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1.
Uptake of d-glucosamine by rat brain synaptosomes occurs via a saturable transport process (Km 2.1 mM, V 3.0 nmol/mg per min) which was clearly distinguishable from simple diffusion. This transport process is highly sensitive to cytochalasin (Ki = 7 · 10?5 mM. d-Glucose competitively inhibits d-glucosamine uptake with a Ki value of 8 · 10?1 mM. 相似文献
2.
The efflux of mitochondrial adenine nucleotide which is induced by addition of PPi to suspensions of rat liver mitochondria has been investigated. This efflux of adenine nucleotide is greatly stimulated by the uncoupler FCCP at 1 μM, Vmax being 6.7 nmol/min per mg protein as compared to 2.0 nmol/min per mg protein in its absence. The depletion process is inhibited by carboxyatractyloside. The Km for PPi of 1.25 mM is essentially unchanged when uncoupler is added. Quantitation of the individual adenine nucleotide species (ATP, ADP and AMP) and their relationship to the rate of efflux suggests that ADP is the predominant species being exchanged for PPi. 相似文献
3.
Florida's red tide organism, Gymnodinium breve, utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a KFD of 11 μM and a Vmax of 1 × 10?10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin (Ki = 40 μM), mannose (Ki = 12O μM), and 2-deoxy-d-glucose (Ki = 190 μM) and non-competitively inhibited by galactose (Ki = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system. 相似文献
4.
Isolated basolateral plasmamembrane vesicles from rat duodenum epithelial cells exhibit ATP-dependent calcium-accumulation and Ca2+-dependent ATPase activity. Calcium accumulation stimulated by ATP is prevented by the calcium ionophore A23187, inhibited 80% by 0.1 mM orthovanadate but is not effected by oligomycin. Calcium accumulation is not observed with the substrate β-γ-(CH2)-ATP, ADP and p-nitrophenyl phosphate. Kinetic studies reveal an apparent Km of 0.2 μM Ca2+ and a Vmax of 5.3 nmol Ca2+/min per mg protein for the ATP-dependent calcium-uptake system. Calmodulin and phenothiazines have no effect on calcium accumulation in freshly prepared membranes, but small effects are inducable after a wash with a 5 mM EGTA. The kinetic parameters of Ca2+-ATPase are: Km = 0.25 μM Ca2+ and Vmax = 19.2 nmol Pi/min per mg protein. Three techniques, osmotic shock, treatment with Triton X-100 or the channel-forming peptide alamethacin, reveal that about 40% of the vesicles are resealed. Assuming that half of the resealed vesicles have an inside-out orientation, the Vmax of ATP-dependent calcium uptake amounts to 25 nmol Ca2+/min per mg protein and of the Ca2+-ATPase to 23 nmol Pi/min per mg protein. The close correlation between kinetic parameters of Ca2+-ATPase and ATP-dependent calcium-transport strongly suggests that both systems are expressions of a Ca2+-pump located in duodenal basolateral plasma membranes. 相似文献
5.
The transport of adenosine was studied in pure cultures of glial cells from chick embryo brain. In order to avoid complications in uptake measurements due to adenosine metabolism, cultures were depleted of ATP by incubation with cyanide and iodoacetate prior to addition of [3H]adenosine. Under the 5- to 25-s periods used for the transport assay, no adenosine metabolism could be detected. Initial rates of adenosine transport under these conditions obeyed the Michaelis-Menten relationship with Km = 370 μM and Vmax = 10.3 nmol/min/mg cell protein. ATP depletion or elimination of Na+ from the assay medium had no significant effect on initial rates of adenosine uptake. However, when assays were carried out under conditions of significant adenosine metabolism (10-min uptake in the absence of metabolic inhibitors), a high-affinity incorporation process could be demonstrated in the glial cells (Km = 12 μM; Vmax = 0.34 nmol/ min/mg protein). The transport activity expressed in ATP-depleted glial cells was most sensitive to inhibition by nitrobenzylthioinosine, dipyridamole, and N6-benzyladenosine. In decreasing order of potency, N6-methyladenosine, 2-chloroadenosine, inosine, and thymidine also blocked adenosine translocation in glial cultures. Thus, adenosine transport by cultured glial cells occurs by means of a low-affinity, facilitated diffusion system which is similar to the nucleoside transporter in cells of nonneural origin. 相似文献
6.
Ibuprofen, one of the nonsteroidal anti-inflammatory drugs, inhibited arylamine N-acetyltransferase activity of Klebsiella pneumoniae both in vitro and in vivo. The NAT activities of Klebsiella pneumoniae were inhibited by ibuprofen in a dose-dependent manner both in vitro and in vivo. In vitro, the NAT activity was 0.675 ±
0.028 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was
0.506 ± 0.002 nmol/min/mg of protein for the acetylation of 2-aminofluorene. In vivo, the NAT activity was 0.279 ± 0.016 nmol/min/1010 colony forming units (CFU) for the acetylation of 2-aminofluorene. In the presence of 8 mM ibuprofen, the NAT activity was
0.228 ± 0.008 nmol/min/1010 CFU for the acetylation of 2-aminofluorene. The inhibition of NAT activity by ibuprofen was shown to persist for at least
4 h. For in vitro examination, the values of apparent K
m
and V
max were 1.08 ± 0.05 mM and 9.17 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen
was added to the reaction mixtures, the values of apparent K
m
and V
max were 1.19 ± 0.01 mM and 6.67 ± 0.11 nmol/min/mg of protein, respectively, for 2-aminofluorene. For in vivo examination, the
values of apparent K
m
and V
max were 1.24 ± 0.48 mM and 4.18 ± 1.06 nmol/min/10 × 1010 CFU, respectively, for 2-aminofluorene. However, when 8 mM of ibuprofen was added to the culture, the values of apparent
K
m
and V
max were 0.95 ± 0.29 mM and 2.77 ± 0.37 nmol/min/mg protein, respectively, for 2-aminofluorene, respectively. This report is
the first finding of ibuprofen inhibition of arylamine N-acetyltransferase activity in a strain of Klebsiella pneumoniae.
Received: 28 January 1997 / Accepted: 12 February 1997 相似文献
7.
Hypoxanthine transport by Chinese hamster lung fibroblasts: Kinetics and inhibition of nucleosides 总被引:1,自引:0,他引:1
The transport of []hypoxanthine was studied in monolayer cultures of mutant Chinese hamster lung fibroblasts lacking hypoxanthine-guanine phosphoribosyltransferase. Initial rates of transport were determined by rapid uptake experiments (8 to 20 s); a Michaelis constant of 0.68 ± 0.09 mm for hypoxanthine was derived from linear, monophasic plots of against v. Nucleosides are competitive inhibitors of this process; adenosine and thymidine give respective Ki values of 86 and 300 μm. The corresponding bases give much higher inhibition constants with adenine and thymine yielding values of 3100 and 1700 μm, respectively. A similar pattern was observed for competitive inhibition of hypoxanthine transport by inosine, adenine arabinoside, uridine, cytidine, and two ribofuranosylimidazo derivatives of pyrimidin-4-one; in every case the nucleoside exhibited a lower Ki value than the corresponding homologous base. The inhibition constants observed for nucleosides are remarkably similar to their Km values for nucleoside transport by cultured cells recently reported by others. Hypoxanthine transport was also blocked by the 6-(2-hydroxy-5-nitrobenzylthio) derivatives of inosine and guanosine and by dipyridamole; these agents are also inhibitors of nucleoside transport. These results indicate a closer relationship between base and nucleoside transport than previously recognized and suggest that these two transport processes may involve identical or very similar transport proteins. 相似文献
8.
The presence of a sodium-dependent, saturable uptake process is described in basolateral membranes of rat renal cortex for L-glutamine. Concentration-dependence studies indicate the presence of multiple transport systems withK
m
1 of 0.032 mM and V1 of 0.028 nmol/mg of protein per min, andK
m
2 of 17.6 mM and V2 of 17.6 nmol/mg of protein per min. Lysine completely inhibits the high-affinity, low-capacityK
m system and partially inhibits the low-affinity, high-capacity system. Cystine and other dibasic amino acids also affect glutamine uptake. 相似文献
9.
The ability of trout to metabolize aniline in vitro in the presence of some divalent metal ions was investigated in the liver microsomes of rainbow trout, Salmo gairdneri. Trout liver microsomes were highly capable of catalyzing aniline hydroxylation to p-aminophenol with a specific activity of 0.068 nmoles/min per mg of microsomal protein in potassium phosphate buffer, pH 7.4 at 25°C. The activity of the aniline hydroxylase system was competitively inhibited by Hg+2, Ni+2, Cd+2, and Zn+2, while Cu+2 and Fe+3 seemed to inhibit the activity noncompetitively at 1 mM aniline concentrations. IC50 values at fixed aniline concentration were estimated to be 0.45 mM for Hg+2, Ni+2, and Cd+2, 1.8 mM for Zn+2 and Fe+3, and 1.3 mM for Cu+2. Eadie-Hofstee plots gave identical Vmax values of approximately 0.046 nmol/min per mg of protein while Km values were increased in the presence of Hg+2, Ni+2, CD+2, and Zn+2, indicating competitive inhibition. Both Km and Vmax values were affected by Fe+3 and Cu+2, suggesting noncompetitive inhibition. Ki values extracted from the Dixon plots were determined t be 0.23, 0.43, and 0.65 mM for Hg+2, Ni+2, and Cd+2, respectively, providing the most effective inhibition on the aniline hydroxylase system among studied metal ions. The Ki values were much higher in the presence of others. The results indicate a selective inhibition of the aniline hydroxylase system of trout liver microsomes by divalent metal ions. © 1997 John Wiley & Sons, Inc. 相似文献
10.
Previous work from this laboratory has shown that 5-thio-d-glucose is a competitive inhibitor for active transport of d-glucose. The present work indicates that the thiosugar analog and its 1-phosphate can also interfere with d-glucose 6-P formation.5-Thio-d-glucose serves as a substrate for yeast hexokinase with a Km of 4 mm, and V of 8.8 nmol/min/μg of protein. The analog competitively inhibits d-glucose phosphorylation with a Ki of 20 mm.5-Thio-d-glucose 1-P can act as a substrate for rabbit skeletal muscle phosphoglucomutase with a Km of 60 μm and V of 0.17 μmol/min/μg of protein. Thus, 5-thio-d-glucose 1-P behaves as a near metabolic analog of d-glucose 1-P. 5-Thio-d-glucose 1-P is a competitive inhibitor of d-glucose 1-P conversion to the 6-P with a Ki of 16.2 μm.5-Thio-d-glucose 6-P produced by phosphorylation of 5-thio-d-glucose and by conversion from 5-thio-d-glucose 1-P was identified by chromatographic mobility and by color reactions. 相似文献
11.
The uptake of leucine through the tegument of Cotugnia digonopora, a cestode found in the fowl intestine, occurs by a process of active transport. The Kt of transport is 0.87 mM and the Vmax is 0.223 μmol/min/g. Uptake of the amino acid is competitively inhibited by valine (Kt = 1.30 mM). Potassium cyanide and 2,4-dinitrophenol do not completely block the entry of leucine into the parasite. 相似文献
12.
Germlings of Phytophthora palmivora possess at least two systems for the uptake of inorganic phosphate (Pi). The first is synthesized on germination in medium containing 50 M Pi and has a Km of approx. 30 M (Vmax=7–9 nmol Pi/h·106 cells). The second is synthesized under conditions of Pi-deprivation and has a higher affinity for Pi (Km=1–2 M), but a lower Vmax (0.5–2 nmol Pi/h·106 cells). The fungicide phosphite likewise enters the germlings via two different transport systems, the synthesis of which also depends on the concentration of Pi in the medium. The Km of the lower affinity system is 3 mM (Vmax=20 nmol phosphite/h·106 cells) and that of the higher affinity system is 0.6 mM (Vmax=12 nmol/h·106 cells). Pi and phosphite are competitive inhibitors for each other's transport in both systems. However, whereas mM concentrations of phosphite are necessary to inhibit Pi transport, only M concentrations of Pi are required to inhibit phosphite transport. A third system of uptake for Pi also exists, since when phosphate-deprived cells are presented with mM concentrations of Pi, they transport the anion at a very high rate (around 100 nmol/h·106 cells). High rates of transport of phosphite are also observed when these cells are presented with mM concentrations of this anion. 相似文献
13.
Acetate is effectively taken up by whole cells of Corynebacterium glutamicum via a specific carrier with a pH optimum of 8. The K
m of acetate uptake was 50 μM and the V
max 25–35 nmol/mg dw min. The activation energy was determined to be 70 kJ/mol. Acetate uptake was competitively inhibited by
propionate with a K
i of about 30 μM and blocked by addition of sulfhydryl reagents. The transport activity was clearly dependent on the membrane
potential, but independent of the presence of Na+-ions. It is concluded that uptake of acetate proceeds by a secondary, proton coupled mechanism. 相似文献
14.
《BBA》1985,810(2):184-199
(1) Mitochondria were prepared from leaves of spinach, green and etiolated seedlings and roots of pea, potato tuber and rat liver and heart. In the case of leaf mitochondria, an improved isolation procedure resulted in high respiratory rates (460–510 nmol/mg protein per min) and good respiratory control ratio (6.8–9.8) with glycine as substrate. (2) In these mitochondria oxaloacetate transport was studied either by following the inhibitory effect of oxaloacetate on the respiration of NADH-linked substrates or by determining the consumption of [4-14C]oxaloacetate. (3) Studies of the competition by other carboxylates and effect of inhibitors on the oxaloacetate transport demonstrate that mitochondria from spinach leaves, green pea seedlings, etiolated pea seedlings and pea roots contain a specific translocator for oxaloacetate with a very high affinity to its substrate (Km = 3–7 μM) and an even higher sensitivity to its competitive inhibitor phthalonate (Ki = 3–5 μM). The Vmax values ranged from 150 to 180 nmol/mg protein per min for mitochondria from etiolated pea seedlings and pea roots and from 550 to 570 nmol/mg protein per min for mitochondria from spinach leaves and green pea seedlings. In mitochondria from potato tuber, the Km was about one order of magnitude higher (Vmax = 450 nmol/mg protein per min). In mitochondria from rat liver and rat heart, a specific translocator for oxaloacetate was not found. (4) The oxaloacetate translocator enables the functioning of a malate-oxaloacetate shuttle for the transfer of reducing equivalents across the inner mitochondrial membrane. (5) This malate-oxaloacetate shuttle appears to play a role in the photorespiratory cycle in catalyzing the transfer of reducing equivalents generated in the mitochondria during glycine oxydation to the peroxysomal compartment for the reduction of β-hydroxypyruvate. (6) Interaction between the mitochondrial and the chloroplastic malate oxaloacetate shuttles would make it possible for surplus-reducing equivalents, generated by photosynthetic electron transport, to be oxidized by mitochondrial electron transport. 相似文献
15.
Elise M. Rosen-Levin Kenneth W. Smithson Gary M. Gray 《Biochimica et Biophysica Acta (BBA)/General Subjects》1980,629(1):126-134
The small intestine is known to possess mechanisms for intact transport and membrane hydrolysis of oligopeptides. To determine the relative role of these processes in peptide assimilation the fate of two model peptides known to be high-affinity substrates for the brush border aminooligopeptidase were studied in rat small intestine in vivo. Both 20 mM Gly-L-Pro, a potent inhibitor of peptide transport, and specific inhibitors of the aminopeptidase, 10 mM L-Ala-β-naphthylamide or the phthalimido derivative of 0.1 mM L-leucine bromomethyl ketone, reduced assimilation of L-Leu-Gly-Gly and L-Leu-L-Leu. Further inhibition was found when both transport and peptidase inhibitors were included in the intestinal perfusate suggesting that the model di- and tripeptides utilize both intact transport and surface hydrolysis for their assimilation. Although comparative kinetic parameters of intact transport (Km = 22 mM; V = 1.9 · 10?3μmol · s?1 · cm?2) and surface hydrolysis (Km = 8.7; V = 1.1 · 10?3) for l-Leu-l-Leu differed markedly, the relationship of peptide concentration to assimilation rate was nearly identical for intact transport and surface hydrolysis in the physiological range of 1–10 mM substrate. Both intact peptide transport and surface hydrolysis appear to be efficient and complementary processes that promote efficient assimilation of dipeptides and tripeptides. The relative importance of each assimilation process appears to depend upon the amino acid composition of the peptide nutrient. 相似文献
16.
Adenine uptake into human blood platelets is a carrier-mediated process with a Km of 159±21 nM and a V of 100±10 pmoles/min per 109 platelets (in citrated platelet-rich plasma). The Q10 was 2.53±0.22. A pH optimum was found at 7.5. Washing of the platelets increased the Km to 453±33 nM and V to 397±38 pmoles/min per 109 platelets. The change in shape induced in platelets by ADP was accompanied by an increase in V (2 times) and Km (1.5 times).Guanine (Ki 50 μM), hypoxanthine (Ki 390 μM), adenine-N′-oxide (Ki 40 μM), adenosine (Ki 100 μM), RA 233 (Ki 75 μM) and papaverine (Ki 15 μM) acted as competitive inhibitors. Adenosine at low concentrations, and prostaglandin E1 gave inhibition at only high adenine levels. A similar inhibition was obtained with 2-deoxy-d-glucose. Sulfhydryl-group inhibitors, pyrimidines and ouabain had no effect. 相似文献
17.
Dicarboxylate transport in maize mesophyll chloroplasts 总被引:7,自引:0,他引:7
Evidence is presented for high rates of carrier-mediated dicarboxylate anion transport in maize mesophyll chloroplasts. Radioactively labeled malate is transported across the chloroplast envelope leading to accumulation in the stroma. Malate in the stroma will exchange for external malate, oxaloacetate, glutamate, aspartate, and oxoglutarate. At 4 °C the V of malate uptake is 50 μmol·h?1·mg Chl?1 and the Km for malate is 0.5 mm. Oxaloacetate competitively inhibits malate uptake with a Ki estimated to be 0.3 mm. The temperature dependence of malate uptake indicates an activation energy of 12 kcal/mol, and extrapolation using this value gives a rate of transport at 30 °C of approximately 300 μmol·h?1·mg Chl?1. This rate approximates the rates of photosynthetic malate production by these chloroplasts. 相似文献
18.
A glucose-tolerant β-glucosidase was purified to homogeneity from prune (Prunus domestica) seeds by successive ammonium sulfate precipitation, hydrophobic interaction chromatography and anion-exchange chromatography. The molecular mass of the enzyme was estimated to be 61 kDa by SDS-PAGE and 54 kDa by gel permeation chromatography. The enzyme has a pI of 5.0 by isoelectric focusing and an optimum activity at pH 5.5 and 55 °C. It is stable at temperatures up to 45 °C and in a broad pH range. Its activity was completely inhibited by 5 mM of Ag+ and Hg2+. The enzyme hydrolyzed both p-nitrophenyl β-d-glucopyranoside with a Km of 3.09 mM and a Vmax of 122.1 μmol/min mg and p-nitrophenyl β-d-fucopyranoside with a Km of 1.65 mM and a Vmax of 217.6 μmol/min mg, while cellobiose was not a substrate. Glucono-δ-lactone and glucose competitively inhibited the enzyme with Ki values of 0.033 and 468 mM, respectively. 相似文献
19.
I. L. Bazukyan A. E. Vardanyan A. A. Hambardzumyan P. V. Tozalakyan Yu. G. Popov 《Applied Biochemistry and Microbiology》2009,45(1):17-21
L-Phenylalanine ammonia-lyase (PAL, EC 4.3.1.5) of the Rhodotorula aurantiaca strain KM-1 deaminates L-phenylalanine according to the Michaelis-Menten kinetics with K M 1.75 ± 0.44 mM and V max 3.01 ± 0.43 units/mg. The enzyme is competitively inhibited by D-phenylalanine with K i 3.38 ± 0.32 mM. The Michaelis-Menten kinetics was analyzed, the inhibition type (competitive, noncompetitive, and mixed) was identified, and corresponding kinetic parameters were calculated using the computer programs written in Gauss 4.0. PAL was most stable at pH 6.55 and lacked approximately 50% of its activity after incubation at 57°C for 15 min. The yield of L-phenylalanine increased in the presence of mercaptoethanol, sodium ethylenediaminetetraacetate (EDTA), and ascorbic acid. The effects of EDTA and ascorbic acid were additive. 相似文献
20.
A. A. Hambardzumyan A. V. Mkhitaryan A. M. Paloyan S. A. Dadayan A. S. Saghyan 《Applied Biochemistry and Microbiology》2016,52(3):250-255
Studies of substrate specificity revealed that the D-aminoacylase of Rhodococcus armeniensis AM6.1 strain exhibits absolute stereospecificity to the D-stereoisomers of N-acetyl-amino acids. The enzyme is the most active reacted with N-acetyl-D-methionine, as well as with aromatic and hydrophobic N-acetylamino acids and interacts weakly with the basic substrates. It is practically not reacted with acidic and hydrophilic N-acetyl-amino acids. Michaelis constants (Km) and maximum reaction velocities (Vmax) were calculated, using linear regression analysis, for the following substrates: N-acetyl-D-methionine, N-acetyl-D-alanine, N-acetyl-D-phenylalanine, N-acetyl-D-tyrosine, N-acetyl-D-valine, N-acetyl-D-oxyvaline, N-acetyl- D-leucine. Substrate inhibition of D-aminoacylase was displayed with N-acetyl-D-leucine (Ks = 35.5 ± 28.3 mM) and N-acetyl-DL-tyrosine (Ks = 15.8 ± 4.5 mM). Competitive inhibition of the enzyme with product–acetic acid (Ki = 104.7 ± 21.7 mM, Km = 2.5 ± 0.5 mM, Vmax = 25.1 ± 1.5 U/mg) was observed. 相似文献