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1.
N. L. Biddington P. A. Brocklehurst A. S. Dearman Jane Dearman 《Physiologia plantarum》1982,55(4):407-410
Celery seeds ( Apium graveolens L.) given a germination induction period (3 days imbibition at 17°C in the light) could be prevented from germinating by up to 14 days subsequent exposure to high temperature (32°C), polyethylene glycol (PEG), abscisic acid (ABA) or dark (22°C). When the seeds were returned to 17°C in the light, germination occurred and, except for the high temperature treatment, was more rapid compared to seeds given a germination induction period only.
Celery seeds incubated for 3 days at 17°C in the light and then air-dried at 20°C germinated slowly when re-sown at 17°C in the light, and achieved only 19% germination after 21 days. Exposing the seeds to high temperature, PEG, ABA or dark for up to 14 days before drying maintained seed viability and subsequent germination was faster. The longer treatment periods gave increased benefit, and PEG was the most effective treatment. It is suggested that the effectiveness of the treatments in inducing dehydration tolerance relates to their ability to inhibit germination possibly via their prevention of cell expansion. 相似文献
Celery seeds incubated for 3 days at 17°C in the light and then air-dried at 20°C germinated slowly when re-sown at 17°C in the light, and achieved only 19% germination after 21 days. Exposing the seeds to high temperature, PEG, ABA or dark for up to 14 days before drying maintained seed viability and subsequent germination was faster. The longer treatment periods gave increased benefit, and PEG was the most effective treatment. It is suggested that the effectiveness of the treatments in inducing dehydration tolerance relates to their ability to inhibit germination possibly via their prevention of cell expansion. 相似文献
2.
Differences in the chemical and catalytic characteristics of two crystallographically ''identical'' enzyme catalytic sites. Characterization of actinidin and papain by a combination of pH-dependent substrate catalysis kinetics and reactivity probe studies targeted on the catalytic-site thiol group and its immediate microenvironment. 总被引:5,自引:5,他引:0
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E Salih J P Malthouse D Kowlessur M Jarvis M O''Driscoll K Brocklehurst 《The Biochemical journal》1987,247(1):181-193
The characteristics of actinidin (EC 3.4.22.14) and papain (EC 3.4.22.2), two cysteine proteinases whose catalytic-site regions appear to superimpose to a degree that approaches atomic co-ordinate accuracy of both crystal structures, were evaluated by determining (a) the pH-dependence in acid media of the acylation process of the catalytic act (k+2/Ks) using N alpha-benzoyl-L-arginine p-nitroanilide (L-Bz-Arg-Nan) as substrate and (b) the sensitivity of the reactivity of the catalytic-site thiol group and its pH-dependence to structural change in small, thiol-specific, two-protonic-state reactivity probes (2,2'-dipyridyl disulphide and methyl 2-pyridyl disulphide) where enzyme-probe contacts should be restricted to areas close to the catalytic site. Distortion of the catalytic sites of the two enzymes at pH less than 4 was evaluated over time-scales appropriate for both stopped-flow reactivity probe kinetics (less than or equal to 1-2 s) and steady-state substrate catalysis kinetics (3-5 min) by using the 2,2'-dipyridyl disulphide monocation as a titrant for non-distorted catalytic sites. This permitted a lower pH limit to be defined for valid kinetic analysis of both types. The behaviour of the enzymes at pH less than 4 requires a kinetic model in which the apparently biomolecular reaction of enzyme with probe reagent is separated from the process leading to loss of conformational integrity by a potentially reversible step. The acylation of actinidin with L-Bz-Arg-Nan in acidic media occurs in two protonic states, one produced by raising the pH across pKa less than 4 which probably characterizes the formation of -S-/-ImH+ ion pair (pKa approx. 3) and the other, of higher reactivity, produced by raising the pH across pKa 5.5, which may characterize rearrangement of catalytic-site geometry. The pH-dependence of the acylation of papain by L-Bz-Arg-Nan is quite different and is not influenced by protonic dissociation with pKa values in the range 5-6. The earlier conclusion that the acylation of papain depends on two protonic dissociations each with pKa approx. 4 was confirmed. This argument is now more firmly based because titration with 2,2'-dipyridyl disulphide permits the loss of conformational integrity to be taken into account in the analysis of the kinetic data at very low pH. Methyl 2-pyridyl disulphide was synthesized by reaction of pyridine-2-thione with methyl methanethiolsulphonate and its pKa at I = 0.1 was determined by spectral analysis at 307 nm to be 2.8.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
3.
The interplay of electrostatic fields and binding interactions determining catalytic-site reactivity in actinidin. A possible origin of differences in the behaviour of actinidin and papain.
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D Kowlessur M O''Driscoll C M Topham W Templeton E W Thomas K Brocklehurst 《The Biochemical journal》1989,259(2):443-452
1. The pH-dependence of the second-order rate constant (k) for the reaction of actinidin (EC 3.4.22.14) with 2-(N'-acetyl-L-phenylalanylamino)ethyl 2'-pyridyl disulphide was determined and the contributions to k of various hydronic states were evaluated. 2. The data were used to assess the consequences for transition-state geometry of providing P2/S2 hydrophobic contacts in addition to hydrogen-bonding opportunities in the S1-S2 intersubsite region. 3. The P2/S2 contacts (a) substantially improve enzyme-ligand binding, (b) greatly enhance the contribution to reactivity of the hydronic state bounded by pKa 3 (the pKa characteristic of the formation of catalytic-site-S-/-ImH+ state) and pKa 5 (a relatively minor contributor in reactions that lack the P2/S2 contacts), such that the major rate optimum occurs at pH 4 instead of at pH 2.8-2.9, and (c) reveal the kinetic influence of a pKa approx. 6.3 not hitherto observed in reactions of actinidin. 4. Possibilities for the interplay of electrostatic effects and binding interactions in both actinidin and papain (EC 3.4.22.2) are discussed. 相似文献
4.
5.
Growth of food-borne pathogenic bacteria in oil-in-water emulsions: I—Methods for investigating the form of growth 总被引:1,自引:1,他引:0
Mary L. Parker T.F. Brocklehurst P.A. Gunning Heather P. Coleman Margaret M. Robins 《Journal of applied microbiology》1995,78(6):601-608
Methods are presented for investigating the site and form of growth of bacteria in model oil-in-water emulsions and in dairy cream. Following growth of the bacteria, the continuous aqueous phase is gelled using agarose and the oil phase removed using a mixture of chloroform and methanol. Using this method, the authors have found that Listeria monocytogenes, Salmonella typhimurium and Yersinia enterocolitica grow in the form of colonies in concentrated oil-in-water emulsions. Colonies of L. monocytogenes and Y. enterocolitica also form in artificially-inoculated fresh and tinned dairy cream. If information about the precise site of growth is not required, the authors have discovered that intact colonies can be liberated from the model emulsions by dissolving away the oil phase with chloroform: methanol. 相似文献
6.
J C Brocklehurst 《BMJ (Clinical research ed.)》1993,306(6881):832-834
OBJECTIVES--To investigate the prevalence of urinary incontinence among people living at home, their responses to it, and its emotional and social effects. DESIGN--Random sample of 4007 adults interviewed in their own homes. SETTING--Random sample of 178 constituency sampling points throughout Great Britain. SUBJECTS--1883 men, 2124 women aged 30 and over. MAIN OUTCOME MEASURES--Responses to questionnaire. RESULTS--6.6% (125) men and 14.0% (297) women had been incontinent of urine at some time--2.8% (52) men and 7.5% (159) women in the previous two months and 61% (124) of these for more than four years. 52% (108) had consulted their general practitioner at the onset of incontinence and a further 31% (65) later. Doctors commonly took a urine sample (163, 54%), referred the patient to a specialist (127, 42%), and prescribed tablets (109, 36%); only 22% (66) carried out an abdominal, rectal, or vaginal examination. Patients were not embarrassed in seeing their doctor and most thought they were treated sympathetically. 60% (265) of all those affected were concerned or worried about their incontinence, and in almost half incontinence limited their daily social activities. CONCLUSION--More people with incontinence seem to be consulting their doctors about it than has been found in previous studies, but the procedures carried out by general practitioners still seem to be suboptimal. Urinary incontinence has a profound effect on the day to day lives of most of those who suffer from it. 相似文献
7.
The pH-dependence of second-order rate constants of enzyme modification may provide free-reactant pKa values.
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1. Reactions of enzymes with site-specific reagents may involve intermediate adsorptive complexes formed by parallel reactions in several protonic states. Accordingly, a profile of the apparent second-order rate constant for the modification reaction (Kobs., the observed rate constant under conditions where the reagent concentration is low enough for the reaction to be first-order in reagent) against pH can, in general, reflect free-reactant-state molecular pKa values only if a quasi-equilibrium condition exists around the reactive protonic state (EHR) of the adsorptive complex. 2. Usually the condition for quasi-equilibrium is expressed in terms of the rate constants around EHR: (formula: see text) i.e. k mod. less than k-2. This often cannot be assessed directly, particularly if it is not possible to determine kmod. 3. It is shown that kmod. must be much less than k-2, however, if kobs. (the pH-independent value of kobs.) less than k+2. 4. Since probable values of k+2 greater than 10(6)M-1.S-1 and since values of kobs. for many modification reactions less than 10(6)M-1.S-1, the equilibrium assumption should be valid, and kinetic study of such reactions should provide reactant-state pKa values. 5. This may not apply to catalyses, because for them the value of kcat./Km may exceed 5 X 10(5)M-1.S-1. 6. The conditions under which the formation of an intermediate complex by parallel pathways may come to quasi-equilibrium are discussed in the Appendix. 相似文献
8.
The absorption of protons with alpha-methyl glucoside and alpha-thioethyl glucoside by the yeast N.C.Y.C. 240. Evidence against the phosphorylation hypothesis.
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1. When yeast N.C.Y.C. 240 was grown with maltose in a complex medium based on yeast extract and peptone, washed cell preparations fermented alpha-methyl glucoside much more slowly than maltose. 2. The yeast absorbed alpha-methyl[14C]glucoside from a 10mM solution in the presence of antimycin and iodoacetamide, producing [14C]glucose, which accumulated outside the cells. The yeast itself contained hexose phosphates, trehalose, alpha-methyl glucoside and other products labelled with 14C, but no alpha-methyl glucoside phosphate. 3. About 1 equiv. of protons was absorbed with each equivalent of alpha-methylglucoside, and 1 equiv. of K+ ions left the yeast. 4. alpha-Thioethyl glucoside was also absorbed along with protons. Studies by g.l.c. showed that the yeast concentrated the compound without metabolizing it. 5. The presence of trehalose, sucrose, maltose, L-sorbose, glucose or alpha-phenyl glucoside in each case immediately stimulated proton uptake, whereas fructose, 3-O-methylglucose and 2-deoxyglucose failed to do so. 6. The observations support the conclusion that alpha-thioethyl glucoside, alpha-methyl glucoside and maltose are substrates of one or more proton symports, whereas they seem inconsistent with the notion that the absorption of alpha-methyl glucoside involves the phosphorylation of the carbohydrate [Van Stevenick (1970) Biochim. Biophys. Acta 203, 376-384]. 相似文献
9.
Evolution of enzyme catalytic power. Characteristics of optimal catalysis evaluated for the simplest plausible kinetic model 总被引:3,自引:3,他引:0
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Keith Brocklehurst 《The Biochemical journal》1977,163(1):111-116
1. Evolutionary changes in the structure of an enzyme that provide an increase in its Km value are considered. Provided that Km increases as a result of increases in the forward rate constants of the catalysis relative to the reverse rate constants, the enzyme catalyses the conversion of a fixed concentration of its substrate more rapidly when its structure provides that Km>[S] than when Km<[S]. 2. Catalytic efficiency of enzymes is discussed in terms of the simplest plausible model, the Haldane [(1930) Enzymes, Longmans, London] reversible three-step model: [Formula: see text] The rate equation for the forward reaction of this model (formation of P) may be written in the simple form: [Formula: see text] Keq. is the equilibrium constant (=[P]eq./[S]eq.), and kcat.=V/[E]T, where [E]T is the total enzyme concentration. 3. To assess the effectiveness of an enzyme, it is necessary only to determine the extent to which the constraints of a particular kinetic mechanism permit v2 (v when Km»[S]) to approach vd (the diffusion-limited rate). 4. The value of the optimal rate of catalysis (vopt., the maximal value of v2) is dictated by the equilibrium constant for the reaction, Keq.; v2=vd/a, where [Formula: see text] when k+1 is assumed equal to k−3, and vopt.=vd/amin.. When Keq.≥1, it is necessary that k+2»k−1 for a to take its minimum value, amin.; when Keq.«1, it is necessary only that k+2»Keq.·k−1, i.e. a can equal amin. even if k+2<k−1. When Keq.»1, vopt.=vd; when Keq.=1, vopt.=vd/2, and when Keq.«1, vopt.=Keq.·vd. 5. The analysis, together with predicted effects of evolutionary pressure, suggests that in practice the rates of the fastest enzyme-catalysed freely reversible reactions might be expected to be lower than the value of k+1[E]T[S] by about an order of magnitude, particularly if Keq.<1. 6. The existing literature suggests that, in general, appropriate values of Km have evolved for the provision of high rates of catalysis but that many values of kcat. are not large enough to provide optimal rates of catalysis unless the value of k+1 in vivo is lower than its value in free solution. 相似文献
10.
Evidence for a close similarity in the catalytic sites of papain and ficin in near-neutral media despite differences in acidic and alkaline media. Kinetics of the reactions of papain and ficin with chloroacetate. 总被引:3,自引:3,他引:0
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1. The pH-dependences of the second-order rate constants (k) for the alkylation by chloroacetate of the active-centre thiol groups of papain (EC 3.4.22.2) and ficin (EC 3.4.22.3) were determined over a wide range of pH at 25 degrees C at I 0.1. 2. The main feature of both pH-k profiles is a striking rate maximum at pH6 (characterizing parameters in both cases pKI approx. 3.5, pKII approx. 8.4 and pH-independent rate constant approximately kXH 2.5-3.0 M-1 . s-1). 3. The profile for the ficin reaction contains a plateau at high pH, with approximately kX 0.10 M-1 . s-1; if an analogous plateau exists in the papain reaction, approximately kX ix much lower, less than 0.02 M-1 . s-1. 4. Both enzymes appear to contain closely similar thiolate-imidazolium interactive systems at pH6, but differences in their behaviour in more-acidic media and in alkaline media suggest differences in interaction with the postulated carboxylate component of the putative catalytic triad. 相似文献