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J E Thompson

Publications and source records attributed to J E Thompson.

At least 37 records · Page 2Linked to original sources
PubMed2000

An ethylene-induced cDNA encoding a lipase expressed at the onset of senescence.

A cDNA clone encoding a lipase (lipolytic acyl hydrolase) expressed at the onset of petal senescence has been isolated by screening a cDNA expression library prepared from carnation flowers (Dianthus caryophyllus). The cDNA contains the lipase consensus sequence, ITFAGHSLGA, and encodes a 447-amino acid polypeptide with a calculated molecular mass of 50.2 kDa that appears to be a cytosolic protein. Over-expression of the clone in Escherichia coli yielded a protein of the expected molecular weight that proved capable of deesterifying fatty acids from p-nitrophenylpalmitate, tri-linolein, soybean phospholipid, and Tween in both in vitro and in situ assays of enzyme activity. The abundance of the lipase mRNA increases just as carnation flowers begin to senesce, and expression of the gene is also induced by treatment with ethylene. Southern blot analyses of carnation genomic DNA have indicated that the lipase is a single copy gene. The lipase gene is also expressed in carnation leaves and is up-regulated when the leaves are treated with ethylene. Deesterification of membrane lipids and ensuing loss of membrane structural integrity are well established early events of plant senescence, and the expression pattern of this lipase gene together with the lipolytic activity of its cognate protein indicate that it plays a fundamentally central role in mediating the onset of senescence.

Amino Acid Sequence↗
PubMed2000

Production of tomato flavor volatiles from a crude enzyme preparation using a hollow-fiber reactor.

In recent years there has been an increase in the interest in the production of compounds by isolation from natural sources or through processes that can be deemed "natural". This is of particular interest in the food and beverage industry for flavors and aromas. Hexanal, organoleptically known to possess "green character", is of considerable commercial interest. The objective of this study was to determine if the enzyme template known to be responsible for the synthesis of hexanal from linoleic acid (18:2) in tomato fruits could be harnessed using a hollow-fiber reactor. A hollow-fiber reactor system was set up and consisted of a XAMPLER ultrafiltration module coupled to a reservoir. The enzyme template was extracted from ripe tomato fruits and processed through an ultrafiltration unit (NMWC of 100 kDa) to produce a retentate enriched in soluble and membrane-associated lipoxygenase (LOX) and hydroperoxide lyase (HPL). This extract was recirculated through the lumen of the hollow-fiber ultrafiltration unit with the addition of substrate in the form of linoleic acid, with buffer addition to the reaction flask to maintain a constant retentate volume. Product formation was measured in the permeate using solid phase microextraction (SPME) developed for this system. At exogenous substrate concentrations of 16 mM and a transmembrane pressure of 70 kPa, hexanal production rates are in the order of 5.1 microg/min. Addition of Triton X-100 resulted in membrane fouling and reduced flux. The reactor system has been run for periods of up to 1 week and has been shown to be stable over this period.

Aldehyde-Lyases↗
PubMed2000

Evidence for covalent linkage between xyloglucan and acidic pectins in suspension-cultured rose cells.

Neutral xyloglucan was purified from the cell walls of suspension-cultured rose (Rosa sp. 'Paul's Scarlet') cells by alkali extraction, ethanol precipitation and anion-exchange chromatography on 'Q-Sepharose FastFlow'. The procedure recovered 70% of the total xyloglucan at about 95% purity in the neutral fraction. The remaining 30% of the xyloglucan was anionic, as demonstrated both by anion-exchange chromatography at pH 4.7 and by high-voltage electrophoresis at pH 6.5. Alkali did not cause neutral xyloglucan to become anionic, indicating that the anionic nature of the rose xyloglucan was not an artefact of the extraction procedure. Pre-incubation of neutral [3H]xyloglucan with any of ten non-radioactive acidic polysaccharides did not cause the radioactive material to become anionic as judged by electrophoresis, indicating that stable complexes between neutral xyloglucan and acidic polysaccharides were not readily formed in vitro. The anionic xyloglucan did not lose its charge in the presence of 8 M urea or after a second treatment with NaOH, indicating that its anionic nature was not due to hydrogen-bonding of xyloglucan to an acidic polymer. Proteinase did not affect the anionic xyloglucan, indicating that it was not associated with an acidic protein. Cellulase converted the anionic xyloglucan to the expected neutral nonasaccharide and heptasaccharide, indicating that the repeatunits of the xyloglucan did not contain acidic residues. Endo-polygalacturonase converted about 40% of the anionic xyloglucan to neutral material. Arabinanase and galactanase also converted appreciable proportions of the anionic xyloglucan to neutral material. These results show that about 30% of the xyloglucan in the cell walls of suspension-cultured rose cells exists in covalently-linked complexes with acidic pectins.

Arabinose↗
PubMed2000

Single-breath CO2 analysis as a predictor of lung volume change in a model of acute lung injury.

OBJECTIVE: To examine the utility of single-breath CO2 analysis as a measure of lung volume change in a model of acute lung injury. SETTING: Animal laboratory in a university-affiliated medical center. DESIGN: Prospective, animal cohort study comparing 21 variables derived from single-breath CO2 analysis with lung volume measurements determined by nitrogen washout. SUBJECTS: Seven lambs with saline lavage-induced acute lung injury. METHODS: Animals were treated with repetitive saline lavage to achieve a uniform degree of acute lung injury (PaO2 < 100 torr [13.32 kPa] on FiO2 of 1.0). Twenty-one derived components of the CO2 expirogram were evaluated as predictors of lung volume change. Lung volume was manipulated by 3-cm H2O incremental increases in positive end-expiratory pressure from 0 to 21 cm H2O and ranged between 90 and 765 mL. MEASUREMENTS AND MAIN RESULTS: Fifty-five measurements of lung volume were available for comparison with derived variables from the CO2 expirogram. Stepwise linear regression identified five variables that were most predictive of lung volume change: a) dynamic lung compliance; b) the slope of phase III; c) the slope of phase II divided by the mixed expired CO2 concentration; d) airway deadspace; and e) PaO2/FIO2 ratio. The multivariate equation was highly statistically significant and explained 94% of the variance (adjusted r2 = .94, p < .0001). The bias and precision of the calculated lung volume were 10.9 and 55.9, respectively. The mean percentage difference for the lung volume estimate derived from the single-breath CO2 analysis station was 3.3%. CONCLUSIONS: Our data indicate that analysis of the CO2 expirogram can yield accurate information about lung volume in animals with saline lavage-induced acute lung injury. Specifically, five variables derived from a plot of expired CO2 concentration vs. expired volume predict changes in lung volume in healthy lambs with an adjusted coefficient of determination of 0.94. We hope to further define the utility of this technique by prospective application of this methodology in the clinical setting.

Animals↗
PubMed2000

Inhaled nitric oxide reduces the need for extracorporeal membrane oxygenation in infants with persistent pulmonary hypertension of the newborn.

OBJECTIVE: We previously reported improved oxygenation, but no change, in rates of extracorporeal membrane oxygenation (ECMO) use or death among infants with persistent pulmonary hypertension of the newborn who received inhaled nitric oxide (NO) with conventional ventilation, irrespective of lung disease. The goal of our study was to determine whether treatment with inhaled NO improves oxygenation and clinical outcomes in infants with persistent pulmonary hypertension of the newborn and associated lung disease who are ventilated with high-frequency oscillatory ventilation (HFOV). DESIGN: Single-center, prospective, randomized, controlled trial. SETTING: Newborn intensive care unit of a tertiary care teaching hospital. PATIENTS: We studied infants with a gestational age of > or =34 wks who were receiving mechanical ventilatory support and had echocardiographic and clinical evidence of pulmonary hypertension and hypoxemia (PaO2 < or =100 mm Hg on FIO2 = 1.0), despite optimal medical management Infants with congenital heart disease, diaphragmatic hernia, or other major anomalies were excluded. INTERVENTIONS: The treatment group received inhaled NO, whereas the control group did not. Adjunct therapies and ECMO criteria were the same in the two groups of patients. Investigators and clinicians were not masked as to treatment assignment, and no crossover of patients was permitted. MEASUREMENTS AND MAIN RESULTS: Primary outcome variables were mortality and use of ECMO. Secondary outcomes included change in oxygenation and duration of mechanical ventilatory support and supplemental oxygen therapy. Forty-two patients were enrolled. Baseline oxygenation and clinical characteristics were similar in the two groups of patients. Infants in the inhaled NO group (n = 21) had improved measures of oxygenation at 15 mins and 1 hr after enrollment compared with infants in the control group (n = 20). Fewer infants in the inhaled NO group compared with the control group were treated with ECMO (14% vs. 55%, respectively; p = .007). Mortality did not differ with treatment assignment. CONCLUSIONS: Among infants ventilated by HFOV, those receiving inhaled NO had a reduced need for ECMO. We speculate that HFOV enhances the effectiveness of inhaled NO treatment in infants with persistent pulmonary hypertension of the newborn and associated lung disease.

Administration, Inhalation↗
PubMed2000

Co-association of cytochrome f catabolites and plastid-lipid-associated protein with chloroplast lipid particles.

Distinguishable populations of lipid particles isolated from chloroplasts of yellow wax bean (Phaseolus vulgaris L. cv Kinghorn Wax) leaves have been found to contain plastid-lipid-associated protein (J. Pozueta-Romero, F. Rafia, G. Houlné, C. Cheniclet, J.P. Carde, M.-L. Schantz, R. Schantz [1997] Plant Physiol 115: 1185-1194). One population is comprised of plastoglobuli obtained from sonicated chloroplasts by flotation centrifugation. Higher density lipid-protein particles isolated from chloroplast stroma by ultrafiltration constitute a second population. Inasmuch as the stromal lipid-protein particles contain plastid-lipid-associated protein, but are distinguishable from plastoglobuli in terms of their lipid and protein composition, they appear to be plastoglobuli-like particles. Of particular interest is the finding that plastoglobuli and the higher density lipid-protein particles both contain catabolites of the thylakoid protein, cytochrome f. These observations support the view that there are distinguishable populations of plastoglobuli-like particles in chloroplasts. They further suggest that the formation of these particles may allow removal of protein catabolites from the thylakoid membrane that are destined for degradation as part of normal thylakoid turnover.

Amino Acid Sequence↗
PubMed2000

The effects of early and repeated prone positioning in pediatric patients with acute lung injury.

STUDY OBJECTIVE: To describe the physiologic changes and to evaluate the safety of placing pediatric patients with acute lung injury (ALI) prone for 20 h/d during the acute phase of their illness. DESIGN: Single-center prospective case series. SETTING: Tertiary-level pediatric ICU. PATIENTS: Consecutive patients with bilateral pulmonary parenchymal disease requiring intubation and mechanical ventilation with a PaO(2)/fraction of inspired oxygen (FIO(2)) ratio </= 300 mm Hg. INTERVENTIONS: Patients were enrolled as soon as possible after meeting criteria and were placed in a prone position for 20 h/d daily until clinical improvement or death occurred. MEASUREMENTS AND RESULTS: Twenty-five pediatric patients who had ALI/ARDS, ranging in age from 2 months to 17 years, were placed in a prone position within 19 h of meeting the study criteria for a median time of 4 days, which accounted for 47% of their time receiving mechanical ventilation. Eighty-four percent of patients (n = 21) were categorized as overall responders to prone positioning because they experienced more days of increases of >/= 20 mm Hg in PaO(2)/FIO(2) ratio or a decrease of >/= 10% in oxygenation index when shifted from a supine to a prone position during the study period. During the 107 patient-days and 214 positioning cycles, no critical incidents occurred. Furthermore, no patient experienced a persistent decrease in oxygen saturation as measured by pulse oximetry (SpO(2)) of > 10% from values obtained when in the supine position, failed to keep their SpO(2) at > 85%, or experienced an increased respiratory rate of > 40 breaths/min when prone. Using the COMFORT score, patients were objectively rated to be equally comfortable in both the supine and prone positions. Patients also were able to resume spontaneous ventilation and to progress toward endotracheal extubation while in the prone position. Iatrogenic injury associated with prolonged prone positioning included stage II pressure ulcers in six patients (24%). CONCLUSIONS: The pediatric patients in this series demonstrated improvements in oxygenation without serious iatrogenic injury after prone positioning. This study provides a foundation for a prospective randomized study investigating the effect of early and repeated prone positioning on clinical outcomes in pediatric patients with ALI.

Adolescent↗
PubMed2000

Prospective evaluation of early versus delayed laparoscopic cholecystectomy for treatment of acute cholecystitis.

Although laparoscopic cholecystectomy (LC) is known to be safe in the treatment of acute cholecystitis (AC), the optimal timing of laparoscopic intervention remains controversial. The objective of this study is to prospectively compare the safety and cost effectiveness of early versus delayed LC in AC. Our study population consisted of 43 patients presenting with AC (localized tenderness, white blood cell count >10.0 or temperature >38.0 degrees C, and ultrasound confirmation) who were prospectively randomized to early versus delayed LC during their first admission. Exclusion criteria included a history of peptic ulcer disease or evidence of gallbladder perforation. All patients were treated with bowel rest and antibiotics (piperacillin 2 g intravenous piggyback every 6 hours). Early treatment patients underwent LC as soon as the operating schedule allowed. Delayed treatment patients received anti-inflammatory medication (indomethacin 50 mg per rectum every 12 hours) in addition to bowel rest and antibiotics and underwent operation after resolution of symptoms or within 5 days if symptoms failed to resolve. Early LC was performed in 21 patients, whereas 22 patients underwent delayed LC. There was no difference in age, temperature, or white blood cell count on admission between groups. Early LC slightly reduced operative time and conversion rate. There was no difference in complications. Estimated blood loss was significantly lower in those receiving early LC. There was also a significant reduction in total hospital stay and hospital charges with early LC. We conclude that delay in operation combined with anti-inflammatory medication showed no advantage with regard to operative time, conversion, or complication rate. Furthermore, early laparoscopic intervention significantly reduced operative blood loss, hospital days, and hospital charges.

Acute Disease↗
PubMed1999

A fully human antibody neutralising biologically active human TGFbeta2 for use in therapy.

Phage display provides a methodology for obtaining fully human antibodies directed against human transforming growth factor-beta (TGFbeta) suitable for the treatment of fibrotic disorders. The strategy employed was to isolate a human single chain Fv (scFv) fragment that neutralises human TGFbeta2 from a phage display repertoire, convert it into a human IgG4 and then determine its TGFbeta binding and neutralisation properties and its physical characteristics. Several scFv fragments binding to TGFbeta2 were isolated by panning of an antibody phage display repertoire, and subsequent chain shuffling of the selected V(H) domains with a library of V(L) domains. The three most potent neutralising antibodies were chosen for conversion to IgG4 format. The IgG4 antibodies were ranked for their ability to neutralise TGFbeta2 and the most potent, 6B1 IgG4, was chosen for further characterisation. 6B1 IgG4 has a high affinity for TGFbeta2 with a dissociation constant of 0.89 nM as determined using the BIAcore biosensor and only 9% cross-reactivity with TGFbeta3 (dissociation constant, 10 nM). There was no detectable binding to TGFbeta1. 6B1 IgG4 strongly neutralises (IC50 = 2 nM) the anti-proliferative effect of TGFbeta2 in bioassays using TF1 human erythroleukaemia cells. Similarly, there was strong inhibition of binding of TGFbeta2 to cell surface receptors in a radioreceptor assay using A549 cells. 6B1 IgG4 shows no detectable cross-reactivity with related or unrelated antigens by immunocytochemistry or ELISA. The 6B1 V(L) domain has entirely germline framework regions and the V(H) domain has only three non-germline framework amino acids. This, together with its fully human nature, should minimise any potential immunogenicity of 6B1 IgG4 when used in therapy of fibrotic diseases mediated by TGFbeta2.

Amino Acid Sequence↗
PubMed1999

Rapid determination of aspartate enantiomers in tissue samples by microdialysis coupled on-line with capillary electrophoresis.

Microdialysis was coupled on-line with derivatization by o-phthalaldehyde and beta-mercaptoethanol and optically gated capillary electrophoresis to determine D- and L-aspartate in tissue samples obtained from rats. The microdialysis probe was inserted into a homogenized tissue sample which allowed generation of a continuous sample stream that was filtered and deproteinated. With 7.5 mM beta-cyclodextrin (CD) in the electrophoresis buffer, the enantiomers of interest could be resolved in 3 s with an electric field of 2500 V/cm over a separation length of 15 mm. Values of D- and L-aspartate in different tissues agreed well with those obtained by an HPLC procedure that required protein precipitation, centrifugation, and extraction. The speed and compatibility with automation of the microdialysis/CE method may make it a general approach for a variety of applications involving high-throughput analysis or sensorlike operation.

Animals↗
PubMed1999

Surgical approach to cecal diverticulitis.

BACKGROUND: Cecal diverticulitis is a rare condition in the Western world, with a higher incidence in people of Asian descent. The treatment for cecal diverticulitis has ranged from expectant medical management, which is similar to uncomplicated left-sided diverticulitis, to right hemicolectomy. STUDY DESIGN: A retrospective chart review was conducted of the 49 patients treated for cecal diverticulitis at Olive View-UCLA Medical Center from 1976 to 1998. This was the largest-ever single-institution review of cecal diverticulitis reported in the mainland US. RESULTS: The clinical presentation was similar to that of acute appendicitis, with abdominal pain, low-grade fever, nausea/vomiting, abdominal tenderness, and leukocytosis. Operations performed included right hemicolectomy in 39 patients (80%), diverticulectomy in 7 patients (14%), and appendectomy with drainage of intraabdominal abscess in 3 patients (6%). Of the 7 patients who had diverticulectomy, 1 required right hemicolectomy at 6 months followup for continued symptoms. Of the three patients who underwent appendectomy with drainage, all required subsequent hemicolectomy for continued inflammation. Of the 39 patients who received immediate hemicolectomies, there were complications in 7 (18%), with no mortality. CONCLUSIONS: We endorse an aggressive operative approach to the management of cecal diverticulitis, with the resection of all clinically apparent disease at the time of the initial operation. In cases of a solitary diverticulum, we recommend the use of diverticulectomy when it is technically feasible. When confronted with multiple diverticuli and cecal phlegmon, or when neoplastic disease cannot be excluded, we advocate immediate right hemicolectomy. This procedure can be safely performed in the unprepared colon with few complications. Excisional treatment for cecal diverticulitis prevents the recurrence of symptoms, which may be more common in the Western population.

Adolescent↗
PubMed1999

A new ventilator improves CO2 removal in newborn lambs with congenital diaphragmatic hernia.

OBJECTIVES: To demonstrate improved ventilation with intratracheal pulmonary ventilation (ITPV) in new-born lambs with congenital diaphragmatic hernia, using a new microprocessor controlled ITPV-specific ventilator. DESIGN: Prospective study, with each animal serving as its own control (paired data). SETTING: Large animal research laboratory. SUBJECTS: Diaphragmatic hernias were created surgically in seven fetal sheep on gestational day 100 (term = 145 days). INTERVENTIONS: Lambs (2.7 to 5.0 kg) were delivered by cesarean section anywhere between gestational days 136 and 140. Arterial and venous catheterizations, bilateral chest tube thoracostomies, and tracheostomies were performed while the lambs received placental bypass. Initially, congenital diaphragmatic hernia lambs were supported on conventional pressure control mechanical ventilation to achieve steady state with measurements of baseline vital signs, arterial blood gases, and ventilatory settings. ITPV was instituted while maintaining constant peak carinal pressures and oxygen saturations. Statistical comparisons were made using the paired t-test. MEASUREMENTS AND MAIN RESULTS: Postductal Paco2 decreased from 110+/-21 (SD) torr (14.7+/-2.8 kPa) to 52+/-24 torr (6.93+/-3.2 kPa; p= .0014) on ITPV. Simultaneously, pH improved from 7.04+/-0.07 to 7.31+/-0.15 (p = .0012) and minute ventilation increased from 0.66+/-0.40 to 4.00+/-1.35 L/min (p = .0016). Peak carinal pressures and postductal Pao2 were unchanged. CONCLUSIONS: ITPV significantly improved CO2 removal in newborn lambs with diaphragmatic hernias without increasing airway pressures or changing oxygenation. Based on these results, we are conducting human clinical trials.

Animals↗
PubMed1999

Pediatric trauma management in a rural Wisconsin trauma center.

OBJECTIVE: To determine the results of pediatric trauma care managed with a cooperative effort by general surgeons and pediatric intensivists in comparison to national standards. DESIGN: Retrospective chart review. SETTING: Referral level II trauma center in rural Wisconsin. PATIENTS: All pediatric trauma patients age 16 and younger admitted to the hospital from 1990 to 1993. METHODS: Demographics, mechanisms of injury, revised trauma score (RTS), injury severity score (ISS), surgical procedures, need for intensive care, and outcome were examined. All patients were primarily managed by the trauma surgery service. Those patients requiring intensive care were managed jointly by the trauma surgery service and pediatric intensivists. Outcome was predicted by TRISS analysis; patients identified as "unexpected deaths" underwent critical clinical review to determine potential for survival. RESULTS: There were 531 pediatric trauma admissions. The mean age was 9.0 +/- 6.2 (SEM) years and two thirds of the patients were boys. Over half of all injuries were from falls, recreational activities, and motor vehicle crashes. There were few penetrating injuries. The mean RTS was 7.58 +/- 0.05, and the majority of patients had an ISS below 10. Sixty-two percent of patients required surgical procedures, most of which were orthopedic. Fourteen percent of patients were admitted to the pediatric intensive care unit. There were 13 deaths for a mortality rate of 2.4%. TRISS analysis identified six deaths as unexpected. Four drownings were not included in TRISS analysis, and there were no unexpected survivors. Of the six unexpected deaths, there were no significant management errors identified on careful review. CONCLUSIONS: Cooperation between general surgeons and pediatric intensivists can result in excellent pediatric trauma care in a rural level II trauma center.

Accidental Falls↗
PubMed1999

Same admission colon resection with primary anastomosis for acute diverticulitis.

Current standard of care for complicated diverticulitis includes urgent resection with colostomy versus antibiotic treatment, followed by delayed resection with primary anastomosis at a second admission. In certain circumstances, it is possible to perform resection and anastomosis on the same admission for acute diverticulitis. A retrospective review was completed for patients undergoing surgery for diverticulitis from 1991 to 1998. Groups included: 1) sigmoid resection with primary anastomosis on same admission (n = 18); 2) resection with protective end colostomy (n = 16); and 3) in-patient antibiotic treatment alone, followed by a second admission for resection with primary anastomosis (n = 5). Four patients initially treated with antibiotics worsened symptomatically or developed radiographic evidence of perforation and required resection with colostomy. Five patients in Group 1 had abscesses or contained perforations based on radiographic studies. Findings on CT scans did not predict treatment. Group 1 patients had uneventful recoveries and few minor complications (wound infections and an incisional hernia). One anastomotic leak occurred in Group 2 after colostomy closure. Although there will continue to be a role for emergent operation for diverticulitis, same admission sigmoid resection with primary anastomosis after antibiotic treatment is safe, uses a shorter course of antibiotics, and has a low complication rate.

Acute Disease↗
PubMed1999

Ticarcillin/clavulanate versus imipenem/cilistatin for the treatment of infections associated with gangrenous and perforated appendicitis.

The objective of this study was to compare ticarcillin/clavulanate given at 3.1 g every 6 hours with imipenem/cilistatin given at 500 mg every 6 hours for the treatment of infections associated with gangrenous or perforated appendicitis. One hundred thirty-seven patients were found to have gangrenous or perforated appendicitis and received the study medication for 3 to 5 days in a double-blinded, randomized manner. Clinical success was similar for the two treatment groups, 96.9 and 95.9 per cent in the ticarcillin/clavulanate and imipenem/cilistatin groups, respectively (P=0.99; 95% confidence interval for the difference was -5.6% to 7.6%). Bacteriologic success at the end of therapy was similar in the two groups, 100 and 98.4 per cent in the ticarcillin/clavulanate and imipenem/ cilistatin groups, respectively (P=0.99; 95% confidence interval for the difference was -1.8% to 4.7%). The occurrence of adverse events related to treatment was similar for the two groups (P=0.31) and led to study withdrawal for four patients (one with ticarcillin/clavulanate and three with imipenem/ cilistatin). Ticarcillin/clavulanate given at 3.1 g every 6 hours is as effective and as safe as imipenem/ cilistatin given at 500 mg every 6 hours for treatment of gangrenous or perforated appendicitis.

Adolescent↗
PubMed1998

His...Asp catalytic dyad of ribonuclease A: conformational stability of the wild-type, D121N, D121A, and H119A enzymes.

Residue His119 acts as an acid/base during the cleavage/hydrolysis reactions catalyzed by bovine pancreatic ribonuclease A (RNase A). In the native enzyme, His119 forms a hydrogen bond with Asp121. This His...Asp dyad is conserved in all homologous pancreatic ribonucleases of known sequence. Yet, replacing Asp121 with an asparagine or alanine residue does not have a substantial effect on either structure or function [Schultz, L. W., Quirk, D. J., and Raines, R. T. (1998) Biochemistry 37, 8886-8898]. Here, the pH dependencies of the conformational stabilities of wild-type RNase A and the D121N, D121A, and H119A variants were determined by monitoring thermal stability over the pH range 1.2-6.0. Replacing Asp121 with an asparagine or alanine residue results in a loss of conformational stability at pH 6.0 of deltadeltaG(o) = 2.0 kcal/mol, from a total of 9.0 kcal/mol. The magnitude of this loss is similar to that to transition-state binding during catalysis. As the pH decreases, the aspartate residue becomes protonated and deltadeltaG(o) decreases. D121N RNase A and D121A RNase A are approximately equivalent in conformational stability. This equivalence arises from compensating changes to enthalpy and entropy. A general analytical method was developed to determine the value of the pKa of a residue in the native and denatured states of a protein by comparing the pH-stability profile of the wild-type protein with that of a variant in which the ionizable residue is replaced with a nonionizable one. Accordingly, Asp121 was found to have pKa values of approximately 2.4 and 3.4 in the native and denatured states, respectively, of wild-type RNase A. This change in pKa can account fully for the differential effects of pH on the conformational stabilities of the wild-type and variant proteins. We conclude that the His...Asp catalytic dyad in pancreatic ribonucleases has two significant roles: (1) to position the proper tautomer of His119 for catalysis and (2) to enhance the conformational stability of the native enzyme. Most enzymic residues contribute to catalysis or stability (or neither). Asp121 of RNase A is a rare example of a residue that contributes equally to both.

Alanine↗
PubMed1998

2,3-Dihydro-2,5-dihydroxy-4H-benzopyran-4-one: a nonphysiological substrate for fungal melanin biosynthetic enzymes.

We have synthesized an alternate substrate for trihydroxynaphthalene reductase (3HNR) and scytalone dehydratase (SD), two enzymes in the fungal melanin biosynthetic pathway. The oxidation of 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) to 4,5-dihydroxy-2H-benzopyran-2-one (DBO) with concomitant reduction of NADP+ is catalyzed by 3HNR. DDBO is dehydrated by SD to 5-hydroxy-4H-1-benzopyran-4-one (HBO). These reactions can be monitored using continuous spectrophotometric assays. DDBO race-mizes rapidly, so chiral synthesis to mimic the natural substrate is not required. DDBO, DBO, and HBO are stable in aerated aqueous solution, in contrast to the rapidly autooxidizing trihydroxynaphthalene, a physiological substrate for 3HNR and product of SD. Unlike the natural substrates, DDBO, DBO, and HBO do not change protonation state between pH's 4 and 9. Oxidation of DDBO is effectively irreversible at pH 7, as DBO deprotonates with a pKa of 2.5. At pH 7.0 and 25 degrees C, the kcat for 3HNR catalyzed DDBO oxidation is 14 s-1 and the K(m) is 5 microM; the kcat for SD catalyzed DDBO dehydration is 400 s-1 and the K(m) is 15 microM. Based on these kinetic constants, DDBO is a better substrate than the natural substrate scytalone for both 3HNR and SD at neutral pH. An explanation for the preference of DDBO over scytalone in the oxidation and dehydration reactions is offered.

Ascomycota↗