SUMMARY.-An assay is described for measuring the concentration of specific, high-affinity oestradiol receptors in the cell supernatant fraction of breast tumour biopsies. The (Gorski et al, 1968;Jensen et al., 1968).The presence of oestrogen receptors has been demonstrated in hormonedependent tumours in experimental animals. notably in some dimethylbenzanthracene-induced mammary tumours in the rat and in oestrogen-induced kidney tumours in the hamster (King, Smith and Steggles, 1970;Mobbs, 1966; Sander and Attran-iadal, 1968

A quantitative method was used to determine the concentration of high-affinity oestradiol-receptor sites in rat uterine supernatant preparations under various physiological conditions. Cyclic changes in concentration were observed during the oestrous cycle, with a maximum occurring in late dioestrus. The changes followed a similar pattern in endometrium and myometrium, although concentrations were higher in the former. In pseudopregnancy the concentration was initially low, rising to a maximum on the tenth day. In early pregnancy a high concentration of receptor was found to be associated with the developing placenta, but this declined in later stages of pregnancy. After ovariectomy or combined ovariectomy and adrenalectomy the receptor concentration remained at a constant low value that could be increased by treatment with oestradiol. The receptor concentration was considerably higher in immature than in adult uteri.

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An assay method was developed for the determination of high-affinity oestradiol receptors in uterine supernatant preparations. When only high-affinity sites are present in such preparations, or when they predominate, the analysis of the equilibrium between oestradiol and receptor sites based on the Scatchard (1949) plot may be used to determine the dissociation constant of the equilibrium and the molar concentration of the high-affinity sites. When both high-affinity and low-affinity sites are present the Scatchard plot is no longer linear and cannot be used directly to determine high-affinity sites. Determination of the reverse velocity constants of the reaction between high-affinity (k(-1)) and low-affinity (k(-2)) receptor sites and [(3)H]oestradiol has shown that these constants differ by at least one order of magnitude. Advantage has been taken of this difference to introduce an additional step into the assay procedure that eliminates oestradiol bound to low-affinity sites and permits the determination of high-affinity sites in different species and under a variety of physiological conditions.

an active-site-directed alkylation of the activating enzymes, owing to their structural resemblance to the substrate. The synthesis was achieved by reaction of N-t-butyloxycarbonyl-amino acids with dicyclohexylcarbodi-imide to form the corresponding anhydrides, which on treatment with diazomethane gave the diazoketones. The latter compounds reacted with HC1 in ether to furnish the chloromethyl ketone hydrochlorides Cl-H3N(CHR)-CO-CH2Cl (R=amino acid side chain). The chloromethyl ketone analogue of leucine was found to inhibit the action of leucine-activating enzyme in a time-dependent fashion at a concentration of 10mM, and the activity was not recovered on dialysis, showing the irreversible nature of the inhibition. It was found that the chloromethyl ketone analogues of both DL-and D-alanine were also effective irreversible inhibitors, as were the chloromethyl ketone analogues of N-trifluoroacetylalanine and bromopyruvate, which lack the specificity requirement of a free amino group.The alkylation observed with these compounds and with classical thiol-group inhibitors (at or near the active site, suggests the presence of a nucleophile at the active centre. This appears to be sufficiently exposed for alkylation to occur without the prior formation of a Michaelis complex. This highlights the need for the synthesis of new types of inhibitors that can react to a significant extent only after the formation of a Michaelis complex.The chloromethyl ketone analogues of DL-leucine and alanine proved to be powerful reversible inhibitors of the activity of leucine aminopeptidase, both producing 70 % inhibition at a concentration of 17,uM. The chloromethyl ketone analogue of Dalanine showed no inhibition at concentrations up to 300Mm. This and other evidence will be presented to suggest that chloromethyl ketone analogues of L-alanine and leucine may inhibit the action of leucine aminopeptidase through a specific active-sitedirected property.