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1.
Structural requirements for antioxidative and anti-inflammatory properties of apolipoprotein A-I mimetic peptides 总被引:1,自引:0,他引:1
Anantharamaiah GM Mishra VK Garber DW Datta G Handattu SP Palgunachari MN Chaddha M Navab M Reddy ST Segrest JP Fogelman AM 《Journal of lipid research》2007,48(9):1915-1923
Recently, attention has been focused on pharmacological treatments that increase HDL cholesterol to prevent coronary artery disease. Despite three decades of extensive research of human apolipoprotein A-I (apoA-I), the major protein component of HDL, the molecular basis for its antiatherogenic and anti-inflammatory functions remain elusive. Another protein component of HDL, apoA-II, has structural features similar to those of apoA-I but does not possess atheroprotective properties. To understand the molecular basis for the effectiveness of apoA-I, we used model synthetic peptides. We designed analogs of the class A amphipathic helical motif in apoA-I that is responsible for solubilizing phospholipids. None of these analogs has sequence homology to apoA-I, but all are similar in their lipid-associating structural motifs. Although all of these peptide analogs interact with phospholipids to form peptide:lipid complexes, the biological properties of these analogs are different. Physical-chemical and NMR studies of these peptides have enabled the delineation of structural requirements for atheroprotective and anti-inflammatory properties in these peptides. It has been shown that peptides that interact strongly with lipid acyl chains do not have antiatherogenic and anti-inflammatory properties. In contrast, peptides that associate close to the lipid head group (and hence do not interact strongly with the lipid acyl chain) are antiatherogenic and anti-inflammatory. Understanding the structure and function of apoA-I and HDL through studies of the amphipathic helix motif may lead to peptide-based therapies for inhibiting atherosclerosis and other related inflammatory lipid disorders. 相似文献
2.
Chien HC Lin LL Chao SH Chen CC Wang WC Shaw CY Tsai YC Hu HY Hsu WH 《Biochimica et biophysica acta》2002,1576(1-2):119-126
Extracellular leucine aminopeptidase (LAP) from Aspergillus sojae was purified to protein homogeneity by sequential fast protein liquid chromatography steps. LAP had an apparent molecular mass of 37 kDa, of which approximately 3% was contributed by N-glycosylated carbohydrate. The purified enzyme was most active at pH 9 and 70 degrees C for 30 min. The enzyme preferentially hydrolyzed leucine p-nitroanilide followed by Phe, Lys, and Arg derivatives. The LAP activity was strongly inhibited by metal-chelating agents, and was largely restored by divalent cations like Zn(2+) and Co(2+). The lap gene and its corresponding cDNA fragment of the A. sojae were cloned using degenerated primers derived from internal amino acid sequences of the purified enzyme. lap is interrupted by three introns and is transcribed in a 1.3-kb mRNA that encodes a 377-amino-acid protein with a calculated molecular mass of 41.061 kDa. The mature LAP is preceded by a leader peptide of 77 amino acids, predicted to include an 18-amino-acid signal peptide and an extra sequence of 59 amino acids. Two putative N-glycosylation sites are identified in Asn-87 and Asn-288. Southern blot analysis suggested that lap is a single-copy gene in the A. sojae genome. The deduced amino acid sequence of A. sojae LAP shares only 11-33.1% identity with those of LAPs from 18 organisms. 相似文献
3.
Leucine aminopeptidases (LAPs) are present in animals, plants, and microbes. In plants, there are two classes of LAPs. The neutral LAPs (LAP-N and its orthologs) are constitutively expressed and detected in all plants, whereas the stress-induced acidic LAPs (LAP-A) are expressed only in a subset of the Solanaceae. LAPs have a role in insect defense and act as a regulator of the late branch of wound signaling in Solanum lycopersicum (tomato). Although the mechanism of LAP-A action is unknown, it has been presumed that LAP peptidase activity is essential for regulating wound signaling. Here we show that plant LAPs are bifunctional. Using three assays to monitor protein protection from heat-induced damage, it was shown that the tomato LAP-A and LAP-N and the Arabidopsis thaliana LAP1 and LAP2 are molecular chaperones. Assays using LAP-A catalytic site mutants demonstrated that LAP-A chaperone activity was independent of its peptidase activity. Furthermore, disruption of the LAP-A hexameric structure increased chaperone activity. Together, these data identify a new class of molecular chaperones and a new function for the plant LAPs as well as suggesting new mechanisms for LAP action in the defense of solanaceous plants against stress. 相似文献
4.
Binding specificity of the periplasmic oligopeptide-binding protein from Escherichia coli. 总被引:6,自引:2,他引:4
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The structural properties required for the binding of peptide substrates to the Escherichia coli periplasmic protein involved in oligopeptide transport were surveyed by measuring the ability of different peptides to compete for binding in an equilibrium dialysis assay with the tripeptide Ala-Phe-[3H]Gly. The protein specifically bound oligopeptides and failed to bind amino acids or dipeptides. Acetylation of the peptide amino terminus of (Ala)3 severely impaired binding, whereas esterification of the carboxyl terminus significantly reduced but did not completely eliminate binding. Peptides composed of L-amino acids competed more effectively than did peptides containing D-residues or glycine. Experiments with a series of alanyl peptide homologs demonstrated a decrease in competitive ability with increasing chain length beyond tripeptide. Competition studies with tripeptide homologs indicated that a wide variety of amino acyl side chains were tolerated by the periplasmic protein, but side-chain composition did affect binding. Fluorescence emission data suggested that this periplasmic protein possesses more than one substrate-binding site capable of distinguishing peptides on the basis of amino acyl side chains. 相似文献
5.
McCarthy E Stack C Donnelly SM Doyle S Mann VH Brindley PJ Stewart M Day TA Maule AG Dalton JP 《International journal for parasitology》2004,34(6):703-714
An array of schistosome endoproteases involved in the digestion of host hemoglobin to absorbable peptides has been described, but the exoprotease responsible for catabolising these peptides to amino acids has yet to be identified. By searching the public databases we found that Schistosoma mansoni and Schistosoma japonicum express a gene encoding a member of the M17 family of leucine aminopeptidases (LAPs). A functional recombinant S. mansoni LAP produced in insect cells shared biochemical properties, including pH optimum for activity, substrate specificity and reliance on metal cations for activity, with the major aminopeptidase activity in soluble extracts of adult worms. The pH range in which the enzyme functions and the lack of a signal peptide indicate that the enzyme functions intracellularly. Immunolocalisation studies showed that the S. mansoni LAP is synthesised in the gastrodermal cells surrounding the gut lumen. Accordingly, we propose that peptides generated in the lumen of the schistosome gut are absorbed into the gastrodermal cells and are cleaved by LAP to free amino acids before being distributed to the internal tissues of the parasite. Since LAP was also localised to the surface tegument it may play an additional role in surface membrane re-modelling. 相似文献
6.
Deuterium nuclear magnetic resonance (2H NMR) was used to study the interaction of amphiphilic model peptides with model membranes consisting of 1,2-dioleoyl-sn-glycero-3-phospho-L-serine deuterated either at the beta-position of the serine moiety ([2-2H]DOPS) or at the 11-position of the acyl chains ([11,11-2H2]DOPS). The peptides are derived from the sequences H-Ala-Met-Leu-Trp-Ala-OH (AX, one-letter code with X = MLWA) and H-Arg-Met-Leu-Trp-Ala-OH (RX+) and contain a positive charge of +1 (AXme+) or +2 (RXme2+) at the amino terminus or one positive charge at each end of the molecule (AXetN2+). Upon titration of dispersions of DOPS with the peptides, the divalent peptides show a similar extent of binding to the DOPS bilayers, which is larger than that of the single charged peptide. Under these conditions the values of the quadrupolar splitting (delta vq) of both [2-2H]DOPS and [11,11-2H2]DOPS are decreased, indicating that the peptides reduce the order of both the DOPS headgroup and the acyl chains. The extent of the decrease depends on the amount of peptide bound and on the position of the charged moieties in the peptide molecule. The effects exerted by the peptides on the delta vq value of [2-2H]DOPS are consistent with the PS headgroup responding as a molecular electrometer to the surface charge resulting from the presence of the peptides in the lipid-water interface. The effects on the acyl chain deuterons are in agreement with a localization of the peptides intercalated in between the lipid headgrouops.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
7.
The energy of hydrolysis of phosphate compounds varies depending on whether they are in solution or bound to the catalytic site of enzymes. With the purpose of simulating the conditions at the catalytic site, the observed equilibrium constant for pyrophosphate hydrolysis (Kobsd) was measured in aqueous mixtures of dimethyl sulfoxide, ethylene glycol, or polymers of ethylene glycol. The reaction was catalyzed by yeast inorganic pyrophosphatase at 30 degrees C. All the cosolvents used promoted a decrease of Kobsd. Polymers of ethylene glycol were more effective than dimethyl sulfoxide or ethylene glycol in decreasing Kobsd. The higher the molecular weight of the polymer, the lower the value of Kobsd. A decrease in Kobsd from 346 M (delta G degree obsd = -3.5 kcal mol-1) to 0.1 M (delta G degree obsd = 1.3 kcal mol-1) was observed after the addition of 50% (w/v) poly(ethylene glycol) 8000 to a solution containing 0.9 mM MgCl2 and 1 mM Pi at pH 8.0. The association constants of Pi and pyrophosphate for H+ and Mg2+ were measured in presence of different ethylene glycol concentrations in order to calculate the Keq for hydrolysis of different ionic species of pyrophosphate. A decrease in all the Keq was observed. The results are interpreted according to the concept that the energy of hydrolysis of phosphate compounds depends on the different solvation energies of reactants and products. 相似文献
8.
Leucine aminopeptidases: diversity in structure and function 总被引:2,自引:0,他引:2
9.
W D Blackburn J G Dohlman Y V Venkatachalapathi D J Pillion W J Koopman J P Segrest G M Anantharamaiah 《Journal of lipid research》1991,32(12):1911-1918
Neutrophils participate in the acute phase response and are often associated with tissue injury in a number of inflammatory disorders. The acute phase response is accompanied by alterations in the metabolism of apolipoprotein A-I and high density lipoprotein (HDL). Structural considerations led to studies investigating the effect of purified HDL and apolipoprotein A-I on neutrophil degranulation and superoxide production. Apolipoprotein A-I but not HDL inhibited IgG-induced neutrophil activation by about 60% as measured by degranulation and superoxide production. This suggests that the lipid-associating amphipathic helical domains of apolipoprotein A-I mediate this effect. In support of this was finding inhibitory effects with two synthetic model lipid-associating amphipathic helix peptide analogs. Apolipoprotein A-I, containing tandem repeating amphipathic helical domains, was approximately ten times more effective than the two peptide analogs and inhibited neutrophil activation at well below physiologic concentrations. Competitive binding studies indicate that resting neutrophils have approximately 190,000 (Kd = 1.7 x 10(-7)) binding sites per cell for apolipoprotein A-I, consistent with a ligand-receptor interaction. These observations suggest that apolipoprotein A-I may play an important role in regulating neutrophil function during the inflammatory response. 相似文献
10.
Huazhang Huang 《Analytical biochemistry》2009,391(1):11-2405
l-Leucine aminopeptidases (LAPs) are implicated in the progress of many pathological disorders and play some regulatory roles in tumor cell proliferation, invasion, and/or angiogenesis. Thus, LAPs not only could become new diagnostic or prognostic biomarkers but also may have potential as novel molecular targets for the treatment of several cancers. Highly sensitive assays are critical for early detection of changes in LAP activity and for screening potent LAP inhibitors. In this study, we developed a novel and highly sensitive fluorescent assay for LAPs based on substituted aminopyridines as fluorescent reporters. This assay was at least 100- and 20-fold more sensitive than commercial colorimetric and fluorescent LAP substrates, respectively. We also showed that this assay was a useful tool for monitoring LAP activities in extracts from cancer cell lines, as well as for the high-throughput screening of inhibitors, which could lead to new cancer treatments. 相似文献
11.
Extension of the fragment method to calculate amino acid zwitterion and side chain partition coefficients 总被引:6,自引:0,他引:6
The fragment method of calculating partition coefficients (P) has been extended to include the common amino acids (AAs). The results indicate that polar and charged side chains influence the hydrophobicity of atoms in the side chain in a predictable manner. Field effects, as evidenced through polar proximity factors and bond factors, need to be considered for accurate estimation of transfer phenomena. The calculated log P and delta G degree ' values of the 20 AAs agree well with the observed values. Pro calculates to be more hydrophilic than the observed log P. Hydrophobicity scales for peptide side chain residues are compared and evaluated in terms of suitability. Calculated pi values for nonpolar side chain residues agree well with the observed values; calculated values for uncharged polar side chain residues deviate by about 0.6 log units except for Gln and Cys; and polar side chain residues with charged side chains calculate as too hydrophilic. Reasons for the differences are explored. We also suggest that tightly bound water to polar moieties in amino acids and peptides may be transferred into the octanol phase during partitioning experiments. A quantitative methodology is presented which characterizes the thermodynamic partitioning of groups and individual atoms in amino acids and proteins. 相似文献
12.
Mechanism of energy coupling to entry and exit of neutral and branched chain amino acids in membrane vesicles of Streptococcus cremoris 总被引:13,自引:0,他引:13
A J Driessen K J Hellingwerf W N Konings 《The Journal of biological chemistry》1987,262(26):12438-12443
The energetics of neutral and branched chain amino acid transport by membrane vesicles from Streptococcus cremoris have been studied with a novel model system in which beef heart mitochondrial cytochrome c oxidase functions as a proton-motive force (delta p) generating system. In the presence of reduced cytochrome c, a large delta p was generated with a maximum value at pH 6.0. Apparent H+/amino acid stoichiometries (napp) have been determined at external pH values between 5.5 and 8.0 from the steady state levels of accumulation and the delta p. For L-leucine napp (0.8) was nearly independent of the pH. For L-alanine and L-serine napp decreased from 0.9-1.0 at pH 5.5 to 0-0.2 at pH 8.0. The napp for the different amino acids decreased with increasing external amino acid concentration. At pH 6.0, first order rate constants for amino acid exit (kex) under steady state conditions for L-leucine, L-alanine, and L-serine were 1.1-1.3, 0.084, and 0.053 min-1, respectively. From the pH dependence of kex it is concluded that amino acid exit in steady state is the sum of two processes, pH-dependent carrier-mediated amino acid exit and pH-independent passive diffusion (external leak). The first order rate constant for passive diffusion increased with increasing hydrophobicity of the side chain of the amino acids. As a result of these processes the kinetic steady state attained is less than the amino acid accumulation ratio predicted by thermodynamic equilibrium. The napp determined from the steady state accumulation represents, therefore, a lower limit. It is concluded that the mechanistic stoichiometry (n) for L-leucine, L-alanine, and L-serine transport most likely equals 1. 相似文献
13.
Modulation of metarhodopsin formation by cholesterol-induced ordering of bilayer lipids 总被引:7,自引:0,他引:7
The effect of lipid ordering on the kinetics and extent of metarhodopsin II (meta II) formation was evaluated in bovine rhodopsin which had been reconstituted into phosphatidylcholine vesicles containing 0, 15, and 30 mol% cholesterol. The rate of establishment of the dynamic equilibrium between metarhodopsin I (meta I) and the two kinetically distinguished forms of meta II in the branched meta II model [meta IIfast and meta IIslow; Straume, M., Mitchell, D. C., Miller, J. L., & Litman, B. J. (1990) Biochemistry (preceding paper in this issue)] is derived from kinetic measurements of rhodopsin photolysis in these vesicle systems at several temperatures. Values of the meta I in equilibrium with meta IItotal equilibrium constant, Keq, are calculated from the derived model-dependent rate constants, and are shown to be equivalent to those derived from rapidly acquired absorbance spectra. The presence of 30 mol% cholesterol reduces Keq by approximately 50% between 10 and 37 degrees C. Analysis of the model-dependent parameters in terms of delta H and delta S reveals that cholesterol raises the free energy of meta IIslow, relative to meta I, by increasing delta H whereas it raises the relative free energy of meta IIfast by making delta S meta IIfast relative to meta I less positive. The reduction in Keq by both temperature and cholesterol is found to be directly correlated with a parameter that reflects the free volume available for molecular motion in the hydrophobic core of the bilayer [Straume, M., & Litman, B. J. (1988) Biochemistry 27, 7723-7733].(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
T Niidome M Urakawa K Takaji Y Matsuo N Ohmori A Wada T Hirayama H Aoyagi 《The journal of peptide research》1999,54(4):361-367
For the purpose of achieving gene transfer into cells mediated by peptides with a short chain length, we employed two kinds of amphiphilic alpha-helix peptides, mastoparan (INLK-ALAA-LAKK-IL-NH2) obtained from wasp venom and an alpha-helix model peptide (LARL-LARL-LARL-NH2). Furthermore, to strengthen the hydrophobicity of the peptide required for the formation of the aggregates with the DNA, we modified these peptides using several lipophilic groups, i.e. acyl groups with a single chain, a dialkylcarbamoyl group and a cholesteryloxycarbonyl group. We examined the ability of the peptides and their derivatives to bind and aggregate with plasmid DNA, the structural change in the peptides caused by binding with the DNA and the in vitro gene transfer abilities into COS-7 cells. As a result, mastoparan was found to acquire the DNA binding ability by introduction of the lipophilic group. The conformational change in the peptides depended on the hydrophobicity of the introduced acyl group. The DNA complex of most lipophilic mastoparan derivatives could be incorporated into the cells via the endocytosis pathway. In the case of the helix model peptide, the acyl group with a moderate chain length was required for the formation of the aggregate which is competent for incorporation into the cells. In this study, we succeeded in giving such short peptides sufficient gene transfer ability by modifying them with some lipophilic groups. However, the influence of the modification by the lipophilic groups on the formation of aggregates with DNA and the gene transfer ability depended on the structure of the peptide portion. These results indicate that consideration of total hydrophobicity balance is needed for the design of an efficient gene carrier peptide. 相似文献
15.
R M Epand A Gawish M Iqbal K B Gupta C H Chen J P Segrest G M Anantharamaiah 《The Journal of biological chemistry》1987,262(19):9389-9396
Four peptides capable of forming an amphipathic alpha-helix have been synthesized and their conformational and lipid-binding properties studied. These peptides have been designed to vary the alpha-helix-forming potential as well as the charge distribution of the model peptide. The resulting peptide analogs and their complexes with dimyristoyl phosphatidylcholine were studied by using right angle light scattering, negative stain electron microscopy, nondenaturing gradient gel electrophoresis, circular dichroism, intrinsic tryptophan fluorescence, and differential scanning calorimetry techniques. The four analogs, [Glu4,9, Leu11,17] (reverse-18A, [Glu4,9, Leu5,11,17] reverse-18A, [Glu1,8, Leu11,17] 18A, and [Glu1,8, Leu5,11,17] 18A were derived from a model amphipathic peptide Asp-Trp-Leu-Lys-Ala-Phe-Tyr-Asp-Lys-Val-Ala-Glu-Lys-Leu-Lys-Glu-Ala-Phe (18A) whose lipid-associating properties strongly mimic apolipoprotein A-I or derived from Lys-Trp-Leu-Asp-Ala-Phe-Tyr-Lys-Asp-Val-Ala-Lys-Glu-Leu-Glu-Lys-Ala-Phe (reverse-18A), a peptide with little affinity for lipid and having a reversed charge distribution compared to the 18A peptide. We have shown that by substituting glutamic acid and leucine for aspartic acid and alanine, respectively, in a weak lipid-associating amphipathic helix peptide, the lipid-associating ability can be increased. Thus, peptides with both kinds of charge distribution can associate with the lipid. The ability of the peptide to disrupt phospholipid bilayers, however, is higher for 18A analogs compared to the reverse-18A analogs even after increasing the helix-forming potential and hydrophobicity. In addition to forming smaller lipoprotein particles, the modified 18A analogs were much superior to the modified reverse-18A analogs in their ability to activate the enzyme lecithin:cholesterol acyltransferase. This demonstrates that the positions of charged residues in the amphipathic helix play an important role in lecithin:cholesterol acyltransferase activation. 相似文献
16.
Both the phosphatidylcholine transfer protein (PC-TP) and the phosphatidylinositol transfer protein (PI-TP) act as carriers of phosphatidylcholine (PC) molecules between membranes. To study the structure of the acyl binding sites of these proteins, the affinity of 32 distinct natural and related PC molecular species was determined by using a previously developed fluorometric competition assay. Marked differences in affinity between species were observed with both proteins. Affinity vs lipid hydrophobicity (determined by reverse-phase HPLC) plots displayed a well-defined maximum indicating that the acyl chain hydrophobicity is an important determinant of binding of a phospholipid molecule by these transfer proteins. However, besides the overall lipid hydrophobicity, steric properties of the individual acyl chains contribute considerably to the affinity, and PC-TP and PI-TP respond differently to modifications of the acyl chain structure. The affinity of PC-TP increased steadily with increasing unsaturation of the sn-2 acyl moiety, resulting in high affinity for species containing four and six double bonds in the sn-2 chain, whereas the affinity of PI-TP first increased up to two to three double bonds and then declined. These data, as well as the distinct effects of sn-2 chain double bond position and bromination, indicate that the sn-2 acyl chain binding sites of the two proteins are structurally quite different. The sn-1 acyl binding sites are dissimilar as well, since variation of the length of saturated sn-1 chain affected the affinity differently. The data are discussed in terms of the structural organization of the sn-1 and sn-2 acyl binding sites of PC-TP and PI-TP.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
17.
Actinomycin synthetase I was purified to homogeneiety from actinomycin-producing Streptomyces chrysomallus. The purified enzyme is a single polypeptide chain of M(r) 45,000. It catalyzes the formation of the adenylate of 4-methyl-3-hydroxyanthranilic acid (4-MHA) from the free acid and ATP in an equilibrium reaction. 4-MHA is the precursor of the chromophoric part of actinomycin. By using the 4-MHA analogue, 4-methyl-3-hydroxybenzoic acid, as a model substrate it could be established that the equilibrium constant Keq is independent on enzyme concentration, which suggests that no stoichiometric acyladenylate-enzyme complex is formed in contrast to observations made with aminoacyl adenylates formed by aminoacyl-tRNA synthetases or multifunctional peptide synthetases. Actinomycin synthetase I does not charge itself with substrate carboxylic acid via a covalent thioester bond as is usual for amino acid activation in non-ribosomal peptide synthesis. In addition, the enzyme does not act as an acyl-coenzyme A ligase as revealed by its inability to release AMP in the presence of 4-MHA or other structurally related aromatic carboxylic acids, coenzyme A and ATP. Additional analysis of the activation reaction showed that it is exothermic, whereas the free enthalpy change delta G0 is positive due to a negative entropy change indicating a strong influence of restriction of random motion on the course of the reaction. Determinations of Km and kcat of various substrate carboxylic acids revealed the highest kcat/Km ratio for the natural substrate 4-MHA. From these properties, actinomycin synthetase I represents the prototype of novel chromophore activating enzymes involved in non-ribosomal synthesis of chromopeptide lactones in streptomycetes. 相似文献
18.
In alpha-chymotrypsin-catalyzed acyl-transfer reactions in water the specificity of the enzyme (the nucleophile reactivity of amino acid amides) is correlated with the substrate hydrophobicity and increases as the hydrophobicity of the side chain of the amino acid amides is increased. In a low water system (4% H2O) bulky amino acid amides are less efficient nucleophiles. The specificity of alpha-chymotrypsin towards the amino acid amides in acyl transfer reactions in this case does not depend on the hydrophobicity of the amino acid side chains but correlates with their size. Therefore, different factors can be responsible for the specificity of enzymes in water and in a mainly organic medium. 相似文献
19.
20.
Stearoyl-acyl carrier protein Delta(9)-desaturase (delta9D) catalyzes regio- and stereospecific insertion of cis double bonds into acyl chains attached to acyl carrier protein. Steady-state and stopped-flow fluorescence anisotropy measurements using acylated forms of dansyl- and fluoresceinyl-ACPs revealed equilibrium dissociation constants and dissociation rate constants for 16:0-, 17:0-, and 18:0-ACPs with resting and chemically 4e(-) reduced delta9D. Binding of 1 nM 18:0-fluoresceinyl-ACP to one subunit of the dimeric resting delta9D was observed with K(D1) = 13 +/- 3 nM. No significant difference in the K(D1) value was observed for 4e(-) delta9D. An approximately 4-fold increase in K(D1) per methylene group was observed upon shortening the acyl chain from 18:0 to 17:0 and then 16:0. In different experiments performed with 850 nM 18:0-dansyl-ACP, binding to the second subunit of resting delta9D was estimated to have K(D2) approximately 350 +/- 40 nM. The K(D2) values exhibited a similar dependence on acyl chain length as observed for the K(D1) values. The k(off) values measured by stopped-flow anisotropy measurements for reversal of the enzyme-substrate complex were also acyl-chain length dependent and increased 130-fold for 16:0-ACP (130 s(-)(1)) relative to 18:0-ACP (1 s(-)(1)). Increases in acyl chain length are thus associated with the presently reported increases in the K(D) and k(off) values. These results indicate that acyl chain length selectivity derives in major part from partition of the enzyme-substrate complex between substrate release and subsequent steps in catalysis. 相似文献