Serum immunoglobulin G directed against porcine trypsin in the serum of cystic fibrosis children receiving porcine pancreatic enzyme supplements

Cystic fibrosis (CF) patients require pancreatic enzyme replacement therapy to correct pancreatic insufficiency. These enzymes are derived from porcine pancreas and are known to be antigenic. To determine the possible clinical consequences, a specific ELISA was developed to detect IgG antibody directed against porcine trypsin (PTAb) in the sera of CF patients. The assay was used to evaluate the occurrence of PTAb in a cross sectional study of 103 CF patients in relation to the introduction of porcine enzyme therapy, clinical status and genotype. Antibodies against porcine trypsin were detected in the sera of 63% of patients unrelated to the age of commencement or the duration of enzyme therapy. No differences were observed in the clinical status of CF patients who had developed PTAb (n = 65) and those who had no detectable PTAb (n = 38) as determined from: the current prescribed dose of porcine pancreatic enzyme capsules; Z scores for height and weight; and respiratory function tests. It is suggested that the PTAb commonly found in the sera of CF patients are of doubtful clinical significance but the prospect of PTAb contributing to immune complex disease should be examined further.

Mycobacterium tuberculosis pantothenate kinase: possible changes in location of ligands during enzyme action

The crystal structures of complexes of Mycobacterium tuberculosis pantothenate kinase with the following ligands have been determined: (i) citrate; (ii) the nonhydrolysable ATP analogue AMPPCP and pantothenate (the initiation complex); (iii) ADP and phosphopantothenate resulting from phosphorylation of pantothenate by ATP in the crystal (the end complex); (iv) ATP and ADP, each with half occupancy, resulting from a quick soak of crystals in ATP (the intermediate complex); (v) CoA; (vi) ADP prepared by soaking and cocrystallization, which turned out to have identical structures, and (vii) ADP and pantothenate. Solution studies on CoA binding and catalytic activity have also been carried out. Unlike in the case of the homologous Escherichia coli enzyme, AMPPCP and ADP occupy different, though overlapping, locations in the respective complexes; the same is true of pantothenate in the initiation complex and phosphopantothenate in the end complex. The binding site of MtPanK is substantially preformed, while that of EcPanK exhibits considerabl plasticity. The difference in the behaviour of the E. coli and M. tuberculosis enzymes could be explained in terms of changes in local structure resulting from substitutions. It is unusual for two homologous enzymes to exhibit such striking differences in action. Therefore, the results have to be treated with caution. However, the changes in the locations of ligands exhibited by M. tuberculosis pantothenate kinase are remarkable and novel.

A Genetic Analysis of Lysosomal Enzyme Activities in Brahman Cattle

Analyses of variance and co variance were carried out on the activities of three lysosomal enzymes in mononuclear blood cells from Brahman cattle. These were hexosaminidase (HEX), beta-D-galacto-sidase (GAL) and acid alpha-glucosidase (GLU) which had been measured in blood mononuclear cells from 1752 cattle from 6 herds in a Pompe's disease control programme. Herd of origin and date of bleeding significantly affected the level of activity of all enzymes. In addition, HEX and GAL were affected by age and HEX by the sex of the animal bled. Estimates of heritability from sire variances were 0.29:t 0.09 for HEX, 0.31 :t 0.09 for GAL and 0.44:t 0.09 for GLU. Genetic correlations between all enzymes were positive. The data indicate the existence of a major gene causing Pompe's disease and responsible for 16% of the genetic variation in GLU. One standard deviation of selection differential for high GLU should almost eliminate Pompe's disease from the population. The effi-ciency of selection would be aided by estimating the breeding value for GLU using measurements of HEX and GLU and taking account of an animal's sex, age, date of bleeding and herd of origin.

Enzymic Conversion of Agmatine to Putrescine inL athyrms satiuus seedlings - Purification and properties of a multifunctional enzyme (putrescine synthase)

The participation of a multifunctional enzy(am sein - gle polypeptide with multiple catalytic activities (14)) has been demonstrated in the conversion of agmatine to putrescine in Lathyrus sativus seedlings. This enzyme (putrescine synthase) with inherent activities of agmatine iminohydrolase, putrescine transcarbamylase, ornithine transcarbamylase, and carbamate has been purified to homogeneity anhda s M, = 55,000.

The use of a synthetic prostaglandin e1 analogue (misoprostol) as an adjunct to pancreatic enzyme replacement in cystic fibrosis

Eleven cystic fibrosis children (mean age, 9.6 years) were chosen at random to participate in a study to observe the effects of concurrently stimulating gastric/duodenal bicarbonate secretion and inhibiting gastric acid secretion, using a methylated prostaglandin E1 analogue in patients with pancreatic insufficiency and taking pancreatic enzymes. Percentage fat absorption in 3-day stool collections were calculated before and after commencing therapy with misoprostol, 400 μg/day in divided doses. We found a significant reduction in fat output (14.7 ± 11.7 versus 7.5 ± 3.5 g/day, p < 0.05) in the study group as a whole and a significant reduction in steatorrhoeic level as a percentage of fat intake in all of the patients with abnormal base-line collections (23.1% versus 9.2% p < 0.002). We conclude that misoprostol should be considered in cystic fibrosis patients with steatorrhoea as a means of improving nutrient absorption. © 1988 Informa UK Ltd All rights reserved: reproduction in whole or part not permitted.

The use of a synthetic prostaglandin-E1 analog (misoprostol) as an adjunct to pancreatic-enzyme replacement in cystic-fibrosis

Eleven cystic fibrosis children (mean age, 9.6 years) were chosen at random to participate in a study to observe the effects of concurrently stimulating gastric/duodenal bicarbonate secretion and inhibiting gastric acid secretion, using a methylated prostaglandin E1 analogue in patients with pancreatic insufficiency and taking pancreatic enzymes. Percentage fat absorption in 3-day stool collections were calculated before and after commencing therapy with misoprostol, 400 μg/day in divided doses. We found a significant reduction in fat output (14.7 ± 11.7 versus 7.5 ± 3.5 g/day, p < 0.05) in the study group as a whole and a significant reduction in steatorrhoeic level as a percentage of fat intake in all of the patients with abnormal base-line collections (23.1% versus 9.2%, p < 0.002). We conclude that misoprostol should be considered in cystic fibrosis patients with steatorrhoea as a means of improving nutrient absorption.

Validation of a Competitive Enzyme-Linked Immunosorbent Assay for Detection of Babesia bigemina Antibodies in Cattle

A competitive enzyme-linked immunosorbent assay (cELISA) based on a broadly conserved, species-specific, B-cell epitope within the C terminus of Babesia bigemina rhoptry-associated protein 1a was validated for international use. Receiver operating characteristic analysis revealed 16% inhibition as the threshold for a negative result, with an associated specificity of 98.3% and sensitivity of 94.7%. Increasing the threshold to 21% increased the specificity to 100% but modestly decreased the sensitivity to 87.2%. By using 21% inhibition, the positive predictive values ranged from 90.7% (10% prevalence) to 100% (95% prevalence) and the negative predictive values ranged from 97.0% (10% prevalence) to 48.2% (95% prevalence). The assay was able to detect serum antibody as early as 7 days after intravenous inoculation. The cELISA was distributed to five different laboratories along with a reference set of 100 defined bovine serum samples, including known positive, known negative, and field samples. The pairwise concordance among the five laboratories ranged from 100% to 97%, and all kappa values were above 0.8, indicating a high degree of reliability. Overall, the cELISA appears to have the attributes necessary for international application.

Enzyme-catalysed non-oxidative decarboxylation of aromatic acids: I.Purification and spectroscopic properties of 2,3 dihydroxybenzoic acid decarboxylase from Image Image

In order to understand the molecular mechanism of non-oxidative decarboxylation of aromatic acids observed in microbial systems, 2,3 dihydroxybenzoic acid (DHBA) decarboxylase from Image Image was purified to homogeneity by affinity chromatography. The enzyme (Mr 120 kDa) had four identical subunits (28 kDa each) and was specific for DHBA. It had a pH optimum of 5.2 and Km was 0.34mM. The decarboxylation did not require any cofactors, nor did the enzyme had any pyruvoyl group at the active site. The carboxyl group and hydroxyl group in the Image -position were required for activity. The preliminary spectroscopic properties of the enzyme are also reported.

A free-enzyme catalyst for the bioremediation of environmental atrazine contamination.

Herbicide contamination from agriculture is a major issue worldwide, and has been identified as a threat to freshwater and marine environments in the Great Barrier Reef World Heritage Area in Australia. The triazine herbicides are of particular concern because of potential adverse effects, both on photosynthetic organisms and upon vertebrate development. To date a number of bioremediation strategies have been proposed for triazine herbicides, but are unlikely to be implemented due to their reliance upon the release of genetically modified organisms. We propose an alternative strategy using a free-enzyme bioremediant, which is unconstrained by the issues surrounding the use of live organisms. Here we report an initial field trial with an enzyme-based product, demonstrating that the technology is technically capable of remediating water bodies contaminated with the most common triazine herbicide, atrazine.

Identification of amino acid residues essential for enzyme activity of sheep liver 5,10-methylenetetrahydrofolate reductase

Sheep liver 5,10-methylenetetrahydrofolate reductase was subjected to specific chemical modification with phenylglyoxal, diethyl pyrocarbonate and N-bromosuccinimide. The second-order rate constants for inactivation were calculated to be 54 M-1 X min-1, 103 M-1 X min-1 and 154 M-1 X min-1 respectively. This inactivation could be prevented by incubation with substrates or products, suggesting that the residues modified, namely arginine, histidine and tryptophan, are essential for enzyme activity.

A conformational approach to the study of the dynamics of enzyme inhibition: studies on thermolysin

The preferred conformations of β-phenylpropionyl-Image -phenylalanine (β-PPP) and N-carbobenzoxy-L-phenylalanine (Cbz-Phe), two inhibitors of thermolysin, have been determined by computing potential energy using empirial potential energy functions. Of the 15 to 20 conformations that are favoured for each of these inhibitors only a few have the right conformation to reach the active site of the enzyme. The conformer of β-PPP that initiates binding with the enzyme is different from the bound one, while for Cbz-Phe the bound and initiating conformers are quite similar. Thus, β-PPP favours the ‘induced fit’ model while Cbz-Phe follows the ‘lock and key’ model of binding. The inhibitors differ in their alignment at the active site.

Effect of configuration of the inhibitors on the mode of binding to the enzyme, thermolysin

Preferred conformations of the competitive inhibitors glycyl-L-phenylalanine and glycyl-D-phenylalanine and their mode of binding to thermolysin have been studied. The difference in configuration is shown to affect significantly the mode of binding to thermolysin. Gly-D-Phe prefers to enter the active site in the global minimum conformation whereas Gly-L-Phe may enter in a higher energy conformation. Moreover, D-enantiomer is shown to have a better fit than the L-counterpart in the active site.

A core metabolic enzyme mediates resistance to phosphine gas

Phosphine is a small redox-active gas that is used to protect global grain reserves, which are threatened by the emergence of phosphine resistance in pest insects. We find that polymorphisms responsible for genetic resistance cluster around the redox-active catalytic disulfide or the dimerization interface of dihydrolipoamide dehydrogenase (DLD) in insects (Rhyzopertha dominica and Tribolium castaneum) and nematodes (Caenorhabditis elegans). DLD is a core metabolic enzyme representing a new class of resistance factor for a redox-active metabolic toxin. It participates in four key steps of core metabolism, and metabolite profiles indicate that phosphine exposure in mutant and wild-type animals affects these steps differently. Mutation of DLD in C. elegans increases arsenite sensitivity. This specific vulnerability may be exploited to control phosphine-resistant insects and safeguard food security.

Glycogen debranching enzyme activity in the muscles of meat producing animals

Muscle glycogen exists in two forms: low molecular weight pro-glycogen and high molecular weight macro-glycogen. The degradation of glycogen to glucose 1 phosphate and free glucose is catalysed by glycogen phosphorylase together with glycogen debranching enzyme (GDE). The process in which glycogen is broken down via anaerobic pathways to lactate, results in the acidification of the muscles and has a great influence on meat quality. Thus, the overall aim of this thesis was to characterise the post mortem action of GDE in muscles of meat production animals (pigs, cattle and chickens). Interest was focused on the differences in GDE activity between fast twitch glycolytic muscles and slow twitch oxidative muscles. The effects of pH, temperature, RN genotype (PRKAG3 gene), and of time post mortem on GDE activity were also investigated. This thesis showed that there are differences in GDE activity between animal species and between different muscles of an animal. It was shown that in pigs and cattle, higher GDE activity and phosphorylase activity exists in the fast twitch glycolytic muscles than in slow twitch oxidative muscles of the same animal. Thus, the high activity of these enzymes enables a faster rate of glycogenolysis in glycolytic M. longissimus dorsi compared to oxidative M. masseter. In chicken muscles, the GDE activity was low compared to pig or cattle muscles. Furthermore, the GDE activity in the glycolytic M. pectoralis superficialis was lower than in more oxidative M. quadriceps femoris despite the high phosphorylase activity in the former. The relative ratios between phosphorylase and GDE activity were higher in fast twitch glycolytic muscles than in slow twitch oxidative muscles of all studied animals. This suggests that the relatively low GDE activity compared to the phosphorylase activity in fast twitch glycolytic muscles may be a protection mechanism in living muscle against a very fast pH decrease. Chilling significantly decreased GDE activity and below 15 C porcine GDE was almost inactive. The effect of pH on GDE activity was only minor at the range normally found in post mortem muscles (pH 7.4 to 5.0). The GDE activity remained level for several hours after slaughter. During the first hours post mortem, GDE activity was similar in RN- carrier pigs and in wild type pigs. However, the GDE activity declined faster in M. longissimus dorsi from wild type pigs than in the RN carrier pigs, the difference between genotypes was significant after 24 h post mortem. Pro-glycogen and macro-glycogen contents were higher, pH decrease was faster and ultimate pH was lower in RN- carrier pigs than in wild type pigs. In the RN- carriers, the prolonged high GDE activity level may enable an extended pH decrease and lower ultimate pH in their muscles. In conclusion, GDE is not the main factor determining the rate or the extent of post mortem glycogenolysis, but under certain conditions, such as in very fast chilling, the inhibition of GDE activity in meat may reduce the rate of pH decrease and result in higher ultimate pH. The rate and extent of pH decrease affects several meat quality traits.

Effect of cupric-isonicotinic acid hydrazide complex on avian myeloblastosis virus reverse transcriptase and its associated enzyme activities

Cupric complex of isonicotinic acid hydrazide inhibits DNA synthesis by avian myloblastosis virus reverse transcriptase. This inhibition occurs in the presence of either ribonucleotide or deoxyribonucleotide templates. The inhibition of reverse transcriptase by cupric-INH complex is considerably reduced when stored or proteolytically cleaved enzyme was used in the reaction. The complex also inhibits the reverse transciptase-associated RNase H activity. The cupric-isonicotinic acid hydrazide complex cleaves pBR 322 from I DNA into smaller molecules in the presence or absence of reverse transcriptase-associated endonuclease. However, in the presence of the enzyme the DNA is cleaved to a greater extent.