubules proceeded with of ∼ 1 hr, while degradation in hemolymph was much slower. We injected low doses (1–2 pmol) of [3H]CC-2 into adult female cockroaches and analyzed hemolymph samples at various times; we determined the in vivo to be about 1 hr. We conclude that rate of secretion may be more important in controlling physiological levels of this peptide than rate of degradation. 相似文献
17.
A convective mass transfer model as analyzed and developed for use in determining intestinal wall permeabilities from external perfusion experiments. Analysis of the model indicates that the ratio of the exit to inlet concentration is a function of only two dimensionless independent variables, the wall permeability, and Graetz number, Gz = πDL/2Q. The Graetz number contains the independent variables of interest, length, diflusivity, and flow rate. The radius of the intestine is included implicitly in the flow rate. Since and Gz are the experimental quantities, and the solution to the model system contains Pω1 implicitly, a convenient approximate method is developed which allows a direct calculation of Pω1. This method is in error by 10–20% in the worst cases. The approach is illustrated by application to the determination of the wall permeabilities for two non polar compounds. 相似文献
18.
Quercetin inhibited a dog kidney ( preparation without affecting for ATP or for cation activators, attributable to the slowly-reversible nature of its inhibition. Dimethyl sulfoxide, a selector of E2 enzyme conformations, blocked this inhibition, while the K+-phosphatase activity was at least as sensitive to quercetin as the ( activity, all consistent with quercetin favoring E1 conformations of the enzyme. Oligomycin, a rapidly-reversible inhibitor, decreased the for ATP and the for cation activators, and its inhibition was also diminished by dimethyl sulfoxide. Although oligomycin did not inhibit the K+-phosphatase activity under standard assay conditions, a reaction presumably catalyzed by E2 conformations, its effects are nevertheless accommodated by a quantitative model for that reaction depicting oligomycin as favoring E1 conformations. The model also accounts quantitatively for effects of both dimethyl sulfoxide and oligomycin on , for substrate, and for K+, as well as for stimulation of phosphatase activity by both these reagents at low K+ but high Na+ concentrations. 相似文献
19.
The initial membrane reaction in the biosynthesis of peptidoglycan is catalyzed by (MurNAc)-pentapeptide translocase (UDP-MurNAc-Ala-γ dGlu-Lys-dAla-dAla undecaprenyl phosphate phospho-MurN Acpentapeptide transferase). In addition to the transfer reaction, the enzyme catalyzes the exchange of [3H]uridine monophosphate with the uridine monophosphate moiety of UDP-MurN Ac-pentapeptide. Two distinct discontinuities are observed in the slopes of the Arrhenius plots of the exchange and transfer activities at 22 and 30°C for the enzyme from Staphylococcus aureus Copenhagen. Anisotropy measurements of perylene fluorescence and electron spin resonance measurements of derivatives of 12-and 16-ketostearic acid intercalated into membranes from this organism define the lower () and upper () boundaries of a phase transition. These values correlate with the discontinuities observed for the activity measurements. Thus, it is proposed that the physical state of the lipid micro-environment of phospho-MurN Ac-pentapeptide translocase has a significant effect on the catalytic activity of this enzyme. 相似文献
20.
The mode of interaction of aqueous dispersions of phospholipid vesicles is investigated. The vesicles (average diameter 950 Å) are prepared from total lipid extracts of Escherichia coli composed of phosphatidylethanolamine, phosphatidylglycerol and cardiolipin. One type of vesicle contains , the other type as predominant acyl chain component. The vesicles show order?disorder transitions at transition temperatures, and , respectively. A mixture of these vesicles is incubated at 45° C and lipid transfer is studied as a function of time using the phase transition as an indicator. The system reveals the following properties: Lipids are transferred between the two vesicle types giving rise to a vesicle population where both lipid components are homogeneously mixed. Lipid transfer is asymmetric, i.e. -containing lipid molecules appear more rapidly in the -containing vesicles than vice versa. At a given molar ratio of the two types of vesicles the rate of lipid transfer is independent of the total vesicle concentration. It is concluded that lipid exchange through the water phase by way of single molecules or micelles is the mode of communication of these negatively charged lipid vesicles. 相似文献