Bioluminescence competitive binding assays for biotin based on photoprotein aequorin.
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Aequorin↗
Biomedical subjects
A Witkowski
Publications and source records attributed to A Witkowski.
Fatty acid synthase: in vitro complementation of inactive mutants.
The animal fatty acid synthase is a dimer of identical, multifunctional 272 kDa subunits oriented antiparallel such that two centers for fatty acid synthesis are formed at the subunit interface. In order to clarify the interdomain and intersubunit communications necessary for the operation of the two centers, we have explored the possibility of reassembling catalytically-active fatty acid synthase heterodimers from pairs of inactive dimers carrying mutations in different functional domains. To this end, rat fatty acid synthase mutants, defective in either the beta-ketoacyl synthase, C161T or K326A (KS- FAS), or the acyl carrier protein, S2151A (ACP- FAS), domains, were engineered by site-directed mutagenesis, expressed in insect Sf9 cells using a baculovirus expression system, and purified. A novel procedure was devised to facilitate rapid production and isolation of a population of mixed mutant dimers that had undergone randomization of its constituent subunits. Homodimeric mutants (KS- FAS/KS- FAS and ACP- FAS/ACP- FAS) and KS- FAS heterodimers consisting of paired C161T and K326A mutant subunits were unable to synthesize fatty acids, confirming the essential nature of residues C161, K326, and S2151A. However, KS- FAS/ACP- FAS heterodimers regained partial activity. Formation of these heterodimers necessitated prior dissociation and reassociation of the homodimers, indicating that the rate of spontaneous exchange of subunits in the dimer is negligible. The formation of catalytically-active heterodimers from pairs of inactive, complementary homodimers affords a useful method for testing the validity of the current model for the multifunctional complex.
3-Oxoacyl-(Acyl-Carrier-Protein) Synthase↗
Homogeneous bioluminescence competitive binding assay for folate based on a coupled glucose-6-phosphate dehydrogenase--bacterial luciferase enzyme system.
A homogeneous bioluminescence competitive binding assay for folate was developed by using a coupled enzyme system of glucose-6-phosphate dehydrogenase (G6PDH) and bacterial luciferase. A highly substituted G6PDH-folate conjugate was prepared by employing an N-hydroxysuccinimide/carbodiimide method. Folate binding protein inhibits the activity of the conjugate. In the presence of folate, there is a competition between folate and the G6PDH-folate conjugate for the binding site of the folate binding protein, and the activity of the conjugate is recovered. Thus, the concentration of folate can be related to the activity of the G6PDH-folate conjugate, which is directly related to the bioluminescence produced by the coupled enzyme reaction. Using this assay, dose-response curves with a detection limit of 2.5 x 10(-8) M folate were obtained, which is an improvement of an order of magnitude with respect to an assay that monitors G6PDH activity spectrophotometrically. The assay was validated using vitamin tablets and a cell culture medium.
Binding, Competitive↗
Crystallization and preliminary diffraction studies of thioesterase II from rat mammary gland.
Thioesterase II from rat mammary gland has been crystallized in the presence of decanoic acid by the vapor diffusion method. The crystals belong to the orthorhombic space group P2(1)2(1)2(1), and have cell dimensions, a = 52.7 A, b = 78.0 A, and c = 133.6 A. The asymmetric unit likely consists of two protein monomers based on predictions from its calculated Matthews coefficient. Crystals typically diffract to at least 2.5 A resolution and are suitable for X-ray crystallographic analysis.
Animals↗
Reengineering the specificity of a serine active-site enzyme. Two active-site mutations convert a hydrolase to a transferase.
Two residues are known to play important catalytic roles in fatty acyl-thioester hydrolase, thioesterase II: Ser-101, the site of a covalent acyl-enzyme intermediate, and His-237 which is within hydrogen bonding distance of Ser-101 and facilitates catalysis by increasing the nucleophilicity of this residue. In this study we have examined the effect of mutations at these two residues on the ability of the enzyme to function as a hydrolase and, in the presence of a thiol acceptor, as an acyltransferase. In the hydrolase reaction kcat values for the wild-type, H237R, S101C, and S101C, H237R thioesterase enzymes were 0.11, < 0.002, 0.10, and < 0.002 s-1, respectively, and at steady state, the proportion of each enzyme present as the covalent acyl-enzyme intermediate was 11, 91, 71, and 100%, respectively. In the acyltransferase reaction no activity could be detected for the wild-type or H237R enzymes but the specific activities of the S101C and S101C/H237R thioesterases were 170 and 1300 nmol/min/mg of protein, respectively. From this data we conclude the following: the wild-type enzyme functions exclusively as a hydrolase. The H237R mutant acts ineffectively as a hydrolase primarily because the deacylation reaction is drastically retarded. The S101C enzyme functions well as a hydrolase, even though the rate of deacylation is adversely affected, and this enzyme can also perform as an acyltransferase. Mutation of both catalytic residues leads to a complete loss of hydrolase activity and the S101C,H237R mutant functions as an effective acyltransferase exhibiting kcat values higher then those of the wild-type enzyme acting as a hydrolase. This study reveals that, with only two amino acid replacements, an enzyme capable of functioning exclusively as a hydrolase can be converted into an equally active enzyme performing solely as an acyltransferase.
Acylation↗
Reversal of ischaemic systolic and diastolic left ventricular dysfunction by successful coronary angioplasty in patients with non-Q wave anterior myocardial infarction.
The effect of PTCA on global and regional left ventricular systolic function, isovolumic relaxation, chamber and muscle stiffness were studied in 30 patients with angina pectoris, previous non-Q wave anterior myocardial infarction (AMI) and significant stenosis of the left anterior descending coronary artery (LAD). In 11 of the 30 patients the condition was stable, but it was unstable in 19. Left ventricular angiograms were obtained before and 4.85 +/- 3.67 months after PTCA. The RAO was in the 30 degree projection, with the silhouette of the left ventricle sliced into 90 regions; changes in left ventricular volume, pressure and anterior wall thickness during the full cardiac cycle, together with dp/dt were demonstrated. After PTCA, global ejection fraction increased from 68.77 +/- 5.96% to 76.57 +/- 3.18%, P < 0.001. Impaired contractility was found in 29/90 (32.2%) regions before PTCA and in 5/90 (5.6%) after PTCA, P < 0.001. The time constant of the isovolumic pressure fall decreased after PTCA (52.56 +/- 17.40 ms vs 39.61 +/- 11.26 ms, P < 0.01). Elastic chamber stiffness coefficient decreased (0.022 +/- 0.003 vs 0.008 +/- 0.004, P < 0.001) and peak rate of left ventricular filling increased (319.0 +/- 107.9 ml.min-1 vs 396.8 +/- 201.4 ml.min-1, P < 0.05) after PTCA. The muscle stiffness coefficient was within normal values before and did not change after PTCA. The study findings show that in patients with persistent angina pectoris after non-Q wave AMI, complex systolic and diastolic ischaemic dysfunction occurs. This dysfunction can be reversed after successful PTCA of LAD.
Adult↗
Enzyme-linked immunosorbent assay for an octapeptide based on a genetically engineered fusion protein.
Traditional chemical means of preparing enzyme-ligand conjugates for use in enzyme-linked immunosorbent assays (ELISAs) lead to the production of multisubstituted enzyme-ligand conjugates with a high degree of variability in the site of ligand attachment. A genetically engineered fusion protein was prepared in order to investigate the feasibility of controlled production of conjugates for use in ELISAs. Specifically, a synthetic octapeptide was fused with bacterial alkaline phosphatase. The resulting enzyme-peptide conjugate is monosubstituted (one peptide per subunit), has a single site of attachment, and results in assays with good response characteristics. The use of such fusion proteins, which combine small analyte peptides with enzyme labels, can lead to a new approach to improved assays for numerous biomolecules, including peptide pharmaceuticals, neurotransmitters, hormones, cell surface antigens, etc.
Alkaline Phosphatase↗
[Percutaneous coronary angioplasty and levels of endothelin-1 in blood].
UNLABELLED: The aim of this study was to assess plasma endothelin-1 (ET) concentrations during percutaneous transluminal coronary angioplasty (PTCA) of the left anterio descending coronary artery (LAD). We also examined relations between plasma concentrations of ET and diameter of the PTCA-balloon. Plasma levels of ET determined by radioimmunoassay were measured in 24 patients (pts) without previous myocardial infarction undergoing PTCA of LAD. The pts were divided into two groups. In group 1 (12 pts) plasma ET levels were measured in the right atrium and in the forearm vein 24 hours before PTCA, and 5 minutes after the last balloon deflation. In group 2 (12 pts) concentrations of ET were measured only in venous blood: 24 hours before PTCA, just before PTCA, 5 minutes after last balloon deflation and 24 hours later. We compared the diameter of the inflated balloon with the diameter of the dilated artery [balloon/artery ratio /b/a ratio/] and correlated it with plasma ET concentration rise in every case. [table: see text] The mean b/a ratio correlated with plasma ET concentrations after PTCA (r = 0.677 p < 0.001). CONCLUSIONS: Plasma ET concentrations do not rise after non complicated PTCA procedure. Because plasma ET levels depend on b/t ratio it is very important that the diameter of the dilating balloon should correspond with the diameter of the coronary artery to avoid overdilation.
Angioplasty, Balloon, Coronary↗
[Coronary ultrasonography. More exact than coronary angiography? Description of two case histories].
Coronary angiography as the "golden standard" in diagnostic imaging, guiding and assessing the results of interventional therapy has well known limitations inherent to the two dimensional imaging of three dimensional structures. Intravascular ultrasonography provides cross-sectional images of coronary artery, allowing an accurate calculation of cross-sectional luminal area and assessment of the degree of eccentricity of plaque and its structure including fissures, dissections and intracoronary thrombi. Case 1: Coronary angiography was performed in a 60 y. male patient with unstable angina revealing only a 40% stenosis in proximal LAD. The inconsistency of clinical presentation with angiography findings led to perform an intravascular ultrasound (IVUS) examination of LAD. The angiographic 40% stenosis was found to be over 80% on IVUS. Subsequent PTCA procedure led to a good IVUS and clinical result with no obvious changes in angiography. Case 2: PTCA of 80% Cx stenosis was performed with good immediate angiographic result, without dissection nor residual stenosis. However, IVUS immediately after PTCA assessed a dissection of the plaque with a flap protruding into the lumen. The patient was sent back to coronary angiography several hours later with clinical and ecg symptoms of acute myocardial ischemia. An important, occlusive dissection was found at the PTCA site. Angioplasty was performed again restoring a wide lumen at intervention site. IVUS was able to clarify the ambiguous angiographic findings with an important impact on interventional therapy. The immediate results of invasive procedures were readily and safely assessed by intravascular ultrasound.
Aortic Dissection↗
Utilization of an active serine 101----cysteine mutant to demonstrate the proximity of the catalytic serine 101 and histidine 237 residues in thioesterase II.
Thioesterase II is a 29-kDa monomer which, in certain specialized tissues, acts as a chain terminator in fatty acid synthesis by hydrolyzing medium-chain fatty acids from the fatty acid synthase. As with serine proteases, hydrolysis appears to involve acylation of the active site serine residue (Ser-101) assisted by a histidine, tentatively identified as His-237. To determine whether in the folded protein His-237 is close enough to accept a proton from the Ser-101 hydroxyl, we have made use of a Ser101Cys mutant which retains up to 90% of catalytic activity. Unlike the wild-type enzyme, the S101C thioesterase is inhibited with stoichiometric amounts of the bifunctional alkylating reagent 1,3-dibromopropanone. To facilitate identification of the alkylated residue(s), the keto group introduced into the dibromopropanone-modified S101C mutant was radiolabeled by reduction with sodium [3H] borohydride. The protein was then digested and the radiolabeled peptides analyzed by amino acid sequencing and mass spectrometry. The experimental data unambiguously showed that dibromopropanone cross-linked the active site Cys-101 with His-237, demonstrating that these residues are positioned within 5 A of each other. These data strongly support the hypothesis that in the wild-type thioesterase His-237 accepts a proton from Ser-101, thus increasing its nucleophilic character and improving the catalytic efficiency of the enzyme. The possibility that exchange of cysteine and serine active site residues has occurred in the evolution of thioesterases is discussed.
Amino Acid Sequence↗
Overoxidized polypyrrole films: a model for the design of permselective electrodes.
Studies have been conducted to further characterize overoxidized polypyrrole films grown on carbon electrodes. Differences in apparent diffusion coefficients for various probes are interpreted via a model based on both hydrophobic and electrostatic interactions between the probe and polymer film. These electrode coatings are also compared to Naflon films, and the results of this study are discussed in terms of important considerations for the design of permselective electrodes.
Carbon↗
[Complications of percutaneous transluminal coronary angioplasty (PTCA)].
Between 1981 and 1990, 714 patients underwent 756 percutaneous transluminal coronary angioplasty (PTCA) procedures. A total of 52 patients (6.9%) had major in-hospital complications: 5 patients (0.66%) died, Q-wave or non Q-wave myocardial infarction were observed in 13 patients (1.66%) during procedure and in 8 (1%) outside the catheterization laboratory, before discharge. Because of periprocedural occlusion 11 patients (1.5%) were managed with bypass surgery, 8 (1%) had a transient occlusion that was reopened with PTCA. 21 patients (2.8%) were not ++re-dilated but managed pharmacologically. Dissection, intracoronary thrombus and previous thrombolytic treatment were often associated with occlusion. The risk of dissection was related to lesion morphology. Long-(more than 1 cm) lesion, eccentric stenosis and tortuosity of the vessel segment undergoing dilatation were risk factors for occlusive dissection. There was a high risk of side branch occlusion if its take-off was narrowed and side branch originated from the target lesion. One of the most important risk predictors is the amount of jeopardized myocardium supplied by the target coronary artery. Acute closure of an artery supplying large amount of myocardium may cause abrupt hemodynamic collapse. Hypotension secondary to the artery occlusion may cause a decrease of the flow in the other coronary arteries, leading to cardiogenic shock. Although it is important to note that patients with unstable angina, intracoronary thrombus, long and complex lesion, severe multivessel disease and compromised left ventricular function are at higher risk of acute complication, PTCA is a relatively safe procedure.(ABSTRACT TRUNCATED AT 250 WORDS)
Acute Disease↗
[Percutaneous transluminal coronary angioplasty in patients with high risk bypass surgery].
The purpose of this study was to assess the immediate and long-term results of incomplete percutaneous transluminal coronary angioplasty (PTCA) in high-risk coronary artery bypass surgery (CABG) patients. 24 pts (male-22, female-2, age - 39-60 years) were divided into 2 groups: I-8 pts with unstable angina pectoris who were definitely not CABG candidates because of very low ejection fraction (LVEF < 24%) and/or diffuse coronary atherosclerosis; II-16 pts selected for CABG only after failed PTCA. From this group 12 pts with unstable angina pectoris and history of myocardial infarction were at higher CABG risk because of LVEF < 40% and diffuse coronary atherosclerosis. 4 pts were poor surgical candidates because of coexistent medical disorders. The strategy of PTCA was to dilate first the most critical (culprit) lesion, responsible for the patient symptoms, usually situated in the artery supplying large area of viable myocardium. We did not achieve: complete revascularization in all our pts (incomplete revascularization by intent). Initial success rate of the PTCA in both groups was 100%. There were no serious complications. During follow-up (6 months--4 yrs) long-term clinical improvement was observed in 7 pts from group I (87.5%) and 14 pts from group II (87.5%). We conclude, that in most pts with unstable angina pectoris and with high-risk of CABG good immediate and long-term results of incomplete PTCA can be achieved.
Adult↗
Isolation of a functional transferase component from the rat fatty acid synthase by limited trypsinization of the subunit monomer. Formation of a stable functional complex between transferase and acyl carrier protein domains.
Limited trypsinization of rat fatty acid synthase monomers results in cleavage at sites protected in the native dimer. A 47,000-Da polypeptide containing the transferase component was isolated from the digest and its location in the multifunctional polypeptide established. Both acetyl and malonyl moieties are transferred stoichiometrically from CoA ester to this polypeptide and each can replace the other, confirming that a single common site is utilized in the loading of these substrates onto the fatty acid synthase. Transferase activity of the 47,000-Da polypeptide decreases with increasing acyl donor chain length (malonyl = acetyl greater than butyryl greater than hexanoyl greater than octanoyl). Activity is inhibited by certain thiol-directed reagents, and protection is afforded by substrate suggesting the presence of a sensitive cysteine residue near the substrate binding site. The transferase was also able to utilize as acyl acceptor the Escherichia coli acyl carrier protein and the acyl carrier protein domain of the multifunctional fatty acid synthase. When the fatty acid synthase monomer was trypsinized under milder conditions, the 47,000-Da transferase domain could be isolated in association with the 8,000-Da acyl carrier protein domain. The transferase was capable of translocating substrate moieties from CoA ester donors to the associated acyl carrier protein. The results provide the first direct evidence that, in the head-to-tail oriented fatty acid synthase homodimer, functional communication between the transferase domain located near the end of one polypeptide and the acyl carrier protein domain located at the opposite end of the other polypeptide is facilitated by a stable physical interaction between these domains.
Acyl Carrier Protein↗
A catalytic role for histidine 237 in rat mammary gland thioesterase II.
The involvement of a histidyl residue in the catalytic mechanism of thioesterase II, a serine active-site enzyme that catalyzes the chain terminating reaction in de novo fatty acid synthesis, has been inferred from studies with the inhibitor diethyl pyrocarbonate. Its likely location has been predicted by identification of conserved residues in related thioesterases and ultimately confirmed by site-directed mutagenesis. Diethyl pyrocarbonate inactivated the enzyme with a second-order rate constant of 49 M-1 s-1 at pH 6, 10 degrees C. Data analysis indicated that although several residues reacted with the reagent, modification of a single residue was responsible for the inactivation. Removal of a single ethoxycarbonyl moiety by treatment with neutral hydroxylamine completely restored enzyme activity. Prior ethoxycarbonylation of the histidyl residue blocked the ability of the active-site serine to react with phenylmethanesulfonyl fluoride. Comparison of the amino acid sequences of five structurally related proteins indicated that only 1 histidine has been completely conserved. Replacement of this residue in rat thioesterase II (His-237) with arginine and leucine by mutagenesis reduced the catalytic activity by 2-3 orders of magnitude. The activity of the mutant thioesterases, unlike that of the wild-type enzyme, was relatively insensitive to inhibition by diethyl pyrocarbonate and phenylmethylsulfonyl fluoride. These studies provide strong evidence that His-237 is involved directly in catalysis and suggest that its role is to increase the nucleophilic character of the active-site Ser-101 by acting as a proton acceptor thus facilitating acylation of the seryl residue. The mechanism appears to share certain common features with the charge-relay system characteristic of other esterases.
Animals↗
Structural organization of the multifunctional animal fatty-acid synthase.
The amino acid sequence of the multifunctional fatty-acid synthase has been examined to investigate the exact location of the seven functional domains. Good agreement in predicting the location of interdomain boundaries was obtained using three independent methods. First, the sites of limited proteolytic attack that give rise to relatively stable, large polypeptide fragments were identified; cryptic sites for protease attack at the subunit interface were unmasked by first dissociating the dimer into its component subunits. Second, polypeptide regions exhibiting higher-than-average rates of non-conservative mutation were identified. Third, the sizes of putative functional domains were compared with those of related monofunctional proteins that exhibit similar primary or secondary structure. Residues 1-406 were assigned to the oxoacyl synthase, residues 430-802 to the malonyl/acetyl transferase, residues 1630-1850 to the enoyl reductase, residues 1870-2100 to the oxyreductase, residues 2114-2190 to the acyl-carrier protein and residues 2200-2505 to the thioesterase. The 47-kDa transferase and 8-kDa acyl-carrier-protein domains, which are situated at opposite ends of the multifunctional subunit, were nevertheless isolated from tryptic digests as a non-covalently associated complex. Furthermore, a centrally located domain encompassing residues 1160-1545 was isolated as a nicked dimer. These findings, indicating that interactions between the head-to-tail juxtaposed subunits occur in both the polar and equatorial regions, are consistent with previously derived electron-micrograph images that show subunit contacts in these areas. The data permit refinement of the model for the fatty-acid synthase dimer and suggest that the malonyl/acetyl transferase and oxoacyl synthase of one subunit cooperate with the reductases, acyl carrier protein and thioesterase of the companion subunit in the formation of a center for fatty-acid synthesis.
Alcohol Dehydrogenase↗
Effect of electrode substrate on the morphology and selectivity of overoxidized polypyrrole films.
Polypyrrole (PP) films were electropolymerized on glassy carbon (GC) and rough pyrolytic graphite (RPG) electrodes and treated by overoxidation in sodium hydroxide and phosphate buffer. The effects of treatment on film morphology were investigated, including permselective response of treated films. Films grown on GC and RPG were also compared, and it was found that the substrate plays a definite role in the properties of the polymer layer. Differences in response to ionic probes on treated PP electrodes are shown to arise from the porosity of the membranes, which affects the electron density within the film due to the spacing of carbonyl groups introduced during treatment.
Electrodes↗
Expression in Escherichia coli, purification and characterization of two mammalian thioesterases involved in fatty acid synthesis.
Thioesterase I, a constituent domain of the multifunctional fatty acid synthase, and thioesterase II, an independent monofunctional protein, catalyse the chain-terminating reaction in fatty acid synthesis de novo at long and medium chain lengths respectively. The enzymes have been cloned and expressed in Escherichia coli under the control of the temperature-sensitive lambda repressor. The recombinant proteins are full-length catalytically competent thioesterases with specificities indistinguishable from those of the natural enzymes.
Amino Acid Sequence↗