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K L Peterson

Publications and source records attributed to K L Peterson.

At least 19 recordsLinked to original sources
PubMed2001

A cardiac myocyte vascular endothelial growth factor paracrine pathway is required to maintain cardiac function.

The role of the cardiac myocyte as a mediator of paracrine signaling in the heart has remained unclear. To address this issue, we generated mice with cardiac myocyte-specific deletion of the vascular endothelial growth factor gene, thereby producing a cardiomyocyte-specific knockout of a secreted factor. The hearts of these mice had fewer coronary microvessels, thinned ventricular walls, depressed basal contractile function, induction of hypoxia-responsive genes involved in energy metabolism, and an abnormal response to beta-adrenergic stimulation. These findings establish the critical importance of cardiac myocyte-derived vascular endothelial growth factor in cardiac morphogenesis and determination of heart function. Further, they establish an adult murine model of hypovascular nonnecrotic cardiac contractile dysfunction.

Animals↗
PubMed2001

Improvement of coronary artery endothelial dysfunction with lipid-lowering therapy: heterogeneity of segmental response and correlation with plasma-oxidized low density lipoprotein.

OBJECTIVES: This study assessed coronary artery endothelial function in patients with hypercholesterolemia before and after lipid lowering, using quantitative angiography to examine the acetylcholine (Ach) response along the entire analyzable vessel. BACKGROUND: Lipid lowering reverses endothelial dysfunction, but whether improvement occurs only in some segments and not others has not been established. Statistical correlation of improvement with specific lipid moieties remains undefined. METHODS: Quantitative angiography was performed after Ach (10(-6), 10(-5), 10(-4) M) in 29 patients with coronary atherosclerosis before and 18 +/- 5.2 months after lipid-lowering treatment (statins, bile sequestrant resins). Standard lipid moieties and markers of oxidized low density lipoprotein (LDL) (immunoglobulin G and M autoantibody titers to malondialdehyde-LDL, E06 epitope) were measured serially. RESULTS: Pre-treatment of the vessel diameters at control and with 10(-6)M, 10(-5) M and 10(-4) M Ach were 2.108 +/- 0.085, 2.086 +/- 0.087, 2.069 +/- 0.084 and 1.963 +/- 0.097 mm (M +/- SE), respectively, and increased at follow-up to 2.139 +/- 0.094, 2.119 +/- 0.086, 2.127 +/- 0.084 and 2.080 +/- 0.085 mm (p < 0.0001). Improvement in the most constricted and modest declination in the more dilated segments were observed. Change in the E06 and Apolipoprotein A-1 titers correlated with improved vasomotion (p = 0.027 and 0.005, respectively). The pre- and post-treatment levels of the E06 epitope, as well as the post-treatment IgM autoantibody titer to MDA-low density lipoprotein, also correlated (p < 0.028, < 0.001 and p < 0.004, respectively). CONCLUSIONS: Drug treatment reverses endothelial dysfunction, but the effect is heterogeneous. Most coronary segments show enhancement, while others show declination of dilation, underscoring the importance of assessing the entire analyzable artery. Improvement in vasomotion correlates most significantly with markers of plasma-oxidized low-density lipoprotein.

Acetylcholine↗
PubMed2001

Cardiovascular effects of fentanyl in conscious rats.

The polymicrobial sepsis induced by cecal ligation and puncture (CLP) in the rat is widely used in shock research. For ethical reasons, narcotic analgesics are often administered in this model, with the potential risk of confounding effects. In conscious non-septic rats, we investigated the cardiovascular effects of a continuous i.v. infusion of fentanyl (20 microg/kg per h) administered with fluid loading (10 ml/kg per h) for 24 h, a regimen commonly applied in rat CLP. Animals were randomly allocated to receive analgesia with fluid loading (Fentanyl group), or fluid loading alone (Control). All endpoints were assessed after 24 h of infusion. At that time, Control animals had mild respiratory alkalosis, which was essentially abolished by fentanyl. Analgesia mildly elevated the plasma norepinephrine levels [median (interquartile range): Control 232 pg/ml (0-292), Fentanyl 302 pg/ml (234-676), P=0.045] but was devoid of any effect on blood pressure, heart rate, cardiac output (mean +/-SD: Control 388+/-61 ml/kg per min, Fentanyl 382+/-62 ml/kg per min, P=0.87) and indices of left ventricular function derived from high-fidelity recordings of left ventricular pressure (dP/dtmax: Control 11782+/-2324 mmHg/s, Fentanyl 12107+/-2816 mmHg/s, P=0.77). In ex vivo experiments carried out immediately after animal sacrifice, no differences were noted between the Control and Fentanyl groups in the sensitivity of endothelium-intact aortic rings to norepinephrine-induced vasoconstriction (-logEC50: Control 8.78+/-0.28, Fentanyl 8.83+/-0.26, P=0.52) or acetylcholine-induced vasodilatation (-logEC50: Control 7.00+/-0.37, Fentanyl 7.06+/-0.26+/-0.53, P=0.75). In conclusion, the present data provide no contraindication, and even some support for the ethical use of a high dose i.v. infusion of fentanyl in cardiovascular studies of conscious catheterized rats undergoing CLP or other painful procedures.

Acetylcholine↗
PubMed2001

Adult mice deficient in actinin-associated LIM-domain protein reveal a developmental pathway for right ventricular cardiomyopathy.

Although cytoskeletal mutations are known causes of genetically based forms of dilated cardiomyopathy, the pathways that link these defects with cardiomyopathy are unclear. Here we report that the alpha-actinin-associated LIM protein (ALP; Alp in mice) has an essential role in the embryonic development of the right ventricular (RV) chamber during its exposure to high biomechanical workloads in utero. Disruption of the gene encoding Alp (Alp) is associated with RV chamber dilation and dysfunction, directly implicating alpha-actinin-associated proteins in the onset of cardiomyopathy. In vitro assays showed that Alp directly enhances the capacity of alpha-actinin to cross-link actin filaments, indicating that the loss of Alp function contributes to destabilization of actin anchorage sites in cardiac muscle. Alp also colocalizes at the intercalated disc with alpha-actinin and gamma-catenin, the latter being a known disease gene for human RV dysplasia. Taken together, these studies point to a novel developmental pathway for RV dilated cardiomyopathy via instability of alpha-actinin complexes.

Actinin↗
PubMed2000

A report of two hundred twenty cases of regional anesthesia in pediatric cardiac surgery.

The use of regional anesthesia (ie, epidural, spinal, or caudal) has been reported in a few small series of children undergoing cardiac surgery, but not in larger studies. In this retrospective, descriptive study, we report the results of the use of regional anesthesia in 220 pediatric cardiac operations. We reviewed the records of children receiving a regional anesthetic for cardiothoracic surgery at Stanford Medical Center between January 1993 and February 1997. All patients were targeted for early tracheal extubation. A variety of regional techniques were used. Time to extubation, control of pain, incidence of respiratory depression and other complications, and length of hospital stay were determined. There were no deaths. Eighty-nine percent of the patients were tracheally extubated in the operating room; 4.1% of whom required reintubation within 24 h. Ninety-five percent +/-2.5% of the patients had pain scores < or =4.0 at all intervals postoperatively. Adverse effects of regional anesthesia included emesis (39%), pruritus (10%), urinary retention (7%), postoperative transient paresthesia (3%), and respiratory depression (1.8%). The incidence of peridural hematoma was zero. The rate of adverse effects was lower in the thoracic catheter epidural approach as compared with various caudal, lumbar epidural, and spinal approaches. Hospital duration of stay was not effected by the presence of regional anesthetic complications. In this study, regional anesthesia was safe and effective in the management of pediatric patients undergoing cardiac surgery.

Adolescent↗
PubMed2000

Altered membrane proteins and permeability correlate with cardiac dysfunction in cardiomyopathic hamsters.

A mutation in the delta-sarcoglycan (SG) gene with absence of delta-SG protein in the heart has been identified in the BIO14.6 cardiomyopathic (CM) hamster, but how the defective gene leads to myocardial degeneration and dysfunction is unknown. We correlated left ventricular (LV) function with increased sarcolemmal membrane permeability and investigated the LV distribution of the dystrophin-dystroglycan complex in BIO14.6 CM hamsters. On echocardiography at 5 wk of age, the CM hamsters showed a mildly enlarged diastolic dimension (LVDD) with decreased LV percent fractional shortening (%FS), and at 9 wk further enlargement of LVDD with reduction of %FS was observed. The percent area of myocardium exhibiting increased membrane permeability or membrane rupture, assessed by Evans blue dye (EBD) staining and wheat germ agglutinin, was greater at 9 than at 5 wk. In areas not stained by EBD, immunostaining of dystrophin was detected in CM hamsters at sarcolemma and T tubules, as expected, but it was also abnormally expressed at the intercalated discs; in addition, the expression of beta-dystroglycan was significantly reduced compared with control hearts. As previously described, alpha-SG was completely deficient in CM hearts compared with control hearts. In myocardial areas showing increased sarcolemmal permeability, neither dystrophin nor beta-dystroglycan could be identified by immunolabeling. Thus, together with the known loss of delta-SG and other SGs, abnormal distribution of dystrophin and reduction of beta-dystroglycan are associated with increased sarcolemmal permeability followed by cell rupture, which correlates with early progressive cardiac dysfunction in the BIO14.6 CM hamster.

Animals↗
PubMed1999

Progressive cardiac dysfunction and fibrosis in the cardiomyopathic hamster and effects of growth hormone and angiotensin-converting enzyme inhibition.

BACKGROUND: Growth hormone (GH) improves cardiac function in the rat with myocardial infarction, but its effects in a model of primary dilated cardiomyopathy have not been reported. GH effects were examined at early (4 months) and late (10 months) phases of disease in the cardiomyopathic (CM) hamster, and the combination of GH with chronic ACE inhibition was assessed in late-phase heart failure. METHODS AND RESULTS: CM hamsters (CHF 147 line) at 4 months showed severe systolic left ventricular (LV) dysfunction with normal LV filling pressure, and at 10 months there was more severe systolic as well as diastolic dysfunction with increasing myocardial fibrosis. Recombinant human GH alone for 3 weeks at age 4 months increased LV wall thickness and reduced systolic wall stress without altering diastolic wall stress, whereas at 10 months, wall stress and fractional shortening did not improve. The LV dP/dt(max) was enhanced at both ages by GH, which at 4 months reflected increased contractility, but at 10 months was most likely caused by elevation of the LV filling pressure. The increasing degree of fibrosis correlated inversely with LV function but was unaffected by GH. In other CM hamsters, high-dose ACE inhibition alone (quinapril), started at 8 months and continued for 11 weeks, improved LV function and inhibited unfavorable remodeling, but the addition of GH for 3 weeks at age 10 months produced increased wall thickness with little additional functional benefit and increased the LV filling pressure and diastolic wall stress. CONCLUSIONS: GH treatment alone improved LV dysfunction at 4 months of age in CM hamsters by increasing contractility and reducing wall stress but had few beneficial effects at 10 months in severe LV failure. After chronic ACE inhibition, addition of GH at 10 months had no additional beneficial effects and further increased LV diastolic pressure. These differing effects of GH may relate to the progressive increase of LV fibrosis in the CM hamster.

Angiotensin-Converting Enzyme Inhibitors↗
PubMed1999

The internalization of posterior subcapsular cataracts (PSCs) in Royal College of Surgeons (RCS) rats. I. Morphological characterization.

PURPOSE: To document lens ultrastructure during and after internalization of posterior subcapsular cataracts (PSCs) in Royal College of Surgeons (RCS) rats, a model for human autosomal retinal degenerative disease. METHODS: RCS rat lenses at 2, 2.5, 3, 4, 6, 9, 12, and 15 months old were enucleated and fixed. For light and transmission electron microscopy (TEM), lenses were embedded in epoxy and sectioned along the visual axis. For scanning electron microscopy, lenses were dissected to expose the posterior fibers in concentric growth shells down to the internalized PSC plaques. RESULTS: Overgrowth of the plaque began between 8 and 9 weeks postnatal and proceeded from the periphery to the posterior pole. This is in contrast to PSC formation which begins centrally and enlarges radially between 4-6 weeks postnatal. Peripheral-to-central overgrowth resulted in the formation of a convexo-concave, disk-shaped suture plane oriented parallel to the capsule. The initial fibers overlying the plaque were extremely flattened at their posterior ends. However, by 3 months postnatal, fiber ultrastructure was relatively normal and displayed only minor morphological irregularities. These temporal and structural changes were used to create 3-dimensional computer assisted-drawing (3D-CAD) reconstructions and animations. TEM examination of plaques revealed scattered fiber defects such as membrane whorls, globular aggregates and intracellular voids in both the internalized plaques and the initial overgrowth. The internalized PSC plaques had comparable morphology in all animals, regardless of age. Specifically, the posterior segments of fibers were enlarged and curved abnormally toward the capsule. CONCLUSIONS: PSC plaques are not internalized and broken down in the classical cell biological sense (i. e. via lysosomal degradation). Rather the plaques retain their structure indefinitely as lens growth proceeds (albeit not entirely normally). This demonstrates that the lens has a restricted ability to respond to growth defects and effect a limited recovery after PSC formation.

Age Factors↗
PubMed1999

The internalization of posterior subcapsular cataracts (PSCs) in Royal College of Surgeons (RCS) rats. II. The inter-relationship of optical quality and structure as a function of age.

PURPOSE: The Royal College of Surgeons (RCS) rat is an animal model for human retinal degenerative disease and posterior subcapsular cataracts (PSCs). The purpose of this study was to correlate the structure and optical quality of RCS lenses with PSCs as a function of their internalization, with normal, non-cataractous, age-matched control lenses. METHODS: Correlative light (LM), scanning electron microscopic (SEM), three-dimensional computer assisted drawings (3D-CADs) and low power helium-neon laser scan analysis were used to examine the structure and function of lenses. RESULTS: The optical properties (average focal length variability; sharpness of focus) of RCS rat lenses are quantitatively compromised by PSCs. Correlative LM and SEM analysis of RCS lenses at various stages of PSC internalization (1.5, 3, 6, 9, 12 and 15 months of age), revealed that the sutures formed by additional fiber growth were progressively more abnormal. During PSC internalization, two to nine small suture branches were formed and arranged in modified line to multiple y configurations rather than the normal three branch y sutures. These temporal changes were also chronicled in animated 3D-CAD videos derived from lens reconstructions based on LM and SEM micrographs from the selected time points stated above. However, laser scan analysis also revealed that as the PSCs of RCS rat lenses were progressively internalized, there was a steady improvement in total sharpness of focus that reached normal levels by 12 months of age. The correlation of laser scan and structural data from specific regions of lenses revealed the following: 1. The abnormal posterior sutures of RCS rats with internalized PSCs effect a greater reduction in optical quality than normal posterior sutures of age-matched controls; 2. However, the resulting abnormal suture plane area was cumulatively similar to that of age-matched controls; 3. Thus, total optical quality was similar between RCS lenses with internalized PSCs and age-matched controls by 12 months of age. CONCLUSIONS: The results of this study show that RCS lenses with internalized PSCs can appear grossly, and indeed optically perform, at levels comparable to aged lenses. These findings are consistent with clinical observations of spontaneous recovery from PSC. The results suggest that human PSCs that occur as a consequence of retinal degenerative disease could also be the result of abnormal posterior suture growth. If this is proven to be the case, such PSCs may have some capacity for repair or recovery thereby obviating their surgical removal.

Aging↗
PubMed1999

The utility of an in-hospital observation period after discontinuing intravenous antibiotics.

PURPOSE: To determine whether observing patients overnight in the hospital after intravenous antibiotics have been discontinued is a useful way to identify important clinical events. SUBJECTS AND METHODS: We performed a retrospective chart review of patients admitted during a 6-month period to a tertiary care teaching hospital with a primary diagnosis of either pneumonia, urinary tract infection, or cellulitis who were treated with intravenous antibiotics. Charts were abstracted for patient characteristics, including comorbid illnesses and laboratory values, as well as for evidence of recurrent infection or other adverse events. RESULTS: Of the 374 patients in the study, 63 (17%) were discharged on the day intravenous antibiotics were discontinued. These patients were 10 years younger (P = 0.0009) and had fewer comorbid illnesses (P = 0.02) than those who were observed in the hospital. Recurrent infection was noted in 3 (1%; 95% confidence interval 0.2% to 3%) of the 308 patients who were observed. A mild adverse antibiotic reaction was also noted in three observed patients. The readmission rate to the same institution for recurrent infection was 3% for patients with an observation period and 2% for patients without an observation period (P = 0.70). CONCLUSIONS: Observing patients overnight in the hospital after discontinuing intravenous antibiotics is a common clinical practice. There was an extremely low incidence of adverse events during the observation period, and the events that did occur would have been discovered in an outpatient setting. In-hospital observation after discontinuing intravenous antibiotics is unnecessary for most patients with pneumonia, urinary tract infection, or cellulitis and greatly increases health-care costs.

Adult↗
PubMed1999

Role of motor unit number estimate electromyography in experimental canine laryngeal reinnervation.

Laryngeal electromyography has been used clinically to differentiate neuromuscular pathology from other causes of vocal fold immobility such as arytenoid dislocation, tumor invasion, or cricoarytenoid joint fixation. Electromyography has also been used to predict the prognosis for nerve recovery in laryngeal paralysis. Existing electromyographic techniques either record activity with voluntary motion or study nerve conduction. In this study a new technique, motor unit number estimation, a commercially available quantitative method of electromyographic analysis, is used to study the progress of recovery of vocal fold function after recurrent laryngeal nerve injury. Four dogs underwent transection and immediate reanastomosis of selected branches of the adductor and abductor branches of the recurrent laryngeal nerve on 1 side; the opposite side served as a control. Baseline electromyographic and videolaryngoscopic studies were performed. These measures were then repeated in a longitudinal fashion every 6 weeks after denervation. The motor unit number estimation technique indicated a return of motor unit numbers with time, along with estimates of their size. This was consistent with the expected progress of laryngeal reinnervation. These data and their predictive value for nerve recovery will be discussed.

Anastomosis, Surgical↗
PubMed1999

Outcomes after single injection caudal epidural versus continuous infusion epidural via caudal approach for postoperative analgesia in infants and children undergoing patent ductus arteriosus ligation.

Adequate postoperative analgesia enhances deep breathing and minimizes respiratory complications after thoracotomy. This study compares postoperative outcomes after single injection caudal epidural vs continuous infusion epidural via caudal approach for postoperative analgesia in infants and children undergoing thoracotomy for patent ductus arteriosus (PDA) ligation. A retrospective chart review was performed for 27 children who had undergone PDA ligation. The children were divided into three groups. We compared patient demographics, surgical duration, anaesthesia duration, length of ICU stay, incidence of emesis requiring treatment, time required to establish regular oral intake, requirement for supplemental intravenous opioids during the first postoperative day, and length of hospital stay. For paediatric patients undergoing PDA ligation, postoperative analgesia with continuous infusion epidural via caudal approach produced shorter ICU stay, less occurrence of postoperative emesis, earlier oral intake, elimination of intravenous opioid supplementation, and shorter hospital stay compared with single injection caudal epidural techniques.

Analgesia, Epidural↗
PubMed1999

Comparison of nerve banking techniques in delayed laryngeal reinnervation.

Successful laryngeal transplantation will require adequate reinnervation of the larynx to allow phonation, coordinated swallowing, and respiration. A delay between laryngectomy and transplantation would be necessary in oncology patients because of the need for immunosuppression. In these patients, reinnervation of the donor organ would require "banking" and recovery of dormant recipient recurrent laryngeal nerves (RLNs). This pilot study was undertaken to compare the effectiveness of RLN storage using 1 of 2 techniques: 1) inserting the nerve into a muscle pocket or 2) anastomosing the proximal RLN stump to the ansa cervicalis. Six months following nerve transection and "banking," the proximal anterior branch of the RLN was reanastomosed to the distal anterior segment and the posterior branch was anastomosed directly to the posterior cricoarytenoid muscle. Tensionometry, image analysis, and electromyographic data were collected 1 year later. Results show reinnervation of adductors and abductors with both techniques. Banking of the RLN branches during total laryngectomy is effective and should permit delayed physiological reinnervation following laryngeal transplantation.

Anastomosis, Surgical↗
PubMed1998

Thermodynamics of ligand binding and catalysis in human liver medium-chain acyl-CoA dehydrogenase: comparative studies involving normal and 3'-dephosphorylated C8-CoAs and wild-type and Asn191 --> Ala (N191A) mutant enzymes.

Following our demonstration that the terminal 3'-phosphate group of acyl-CoA substrates (which is confined to the exterior of the protein structure, and is fully exposed to the outside solvent environment) exhibits a functional role in the recombinant human liver medium-chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction [Peterson, K. L., and Srivastava, D. K. (1997) Biochem. J. 325, 751-760], we became interested in delineating its thermodynamic contribution in stabilizing the "ground" and "transition" state structures during enzyme catalysis. Since the 3'-phosphate group of the coenzyme A thiolester has the potential to form a hydrogen bond with the side chain group of Asn-191, these studies were performed utilizing both normal and 3'-dephosphorylated forms of octanoyl-CoA and octenoyl-CoA (cumulatively referred to as C8-CoA) as the physiological substrate and product of the enzyme, respectively, as well as utilizing wild-type and Asn191 --> Ala (N191A) site-specific mutant enzymes. The experimental data revealed that the enthalpic contribution of the 3'-phosphate group was similar in both ground and transition states, and was primarily derived from the London-van der Waals interactions (between the 3'-phosphate group of C8-CoA and the surrounding protein moiety), rather than from the potential hydrogen bonding. The temperature dependence of DeltaH degrees for the binding of octenoyl-CoA and 3'-dephosphooctenoyl-CoA revealed that the deletion of the 3'-phosphate group from octenoyl-CoA increased the magnitude of the heat capacity changes (DeltaCp degrees) from -0.53 to -0.59 kcal mol-1 K-1. Although the latter effect could be attributed to an increase in the relative hydrophobicity of the ligand, the experimentally observed DeltaCp degrees's (for either of the ligands) could not be predicted on the basis of the changes in the solvent-accessible surface areas of the enzyme and ligand species. These coupled with the fact that the DeltaCp degrees for the binding of octenoyl-CoA to pig kidney MCAD (which is believed to be structurally identical to human liver MCAD) is only -0.37 kcal mol-1 K-1 [Srivastava, D. K., Wang, S., and Peterson, K. L. (1997) Biochemistry 36, 6359-6366] prompt us to question the reliability of predicting the DeltaCp degrees values of the enzyme-ligand complexes from their X-ray crystallographic data. Arguments are presented that certain intrinisic limitations of the crystallographic data preclude kinetic and thermodynamic predictions about the enzyme-ligand complexes and enzyme catalysis.

Acyl Coenzyme A↗
PubMed1998

Quantitative angiography of coronary artery dimensions 24 hours after rotational atherectomy.

Rotational atherectomy results in platelet activation and heat generation, which may impact artery size immediately after treatment. In addition, arteries treated with balloon angioplasty may exhibit recoil within 24 hours. In this study, arteries treated with rotational atherectomy, with and without adjunctive balloon angioplasty, were analyzed by quantitative coronary angiography to determine the effect of rotational atherectomy on the dynamic behavior of the arterial wall within 24 hours after the procedure. Quantitative coronary angiography was performed at a core laboratory. Coronary angiogram acquisitions were preceded by intracoronary nitroglycerin injections and were repeated using identical angles of projection. Proximal and distal reference vessel diameters were 2.55 +/- 0.60 and 2.28 +/- 0.51 mm, respectively, and did not change from pre- to postprocedure. Both were larger the following day increasing to 2.72 +/- 0.65 and 2.52 +/- 0.52 mm, respectively, (p <0.001). Minimum luminal diameter (MLD) increased from 0.70 +/- 0.28 mm before to 1.49 +/- 0.34 mm after the procedure and to 1.72 +/- 0.37 mm at 24-hour follow-up (p <0.001). Subset analysis of patients treated with rotational atherectomy alone or rotational atherectomy with adjunctive balloon angioplasty revealed that the increase in luminal diameters occurred in both subsets. Patients treated with adjunctive angioplasty had a smaller initial MLD, a larger postprocedure MLD, and no difference in MLD at 24-hour follow-up compared with stand-alone rotational atherectomy. Subset analysis of 100 patients who had 6-month follow-up angiography revealed that both a calculated acute gain and chronic late loss, based on a 24-hour film, differed significantly from values using a film acquired immediately after the procedure. However, the slope of the linear regression between acute gain and chronic late loss did not differ. Coronary arteries treated with rotational atherectomy with or without adjunctive balloon angioplasty increase significantly in size during the first 24 hours after the procedure. This phenomenon has implications for the calculation of absolute gain and chronic late loss, but not for the linear relation between the 2 quantitative outcomes.

Aged↗
PubMed1998

Discriminatory influence of Glu-376-->Asp mutation in medium-chain acyl-CoA dehydrogenase on the binding of selected CoA-ligands: spectroscopic, thermodynamic, kinetic, and model building studies.

We investigated the influence of Glu-376-->Asp (E376D) mutation on the UV/visible spectral, thermodynamic, and kinetic properties for the interaction of structurally different types of CoA-ligands (viz., octenoyl-CoA, acetoacetyl-CoA, and indoleacryloyl-CoA) to human liver medium-chain acyl-CoA dehydrogenase (MCAD). Whereas the E376D mutation had minimal/negligible effect on the above properties for the binding of octenoyl-CoA to the enzyme, it had pronounced effects (albeit in opposite directions) for the binding of acetoacetyl-CoA and indoleacryloyl-CoA to the enzyme. In the case of acetoacetyl-CoA, the spectrum of the enzyme-ligand complex (in the charge-transfer region; lambdamax = 545 nm) was 1.8-fold more pronounced, and the DeltaH degrees value for the binding of acetoacetyl-CoA to the enzyme was 5.6 kcal/mol more favorable with wild-type as compared to the E376D mutant enzyme. The kinetic data revealed that the above effects were related to an increase in the dissociation "off-rate" of acetoacetyl-CoA from the enzyme-acetoacetyl-CoA complex. In contrast, in the case of IACoA, the resultant UV/visible spectrum of the enzyme-IACoA complex (lambdamax = 416 nm) was 2.7-fold less pronounced, and the DeltaH degrees value of the enzyme-IACoA complex was 6.4 kcal/mol less favorable with the wild-type than the E376D mutant enzyme. The latter effects were supported by the fact that the above mutation impaired the dissociation "off-rate" of IACoA from the enzyme-IACoA complex by 5.7-fold. Molecular model building studies revealed that the discriminatory influence of the E376D mutation on the spectral, thermodynamic, and kinetic properties of the enzyme-ligand complexes is due to ligand-specific changes in the spatial relationship between the FAD and CoA-ligands at the enzyme site. Arguments are presented that the "void" created by excision of a methylene group from Glu-376 (upon Glu-376-->Asp mutation) is adjusted differently upon interaction with structurally different types of CoA-ligands.

Acyl Coenzyme A↗
PubMed1998

Influence of excision of a methylene group from Glu-376 (Glu376-->Asp mutation) in the medium chain acyl-CoA dehydrogenase-catalyzed reaction.

The human liver medium chain acyl-CoA dehydrogenase (MCAD)-catalyzed reaction proceeds via abstraction of an alpha-proton from the acyl-CoA substrates by the carboxyl group of Glu-376. By using the methods of site-directed mutagenesis, we replaced Glu-376 by Asp (E376D mutation), expressed the wild-type and mutant enzymes in Escherichia coli, purified them to homogeneity, and compared their kinetic properties. The steady-state kinetic data revealed that the E376D mutation impaired (by about 15-20-fold) the turnover rate of the enzyme as well as its inactivation by 2-octynoyl-CoA. There was no selective solvent deuterium isotope effect on enzyme catalysis. These results lead to the suggestion that the carboxyl group of Asp-376 does not serve as efficient catalytic base as the carboxyl group of Glu-376. The E376D mutation impaired the octanoyl-CoA-dependent reductive half-reaction such that the rate-limiting step of enzyme catalysis shifted from the product dissociation step (in the case of the wild-type enzyme) to the flavin reduction step, and abolished the previously noted kinetic and thermodynamic correspondences between the octanoyl-CoA-dependent reductive half-reaction and the enzyme-octenoyl-CoA interaction [Kumar, N. R., and Srivastava, D. K. (1994) Biochemistry 33, 8833-8841]. Arguments are presented that the Glu-376-->Asp mutation results in uncoupling between the proton transfer and protein conformational change steps during enzyme catalysis.

Acyl Coenzyme A↗