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1.
Bacillus subtilis aminopeptidase: Specificity toward amino acid amides,dipeptides, and oligopeptides
Mohammed A. Ajabnoor Lee E. Ray Fred W. Wagner 《Archives of biochemistry and biophysics》1980,202(2):540-545
Bacillus subtilis aminopeptidase hydrolyzed amino acid amides with a specificity similar to that determined using amino acyl-β-naphthylamides, but at much greater catalytic rates. Neutral and basic amino acid amides were the best substrates. A series of Leu and Lys NH2-terminal dipeptides hydrolyzed by Co2+-activated aminopeptidase showed that the ratios for the Lys substrates were fourfold greater than the corresponding Leu substrates and that catalytic differences reflected the identity of COOH terminal residues. Greatest catalytic rates were obtained when aromatic residues were in the COOH terminal position of the substrate (Trp, Tyr, Phe); but, significant hydrolysis was achieved when aliphatic residues were COOH-terminal in the dipeptide. The Co2+-activated enzyme would not hydrolyze peptide bonds composed of the imide nitrogen of Pro, thus, bradykinin was not a substrate. However, the Co2+-activated enzyme removed sequentially the first four residues from eledoisin-related peptide and the A chain of bovine insulin. 相似文献
2.
Sensitive methods for the determination of rat mast cell protease I, rat mast cell protease II, human skin chymotrypsinlike enzyme, dog skin chymotrypsinlike enzyme, human leukocyte cathepsin G, and bovine chymotrypsin Aα with peptide thiobenzyl ester substrates are reported. Kinetic constants as well as the maximum sensitivity for the hydrolysis of the peptide substrates succinyl-phenylalanyl-leucyl-phenylalanine thiobenzyl ester and succinyl-alanyl-alanyl-prolyl-phenylalanine thiobenzyl ester were determined. Hydrolysis rates were followed spectrophotometrically at 324 nm by the formation of 4-thiopyridone (? = 19,800 m?1 cm?1), the product of the reaction between benzylthiol, released during hydrolysis of the peptide thiobenzyl esters, and 4,4′-dithiodipyridine present in the assay mixture. Peptide thiobenzyl ester substrates were shown to be very sensitive substrates, predominantly because of the large extinction coefficient of 4-thiopyridone and the high values for these compounds. 相似文献
3.
John A. Jacquez 《生物化学与生物物理学报:生物膜》1973,318(3):411-425
The Michaelis-Menten parameters, and of the initial 1-min fluxes of uptake of l-phenylalanine and of α-aminoisobutyric acid were determined for extracellular concentrations of Na+ ranging from 0.5 to 110 mequiv/l for Ehrlich ascites tumor cells. The maximal initial flux, , decreased with decrease in extracellular Na+ for both α-aminoisobutyric acid and phenylalanine but the for α-aminoisobutyric acid increased markedly as the Na+ concentration fell whereas the for phenylalanine decreased. Cycloleucine behaved like phenylalanine.The data provides strong evidence that the Na+-independent flux of phenylalanine is an exchange diffusion flux that can be varied by changing the intracellular level of amino acids such as phenylalanine. For phenylalanine, cyclolcucine, and methionine this exchange diffusion flux appears to be additive with the Na+-dependent initial flux. α-Aminoisobutyric acid also has an exchange diffusion that is Na+-independent but it has a high and is not additive with the Na+-dependent flux. 相似文献
4.
A complete titration of phosphatidic acid bilayer membranes was possible for the first time by the introduction of a new anaologue, acid, which has the advantage of a high chemical stability at extreme pH values. The synthesis of this phosphatidic acid is described and the phase transition behaviour in aqueous dispersions is compared with that of three ester phosphatidic acids; acid, 1,3-dimyristoylglycerol-2-phosphoric acid and acid.The phase transition temperatures () of aqueous phosphatidic acid dispersions at different degrees of dissociation were measured using fluorescence spectroscopy and 90° light scattering. The values are comparable to the melting points of the solid phosphatidic acids in the fully protonated states, but large differences exist for the charged states.The vs. pH diagrams of the four phosphatidic acids are quite similar and of a characteristic shape. Increasing ionisation results in a maximum value for the transition temperatures at pH 3.5 (). The regions between the first and the second of the phosphatidic acids are characterised by only small variations in the transition temperatures (extended plateau) in spite of the large changes occurring in the surface charge of the membranes. The slope of the plateau is very shallow with increasing ionisation. A further decrease in the H+ concentration results in an abrupt change of the transition temperature. The slope of the vs. pH diagram beyond becomes very steep. This is the 相似文献
5.
The ATP/ADP exchange is shown to be a partial reaction of the by the absence of measurable nucleoside diphosphokinase activity and the insensitivity of the reaction to , -di(adenosine-5′) pentaphosphate, a myokinase inhibitor. The exchange demonstrates an absolute requirement for Mg2+ and is optimal at an ADP/ATP ratio of 2. The high ATP concentration () required for maximal exchange is interpreted as evidence for the involvement of a low affinity form of nucleotide site. The ATP/ADP exchange is regarded as evidence for an ADP-sensitive form of the phosphoenzyme. In native enzyme, pre-steady state kinetics show that the formation of the phosphoenzyme is partially sensitive to ADP while modification of the enzyme by pretreatment with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) in the absence of Mg2+ results in a steady-state phosphoenzyme population, a component of which is ADP sensitive. The ATP/ADP exchange reaction can be either stimulated or inhibited by the presence of K+ as a function of pH and Mg2+. 相似文献
6.
7.
A chromatin-associated histone H1 kinase has been detected in synchronized Novikoff hepatoma cells. Enzyme specific activity increased 4 to 6-fold from late G-2 to mid-metaphase, then decayed exponentially (, 28.5 min) to the interphase level. Extracts of the mitotic kinase retained the ability to decay at 37°C but not at 0°C (, 24 min), under conditions in which interphase activity was stable. Sedimentation rates in sucrose density gradients of interphase and mitotic enzymes (before and after decay) were identical. Purification did not alter the rate of enzyme decay. However, high ionic strength prevented decay of crude but not purified preparations of mitotic enzyme. The results are discussed in terms of an allosteric mechanism for reversible activation of enzyme activity. 相似文献
8.
According to previous authors, cytochrome , when extracted from bovine liver by a detergent method, is called cytochrome . On the other hand, the protein obtained after trypsin action, which eliminates an hydrophobic peptide of about 54 residues, is called cytochrome .Fluorescence polarization of the dansyl phosphatidylethanolamine probe inserted into phospholipid vesicles is very senstive to the binding of proteins, and so is a useful method to study lipid-protein interactions.The chromophore mobility, , decreases markedly when dipalmitoyl phosphatidylcholine vesicles are incubated with cytochrome , whereas does not change for cytochrome and cytochrome . This can be interpreted as a strengthening of the bilayer, only due to the interaction of the hydrophobic peptide tail.Interaction of dipalmitoyl phosphatidylcholine vesicles with cytochrome occurs either below or above the melting temperature of the aliphatic chains (41 °C). Even for a high protein to lipid molar ratio (1 molecule of protein for 40 phospholipid molecules), the melting temperature is apparently unaffected.Phosphatidylserine and phosphatidylinositol do not interact at pH 7.7 with cytochrome , because electrostatic forces prevent formation of complexes. At low pH, the interaction with the protein occurs, but the binding is mainly of electrostatic nature. 相似文献
9.
Spondyloepiphyseal dysplasia, chondroitin sulfate type: a possible defect of PAPS--chondroitin sulfate sulfotransferase in humans 总被引:4,自引:0,他引:4
Paulo A.S. Mourão Setuko Kato Patricia V. Donnelly 《Biochemical and biophysical research communications》1981,98(2):388-396
Four patients with an unusual form of spondyloepiphyseal dysplasia excreted in the urine undersulfated chondroitin 6-sulfate (Biochem. Med. , 415–423, 1973). The sera of these patients show a low activity of PAPS — chondroitin sulfate sulfotransferase, while the undersulfated chondroitin sulfate present in their urine is a much better acceptor of than standard chondroitin sulfate when they are incubated with []PAPS and normal sulfotransferases. These results suggest that in these patients the skeletal lesions are secondary to a defect in the synthesis of chondroitin sulfate involving specifically the sulfotransferase activity. 相似文献
10.
Bacterial bioluminescence-identification of fatty acid as product, its quantum yield and a suggested mechanism 总被引:5,自引:0,他引:5
By using radioactive decanal the direct transformation of this aldehyde to decanoic acid, with a quantum yield of 0.13, has been demonstrated. A mechanism analogous to that of other better understood bioluminescent reactions is proposed, leading to a product, as yet unisolated from the enzymic reaction, whose fluorescence spectrum is an excellent match for that of the luminescence.The extensive examination1,2,3 of the isolated bacterial luminescence system has resulted in the accepted outline shown. We wish to modify it, in accordance with the previous evidence, by suggesting that ’intermediates I and II‘ in Hastings' terminology2 are the same enzyme bound FMNH2 moiety. A lively controversy has surrounded the attempts to determine whether aldehyde exerts a purely catalytic role2 or is transformed in the reaction.4 If the aldehyde reacts, then the simplest product is the corresponding carboxylic acid, perhaps formed via the peracid. The most likely alternative reaction would involve enolistation and oxidation at the α-methylene group. We examined the second alternative fairly carefully, and found no evidence for it. We do not wish to report these results in detail at present, since we have now established that the acid corresponding to that formed in a normal autoxidation of the aldehyde is the product. Some indication of the nature of the products of the reaction is available.5Since the amount of product in the reaction is restricted to a very low level by the concentrations required, we labelled decanal with tritium at C-2 and thus were able to record the yield with some precision. Although recent work6 strongly implies that acid is formed stoichiometrically, the direct measurement of the quantum yield with respect to acid formation is necessary before a mechanism can be written. We have suggested a mechanism compatible with observations in this system, analogous to all cases of bioluminescence for which a mechanism is reasonably well established. This mechanism also leads to a product excited state with excellent agreement around pH7 in fluorescence wavelength to that of the luminescence. 相似文献
11.
Mechanism of action of thrombin on fibrinogen. The reaction of thrombin with fibrinogen-like peptides containing 11, 14, and 16 residues 总被引:4,自引:0,他引:4
J W van Nispen T C Hageman H A Scheraga 《Archives of biochemistry and biophysics》1977,182(1):227-243
The following peptides were synthesized by classical methods in solution: Ac-Gly-Gly- Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH3 (A), Ac-Ala-Glu-Gly-Gly-Gly-Val- Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH3 (B), and Ac-Phe-Leu-Ala-Glu-Gly-Gly- Gly-Val-Arg-Gly-Pro-Arg-Val-Val-Glu-Arg-NHCH3 (C). The rates of hydrolysis of the Arg-Gly bond of these three peptides by thrombin were measured, and the values of were found to be 0.05 × 10?7 (A), 0.02 × 10?7 (B), and 1.6 × 10?7 (C) [(NIH units/ liter)s]?1. The value of for peptide C is less than 1% of that for fibrinogen [although the value of kcat itself, for peptide C (but not for A or B), is comparable to that for fibrinogen]. These results indicate that phenylanine and leucine at positions P9 and P8, respectively, play a key role in the reaction of thrombin with fibrinogen. The data also show that factors outside of the 16 residues of peptide C are important in determining the rate of hydrolysis of fibrogen by thrombin. 相似文献
12.
(1) The ATPase enzyme in untreated chromatophores from Rhodospirillum rubrum is in a low-activity state (designated as E°). It can be activated by application of a transmembrane generated by light-induced electron transport, or by application of acid-base jumps. (2) After rapid dissipation of the light-induced , the active state of the ATPase enzyme decays (in the absence of added substrates or products) to a low-activity state (designated as E′), with a half-time of the order of 2–4 min. This state differs from E° in that E′ (but not E°) can be rapidly reactivated by addition of substrate, but only when the Mg2+ concentration is kept below 20–30 μM. Since this is characteristic of an activated enzyme containing tightly bound ADP (Slooten, L. and Nuyten, A. (1981) Biochim. Biophys. Acta 638, 313–326), it is suggested that release of endogenous, tightly bound ADP is one of the factors involved in activation of the ATPase enzyme. 相似文献
13.
Five species of cockroach were tested on a miniature treadmill at three velocities as O2 consumption () was measured: Gromphadorhina chopardi, Blaberus discoidalis, Eublaberus posticus, Byrsotria fumagata and Periplaneta americana. All cockroaches showed a classical aerobic response to running: increased rapidly from a resting rate to a steady-state on-response varied from under 30 s to 3 min. Recovery after exercise was rapid as well; off-response varied from under 30 s to 6 min. These times are faster or similar to mammalian values. varied directly with velocity as in running mammals, birds and reptiles. during steady-state running was only 4–12 times higher than at rest. Running is energetically much less costly per unit time than flying, but the cost of transport per unit distance is much more expensive for pedestrians. The minimal cost of transport (Mrun), the lowest necessary to transport a given mass a specific distance, is high in cockroaches due to their small size. The new data suggest that insects may be less economical than comparable sized vertebrates. 相似文献
14.
Highly purified divalent and monovalent antibodies against cytochrome , anti- immunoglobulin G (IG) and anti- Fab', were used in elucidating the role of this cytochrome in the drug-oxidizing enzyme system of mouse liver microsomes. Anti- IG strongly inhibited not only NADH-supported but also NADPH-supported oxidation of 7-ethoxycoumarin and benzo(a)pyrene, but had no inhibitory action on the oxidation of aniline. Anti- Fab' also inhibited NADH-supported and NADPH-supported benzo(a)pyrene hydroxylation. These observations indicate an essential role of cytochrome in the transfer of electrons not only from NADH but also from NADPH to cytochrome P-450 in the microsomal oxidation of some drugs, but not of aniline. 相似文献
15.
Glucoamylase (EC 3.2.1.3) was coupled to controlled pore glass by using titanium(IV) chloride. The drying conditions used during the activation step were studied, and the highest activity (237 units/g of matrix) of immobilized enzyme was obtained when the support and the titanium(IV) chloride solution were dried at 45°C in vacuo for 16 h. After several washing cycles, the specific activity of the immobilized enzyme was ~13 units/mg of protein irrespective of the washing cycle used. However, this immobilized enzyme preparation was also the least stable (). Investigation of the possibility of the stabilization of the linkage of the enzyme to the support by crosslinking with bifunctional reagents showed that the stabilization of the enzyme () was achievable by treatment with a 5% glutaraldehyde solution at pH 7.0 for 2 h (product activity 67 units/g of matrix, specific activity 4 units/mg of protein); this product also showed no release of protein during use. A higher activity (296 units/g of matrix was achieved by stabilization by treatment with a 5% tannic acid solution at pH 7.0 for 2 h. The combined use of glutaraldehyde and tannic acid was effective in stabilizing the bound enzyme () with an initial activity of 116 units/g of matrix. When use was made of the same support in presilanized (3-aminopropyltriethoxy silane) form followed by glutaraldehyde coupling a similar initial activity (112 units/g of matrix) was obtained, but the operational stability was much better (. 相似文献
16.
The anomerization of α-d-glucose 6-phosphate has been examined using a spectrophotometric coupled enzyme assay. The pH-rate profile for spontaneous d-glucose 6-phosphate anomerization reveals that the d-glucose 6-phosphate dianion is the species giving rise to the much higher rate of d-glucose 6-phosphate anomerization over that of d-glucose. A deuterium solvent isotope effect of is consistent with the postulated intramolecular general-base catalysis by the phosphate. 相似文献
17.
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
18.
F.M.A.H. Schuurmans Stekhoven H.G.P. Swarts Y.-F. Fu G.A.J. Kuijpers J.J.H.H.M. de Pont S.L. Bonting 《生物化学与生物物理学报:生物膜》1984,774(2):277-287
(1) Treatment of from rabbit kidney outer medulla with the labeled thio-analogue of ATP in the presence of and the absence of K+ leads to thiophosphorylation of the enzyme. The value for [γ-S]ATP is 2.2 μM and for Na+ 4.2 mM at 22°C. Thiophosphorylation is a sigmoidal function of the Na+ concentration, yielding a Hill coefficient . (2) The thio-analogue () can also support overall activity, but at 37°C is only h?1 or 0.09% of the specific activity for ATP (). (3) The thiophosphoenzyme intermediate, like the natural phosphoenzyme, is sensitive to hydroxylamine, indicating that it also is an acylphosphate. However, the thiophosphoenzyme, unlike the phosphoenzyme, is acid labile at temperatures as low as 0°C. The acid-denatured thiophosphoenzyme has optimal stability at pH 5–6. (4) The thiophosphorylation capacity of the enzyme is equal to its phosphorylation capacity, indicating the same number of sites. Phosphorylation by ATP excludes thiophosphorylation, suggesting that the two substrates compete for the same phosphorylation site. (5) The (apparent) rate constants of thiophosphorylation (0.4 s?1 vs. 180 s?1), spontaneous dethiophosphorylation (0.04 s?1 vs. 0.5 s?1) and K+-stimulated dethiophosphorylation (0.54 s?1 vs. 230 s?1) are much lower than those for the corresponding reactions based on ATP. (6) In contrast to the phosphoenzyme, the thiophosphoenzyme is ADP-sensitive (with an apparent rate constant in ADP-induced dethiophosphorylation of 0.35 s?1, ) and is relatively K+-insensitve. The for K+ in dethiophosphorylation is 0.9 mM and in dephosphorylation 0.09 mM. The thiophosphoenzyme appears to be for 75–90% in the ADP-sensitive E1-conformation. 相似文献
19.
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
20.
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