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in phosphate (/ = 0.28, pH 7.8) have been interpreted quantitatively in terms of a monomer-dimer acceptor system, the association constant for the interaction of phenylpropiolate with the two binding sites on the dimeric form of a-chymotrypsin being approximately four-fifths of the corresponding value for the single site on monomeric enzyme. These results, pertaining to the inhibitor-binding properties of a-chymotrypsin in solution correlate well with published X-ray crystallographic data, which indicate that the approach of substrate analogs to the active sites of dimeric -chymotrypsin is subject to steric hindrance.

Rhodopsin (the photosensitive rod visual pigment) has been a model for photobiologic studies of the opsins as well as a structural model for G-protein-coupled receptors. The two palmitate groups attached to cysteines 322 and 323 are thought to serve as membrane anchors for the rhodopsin C-terminus, but the absence of the palmitates does not alter membrane localization. However, removal of the palmitates affects rhodopsin function. Therefore, it is important to quantitate the stability of rhodopsin palmitates to hydroxylamine, which is a widely utilized reagent in biochemical preparations of the apoprotein. We have developed a mass spectrometric method to quantitate the resulting opsin palmitylation. Our data show that both of the bovine rhodopsin palmitates are labile to hydroxylamine, with significant depalmitylation occurring at concentrations of >or=100 mM, with an EC(50) of 220 mM L(-1). The palmitate at position 322 is the more stable to hydroxylamine. Samples prepared in the presence of >50 mM should therefore be considered to be at least partially depalmitylated and the results interpreted accordingly.

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