| Autor: |
Callacher, Gerard, Jackson, Caroline S., Searcey, Mark, Goel, Rajiv, Mellor, Geoffrey W., Smith, Carmen Z., Brocklehurst, Keith |
| Předmět: |
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| Zdroj: |
European Journal of Biochemistry; 5/15/93, Vol. 214 Issue 1, p197-207, 13p |
| Abstrakt: |
1. The hydrolytic activity of IgG purified from (a) 13 samples of ovine antiserum collected from three animals during a two-year immunisation programme using a phosphate immunogen (comprising the amide conjugate bonded through the carboxy group of 4-nitrophenyl 4-carboxymethylphenyl hydrogen phosphate and amino groups of keyhole-limpet haemocyanin) and (b) a sample of ovine antiserum collected from another animal during an 18-week immunisation programme using an analogous sulphone immunogen (comprising the amide conjugate bonded through the amino group of 4-nitrobenzyl, 4-(4-aminobutoxy)benzyl sulphone and carboxyl groups of keyholelimpet haemocyanin) were evaluated kinetically by using 4-nitrophenyl 4-(3-aza-2-oxoheptyl)phenyl carbonate and 4-nitrophenyl 4-(2-hydroxyethoxy)phenyl carbonate as substrates. 2. Catalytic activity was found in all 13 samples of anti-phosphate IgG but was absent in the sample of anti-suiphone IgG as well as in all samples of IgG isolated from the serum of nonimmunised animals. These findings, taken together with the lack of catalytic activity of the antiphosphate IgG towards the 2-nitrophenyl 4-(3-aza-2-oxoheptyl)phenyl carbonate, compel the view that the catalytic activity of the anti-phosphate IgG preparation is entirely antibody-mediated and is not due to contaminant hydrolytic enzymes. The fact that catalytic activity was found in all 13 samples of the anti-phosphate IgG provides the first evidence that it is possible, as a routine, to elicit a catalytic antibody response in a host animal via active immunisation 3. The nature of the, albeit small, variation in the catalytic characteristics of the anti-phosphate IgG (increase in both kcat, the catalytic rate constant calculated as V/2[IgG] and kcat/Km, the apparent second-order rate constant for the overall catalysed conversion of substrate to products, with increase in Km suggests simultaneous improvement in transition state binding and deterioration in substrate binding as predicted from immunogen design and the postulated general mechanistic basis of antibody catalysis. 4. This interpretation is supported by the difference in the values of the dissociation constant Ki for the competitive inhibition by the transition-state analogue 4-methylphenyl 4-nitrophenyl hydrogen phosphate of reactions catalysed by two representative anti-phosphate IgG samples: for the catalysis with Km = 4.5 µM, Ki = 9 nM and for that with Km = 1.3 µM, Ki = 80 nM. 5. 4-Nitrobenzyl 4-(4-aminobutoxy)benzyl sulphone, the hapten that was used to prepare the sulphone immunogen mentioned in (1), failed to inhibit the hydrolysis of 4-nitrophenyl 4-(3-aza-2oxoheptyl)phenyl carbonate catalysed by anti-phosphate IgG. The sulphone moiety, therefore, does not appear to mimic adequately the carbonate in either ground-state or transition-state complexes. [ABSTRACT FROM AUTHOR] |
| Databáze: |
Complementary Index |
| Externí odkaz: |
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