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Nicotinic acid–adenine dinucleotide phosphate (NAADP) is fast emerging as a new intracellular Ca2+-mobilizing messenger. In sea urchin egg homogenates, binding of NAADP to its receptor is not readily reversible; hence, prior incubation with low concentrations of NAADP is more effective in inhibiting subsequent binding of radiolabelled NAADP than incubating the preparation with the two ligands simultaneously [Patel, Churchill and Galione (2000) Biochem. J. 352, 725–729]. We extend this finding to show that NAADP is more effective still in inhibiting the subsequent radioligand binding at lower homogenate concentrations, an effect again quite probably due to the non-reversible nature of the receptor–ligand interaction. Enhanced sensitivity of the preparation to NAADP afforded by simple manipulation of the experimental conditions has been applied to determine low levels of NAADP in acid extracts from human red blood cells, rat hepatocytes and Escherichia coli without interference from NADP breakdown. Our improved method for the quantification of NAADP should prove useful in the further assessment of its signalling role within cells.

CAD, the key enzyme in pyrimidine biosynthesis The first step dedicated to pyrimidine biosynthesis in mammals is catalysed by carbamoylphosphate synthase I1 (CPS 11; EC6.3.5.5). This activity is regulated through feedback inhibition by UTP, the end-product of the pathway, and activated by phosphoribosylpyrophosphate (PRib-PP), a substrate of a later step in the pathway [ l ] . The CPS I1 activity is part of the 240 kDa multienzyme polypeptide CAD, named after the activities it contains in discretely folded domains: CPS 11, aspartate transcarbamoylase (ATCase; EC 2.1.3.2) and dihydro-orotase (EC 3.5.2.3).

Reduced and ‘scrambled’derivatives of NO2-ribnuclease appear to be less stable than the corresponding derivatives of unmodified ribonuclease. In our hands, reduced ribonuclease is stable in solutions of pH4 for 4-5 days at 4”C, whereas reduced NO,-ribonuclease is reoxidized with a half-time of several hours. Likewise, the background enzymic activity of ‘scrambled‘ NO,-ribonuclease increases during storage in solution at pH8.0, 4“C, whereas scrambled ribonuclease is comparatively stable. Although this instability may be a limitation, the nitrotyrosine residue in these derivatives is a useful probe of the conformational changes accompanying disulphide-formation and interchange. The conversion from ‘scrambled‘ to ‘native’ NO,-ribonuclease corresponds to a vertical transition between the titration curves of Fig. 1. As a result of the shift in PIC., if this conversion is carried out between pH6.0 and 7.4, it should be accompanied by a significant change in A428. We find that this conversion is catalysed by protein disulphide isomerase (EC 5.3.4.1), an enzyme catalysing thiol-disulphide interchange, and hence disulphide-bond isomerization in proteins (Freedman & Hawkins, 1977). Incubation of ‘scrambled‘ NO,-ribonuclease with partially purified protein disulphide isomerase (Hawkins & Freedman, 1976) in the standard conditions for assay of this enzyme (Ibbetson & Freedman, 1976) leads to the appearance of increased ribonuclease activity and an increase in A42s. The rate of increase in ribonuclease activity with ‘scrambled’ NO,-ribonuclease is 2-10 % that found with scrambled unmodified r i b nuclease. With ‘scrambled’ NO,-ribonuclease in the range O.l-O.Smg/ml, rates of increase in absorbance were 0.0035-0.0125 AA428 units/min per mg of enzyme. So nitrated ribonculease derivatives may provide useful probes for the kinetic characterization of disulphide-bond formation and isomerization processes, including those catalysed by protein disulphide isomerase.