Substrate inactivation of enzymes in vitro and in vivo

Some enzymes are inactivated by their natural substrates during catalytic turnover, limiting the ultimate extent of reaction. These enzymes can be separated into three broad classes, depending on the mechanism of the inactivation process. The first type is enzymes which use molecular oxygen as a substrate. The second type is inactivated by hydrogen peroxide, which is present either as a substrate or a product, and are stabilized by high catalase activity. The oxidation of both types of enzymes shares common features with oxidation of other enzymes and proteins. The third type of enzyme is inactivated by non-oxidative processes, mainly reversible loss of cofactors or attached groups. Sub classes are defined within each broad classification based on kinetics and stoichiometry. Reaction-inactivation is in part a regulatory mechanism in vivo, because specific proteolytic systems give rapid turnover of such labelled enzymes. The methods for enhancing the stability of these enzymes under reaction conditions depends on the enzyme type. The kinetics of these inactivation reactions can be used to optimize bioreactor design and operation.     

The purification and immunocharacterisation of N-methylputrescine oxidase from transformed root cultures of Nicotiana tabacum L. cv SC58

The enzyme N-methylputrescine oxidase which catalyses the conversion of N-methylputrescine to N-methylpyrrolinium salt has been purified to homogeneity from transformed roots of Nicotiana tabacum L. cv SC58. The enzyme has an apparent sub-unit molecular weight of 53 kDa by sodium dodecyl sulphate-polyacrylamide gel electrophoresis with gel-filtration studies, indicating that the native form is a dimer. The K _m of the enzyme for N-methylputrescine has been estimated to be 0.1 mM. Polyclonal antibodies raised to the purified protein recognise one product in an immunoblot of a crude extract of transformed root tissue and will immunoprecipitate N-methylputrescine oxidase activity from such an extract. The antibodies also show a high degree of specificity in immunoblots of crude extracts of transformed root cultures from a range of other solanaceous and non-solanaceous species but do not cross-react with a partially purified preparation of pea-seedling diamine oxidase.Abbreviations MPO N-methylputrescine oxidase - PVDF polyvinylidene difluoride - SDS-PAGE sodium dodecyl sulphate-polyacrylamide gel electrophoresis We would like to thank members of the Plant Cell Biotechnology Group, Institute of Food Research, Norwich Laboratory, for their helpful discussions during the preparation of this paper.     

A potent specific inhibitor of 6-phosphogluconate dehydrogenase ofCryptococcus neoformans and of certain other fungal enzymes

A particular lot of the zwitterionic buffer, 2(N-morpholino) ethane sulfonic acid (MES), contained a contaminant that inhibited a number of fungal NADP-dependent dehydrogenases. Enzymes that were particularly sensitive include 6-phosphogluconate dehydrogenases fromCryptococcus neoformans andSchizophyllum commune and glucose-6-phosphate dehydrogenase fromSchizophyllum commune. A number of NADP-dependent dehydrogenases of animal origin were tested and all were completely insensitive to inhibition except for rat liver 6-phosphogluconate dehydrogenase, which was 10-fold less sensitive than theCryptococcal enzyme. The pattern of inhibition in all cases was linear competitive versus NADP. The inhibitor has been purified and identified as an ethylenesulfonic acid oligomer. This inhibitor holds promise as a model compound for the development of a specific antifungal agent.     

The Nucleotide Sequence of Human Acylamino Acid-Releasing Enzyme

The nucleotide sequence of a cDNA coding for the human acylaminoacid-releasing enzyme (AARE, also known as acylpeptide hydrolase)[EC 3.4.19.1] subunit has been determined. The amino acid sequenceof human AARE subunit deduced from its cDNA nucleotide sequenceshowed a high degree of identity (91.5%) with both the correspondingproteins from the pig and the rat. The AARE cDNA shows 99.2%identity with a 3.3 kb cDNA transcribed from a locus (DNF15S2)on the short arm of human chromosome 3, whose deletion is associatedwith small cell lung cancer, taking into consideration thatthe sequence of the 3.3-kb cDNA previously reported was causedby misreading.     

The control of respiration in wheat (Triticum aestivum L.) leaves

The aim of this work was to discover whether the respiration of wheat (Triticum aestivum L. cv. Huntsman) leaves, transferred to darkness after 7 h photosynthesis, showed an initial period of wasteful respiration. For young and old leaves, CO₂ production and O₂ uptake after 7 h photosynthesis were up to 56% higher than at the end of an 8-h night. The maximum catalytic activities of citrate synthase (EC 4.1.3.7), aconitase (EC 4.2.1.3), fumarase (EC 4.2.1.2) and cytochrome-c oxidase (EC 1.9.3.1) at the end of the day did not differ from those at the end of the night. Changes in the contents of glucose 6-phosphate, fructose-1,6-bisphosphate, dihydroxyacetone phosphate, and -ketoglutarate did not as a group parallel the changes in the rate of respiration. The detailed distribution of label from [U-¹⁴C] sucrose supplied to leaves in the dark was similar at the end of the day and the end of the night. No correlation was observed between the rates of leaf respiration and extension growth. It is argued that the higher rate of respiration at the beginning of the night cannot be attributed to wasteful respiration.Abbreviation RQ respiratory quotient We thank Dr H. Thomas and Professor C.J. Pollock, Institute for Grassland and Environmental Research, Plas Gogerddan, Aberystwyth, UK for their generous help in measuring leaf extension. R.H.A. thanks the Science and Engineering Research Council for a studentship.     

Enzyme activity measurements on isolated organs of Brachionus plicatilis (Rotifera)

A method is described by which the integument of Brachionus plicatilis, together with its intracellular lamina, is quickly dissolved before other parts or tissues of the animal are destroyed. After removing the integument several parts of the body can be separated and fractionated in a more or less intact state by centrifugation in a Percoll gradient. The measurement of enzyme activities has indicated that this procedure might provide a way of localizing enzymes within the rotifer body.     

Tritium NMR studies of the human carbonic anhydrase I–benzenesulfonamide complex

Tritium NMR spectroscopy has been used to examine the complexformed by [4-³H]benzenesulfon-amide and human carbonicanhydrase I. The results show that in solution the inhibitor forms a 1:1complex with the enzyme. A 100-spin computational model of the system,constructed with reference to crystallographic results, was used tointerpret tritium relaxation behavior and ³H{¹H}NOEs. The analysis shows that the rate of dissociation of theenzyme–sulfonamide complex is 0.35 s^–1 and thatthe aromatic ring of the inhibitor undergoes rapid rotation while complexed.     

The diffusion of gibberellins into agar and water during early germination of Pharbitis nil Choisy

Agar diffusion of imbibed seeds yielded significant amounts of diffusible Gibberellin-like substances. An analysis of the extractable and diffusible gibberellin-like substance, including an analysis of the remaining imbibition water of the seeds, indicated that a significant part of these gibberellin-like substances could be attributed to a net biosynthesis of these substances in the imbibing seeds. At the same time it was found that water diffusion yielded considerably more gibberellin-like activities than comparable agar diffusions i.e. 10 to 12 fold in general.Agar as well as water diffusion showed a temperature effect with regard to the yield of gibberellin-like substances particularly during the first 6 h of diffusion. The yield of these substances is lower at 10°C, and remains lower as shown with consecutive diffusions, in comparison with the yields at 20°C or 30°C.With both agar and water diffusion the sum of activities obtained with consecutive diffusions is always higher, often considerably higher, than equal periods of continuous diffusion which is probably due to inactivation and/or interference of inhibitory substances with the bioassay responses. Finally, water diffusates of both seeds and seedlings of the normal growing cv. Violet of Japanese morning glory contained considerably more gibberellin-like activities than those of the dwarf cv. Kidachi which indicated that normals synthesize more gibberellins than dwarfs.     

Enzyme-Linked Immunosorbent Assay of Substance P: A Study in the Eye

A solid phase enzyme-linked immunosorbent assay for quantitation of substance P is presented. The assay measures the capacity of soluble substance P to compete with the solid phase antigen for a limited quantity of specific substance P antibody. The solid-phase antigen consists of a synthetic substance P.poly-D-glutamic acid conjugate coated to polystyrene micro-ELISA plate wells. Soluble substance P and antibodies to substance P are first preincubated together and then added to the wells containing solid-phase antigen. Subsequently the wells are incubated with anti-antibodies conjugated to alkaline phosphatase. The wells are finally incubated with p-nitrophenyl phosphate an the absorbance is read in a spectrophotometer 16--24 hr after the start of the assay. The threshold for detection of substance P was 5--10 pg per well (0.25 ml). Substance P was extracted from rabbit eyes and the values obtained with the present method are compared with previously reported values based on radioimmunoassay.