Many proteins are S-acylated, affecting their localization and function. Dynamic S-acylation in response to various stimuli has been seen for several proteins in vivo. The regulation of S-acylation is beginning to be elucidated. Proteins can autoacylate or be S-acylated by protein acyl transferases (PATs). Deacylation, on the other hand, is an enzymatic process catalyzed by protein thioesterases (APT1 and PPT1) but only APT1 appears to be involved in the regulation of the reversible S-acylation of cytoplasmic proteins seen in vivo. PPT1, on the other hand, is involved in the lysosomal degradation of S-acylated proteins and PPT1 deficiency causes the disease infant neuronal ceroid lipofuscinosis.

1. 4-Nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate (I), an amide conjugate (XI) involving the carboxy group of 4-nitrophenyl 4'-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin, and a fluorescein derivative (XVII) were synthesized. 2. The conjugate (XI) was used as an immunogen with which to raise polyclonal antibodies in multigeneration cross-bred sheep; the fluorescent derivative (XVII) was used for the initial assessment of the antisera via binding assays monitored by fluorescence polarization; the carbonate ester (I) was used as a chromogenic substrate for the investigation of catalytic activity. 3. The IgG from the antiserum of sheep no. 270 was isolated by Na2SO4 precipitation and chromatography on Protein G-Sepharose. 4. This preparation of IgG catalysed the hydrolysis of the carbonate ester (I); the catalysis at pH 8.0 and 25 degrees C obeyed Michaelis-Menten kinetics with at least 25 turnovers, Km = 3.34 microM, and lower limits for kcat. of 0.029 s-1 and for kcat./Km of 8.77 x 10(3) M-1.S-1, on the unlikely assumption that the concentration of catalytic antibody is provided by twice the total IgG concentration (two sites per molecule); probable estimates of the fraction of the total IgG that is anti-haptenic IgG and of the fraction of this that is catalytically active suggest that the values of kcat./Km are actually very much larger than these lower limits. 5. The failure of the antibody preparation to catalyse the hydrolysis of the isomeric 2-nitrophenyl carbonate (II), which differs from compound (I) only in the position of the nitro substituent in the leaving group, compels the view that catalytic activity is due to antibody rather than contaminant enzyme; this conclusion is supported by (a) the failure of the following to discriminate effectively between the isomeric substrates (I) and (II): pig liver carboxylesterase, rabbit liver carboxylesterase (collectively EC 3.1.1.1), whole serum from a non-immunized sheep and whole serum from a sheep immunized with a derivative of 3-O-methylnoradrenaline and (b) the lack of catalytic activity in IgG preparations from sheep immunized with sulphoxide or sulphone analogues of immunogen (XI). 6. The various parameters used for the comparison of the kinetic characteristics of hydrolytic catalytic antibodies are discussed. 7. The characteristics of hydrolysis of compound (I) catalysed by the present polyclonal antibody preparation are shown to be substantially better in most respects than those of analogous reactions of two other carbonate esters catalysed by monoclonal antibodies.

) -EJB 92 1711121. 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-nitrophenyl4-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 imunogen (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-nitrophenyl4-(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-sulphone 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 k,,,, the catalytic rate constant calculated as V/2[IgG] and k,,,/Km, the apparent second-order rate constant for the overall catalysed conversion of substrate to products, with increase in K,,, 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 K, for the competitive inhibition by the transition-state analogue 4-methylphenyl4-nitrophenyl hydrogen phosphate of reactions catalysed by two representative anti-phosphate IgG samples : for the catalysis with K,,, = 4.5 yM, K, = 9 nM and for that with K, = 1.3 yM, K, = 80 nM.5. 4-Nitrobenzyl 4-(4-aminobutoxy)benzyl sulphone, the hapten that was used to prepare the sulphone immunogen mentioned in (l), failed to inhibit the hydrolysis of 4-nitrophenyl 4-(3-aza-2-oxohepty1)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.Since the discovery of catalytic antibodies by Tramontan0 et al. (1986) and Pollack et al. (1986), progress in this new field has developed rapidly (for a review see Lerner et al., 1991). Most of the sub...

1. The activated amide (4-nitroanilide), N-(4-nitrophenyl) N'-butyl-1,4-phenylenediacetamide (III) was synthesized. 2. A polyclonal antibody preparation (PCA 270-29) was elicited in a multigeneration cross-bred sheep (no. 270) and isolated 29 weeks into the immunization schedule by procedures described previously for PCA 270-22 [Gallacher, Jackson, Searcey, Badman, Goel, Topham, Mellor & Brocklehurst (1991) Biochem J. 271, 871-881]. These involved the use of an amide conjugate bonded through the carboxy group of 4-nitrophenyl 4'-carboxymethylphenyl phosphate and an amino group of keyhole-limpet haemocyanin as the immunogen. 3. PCA 270-29 was shown to catalyse the hydrolysis of both the carbonate ester substrate 4-nitrophenyl 4'-(3-aza-2-oxoheptyl)phenyl carbonate (I) and the amide substrate (III). Both catalyses obeyed the Michaelis-Menten equation with the following values of the parameters at 25 degrees C: for the hydrolysis of (I) at pH 8.0, Km = 3.96 +/- 0.28 microM and k(cat.) = 0.135 +/- 0.004 s-1 (k(non-cat.) = 1.99 x 10(-4) s-1); for the hydrolysis of (III) at pH 9.0, Km = 5.4 +/- 1.4 microM and k(cat.) = (5.95 +/- 0.75) x 10(-5) s-1 (k(non-cat.) = approx. 2 x 10(-7) s-1). 4. The finding that PCA 270-29 is almost equally effective as a catalyst for the hydrolysis of the amide (III) as for that of the carbonate ester (I) when allowance is made for the different intrinsic reactivities of the two types of substrate is discussed. The catalytic characteristics of PCA 270-29, the first example of a polyclonal catalytic antibody preparation shown to catalyse the hydrolysis of an amide and the first example of an antibody preparation (monoclonal or polyclonal) with such catalytic character to be produced by use of a phosphate immunogen, are compared with those of the small number of other antibody-mediated hydrolyses of amides in the literature.

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Covalent attachment of a variety of lipid groups to proteins is now recognized as a major group of post-translational modifications. S-acylation of proteins at cysteine residues is the only modification considered dynamic and thus has the potential for regulating protein function and/or localization. The activities that catalyse reversible S-acylation have not been well characterized and it is not clear whether both the acylation and the deacylation steps are regulated, since in principle it would be sufficient to control only one of them. Both apparently enzymatic and non-enzymatic S-acylation of proteins have previously been reported. Here we show that a synthetic myristoylated c-Yes protein tyrosine kinase undecapeptide undergoes spontaneous S-acylation in vitro when using a long chain acyl-CoA as acyl donor in the absence of any protein. The S-acylation was dependent on myristoylation of the substrate, the length of the incubation period, temperature and substrate concentration. When COS cell fractions were added to the S-acylation reaction no additional peptide:S-acyltransferase activity was detected. These results are consistent with the possibility that membrane-associated proteins may undergo S-acylation in vivo by non-enzymatic transfer of acyl groups from acyl-CoA. In this case, the S-acylation-deacylation process could be controlled by a regulated depalmitoylation mechanism.

Protein acylation is the covalent attachment of fatty acids to a protein; the most commonly added fatty acids are myristate (14:0) and palmitate (16:0). In this unit, protocols describe the use of radiolabeled fatty acids to label eukaryotic cells in vitro. The radiolabeled material produced can then be analyzed by the various methods described here: determination of the type of fatty acid linkage, checking for interconversion by determining the nature of the protein-bound label, and identification of the protein-bound fatty acid.