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gamma-Secretase is a membrane-bound protease that cleaves within the transmembrane region of amyloid precursor protein to generate the C-termini of the Abeta peptides which are believed to play a central role in the neuropathology of Alzheimer's disease. An in vitro gamma-secretase assay using a recombinant substrate C100Flag has been developed to facilitate the characterization and identification of this enigmatic protease. Biochemical studies establish that gamma-secretase activity is catalyzed by a PS1-containing macromolecular complex. Moreover, the fact that the photoreactive active gamma-secretase inhibitor directed to the active site labels PS1 suggests that PS1 contains the active site of the protease. Presenilin/gamma-secretase as a potential target for AD therapy and its role in regulated intramembrane proteolysis are discussed.

Anticoagulant therapy has changed dramatically during the past two years. Low molecular weight heparin has substantially replaced unfractionated heparin as the parenteral anticoagulant of choice. The use of warfarin has substantially increased, especially for prevention of stroke in the setting of atrial fibrillation. It has become clear that warfarin cannot be administered effectively in an unmonitored fixed-dose fashion. The parenteral direct thrombin inhibitor desirudin was shown to be efficacious in the prevention of deep vein thrombosis in man. Small thrombin and factor Xa inhibitors with in vivo oral anticoagulant activity have been identified.

Crystals of human alpha-thrombin complexed with hirugen and the alpha-keto acid thrombin inhibitor APPA (p-amidinophenylpyruvate) that diffract to 1.6 A resolution were obtained by soaking an alpha-thrombin-hirugen crystal in a solution of APPA. The crystal structure was determined using the difference Fourier method and refined to an R factor of 18.7% at 1.6 A resolution. This structure is the highest resolution structure of the thrombin molecule that is currently available. With the exception of the region near Arg77A-Asn78, the structures of the thrombin and hirugen molecules in the ternary complex are similar to those reported for the thrombin-hirugen binary complex. As previously determined for the APPA-trypsin complex, the carbonyl carbon atom of APPA forms a covalent bond with O gamma of Ser195 of thrombin to yield a "transition-state" analog of the tetrahedral intermediate. Comparison of the specificity pocket of the APPA complexes of thrombin and trypsin reveals differences in hydrogen bonding and shows for the first time that the S1 site of thrombin is larger than that of trypsin and as a result thrombin may be able to accommodate a bulkier P1 group than trypsin.

Site-directed mutagenesis was employed to assess the importance of the Arg-Gly-Asp triplet that comprises residues 197 to 199 in the B-chain of thrombin. Properties of the R197E and the D199E variants were compared with those of zeta-thrombin and the inactive S205A variant wherein the active site Ser is replaced by Ala. Relative to zeta-thrombin, the R197E thrombin variant under the assay conditions used exhibits 26% activity toward a small chromogenic substrate, 13% activity in the activation of protein C in the presence of thrombomodulin, < 3% activity in processing fibrinogen, and 1% activity in inducing platelet activation. Thus, the substrate specificity of thrombin was altered by the R197-->E replacement. The D199E variant was essentially inactive. It exhibited only 0.02% of the activity of thrombin toward the chromogenic substrate and its reactivity toward the active site-directed alkylating agent D-Phe-Pro-Arg-CH2Cl was 10,000-fold lower than that of thrombin. Like the inactive S205A thrombin variant, the D199E variant antagonized the interactions of thrombin with hirudin and thrombomodulin, but was a less effective antagonist. The dependence of the antagonism of the thrombin-thrombomodulin interaction on the concentration of D199E thrombin variant provided evidence suggesting the presence of two or more domains in thrombin that independently interact with their counterparts in thrombomodulin. Although the S205A thrombin variant antagonized the action of thrombin on platelets no such activity could be demonstrated for the D199E variant in the concentration range studied (< 800 nm). Comparison of the circular dichroism spectra of zeta-thrombin, the D199E, R197E, and S205A variants indicated that subtle differences in conformation exist between the D199E variant and the other thrombins. These differences in conformation might well account for the altered behavior of the D199E variant with respect to its interactions toward thrombomodulin, hirudin, and platelets.

31P NMR studies were undertaken to determine how potassium ion increases the cofactor affinity of Escherichia coli D-serine dehydratase, a model pyridoxal 5'-phosphate requiring enzyme that converts the growth inhibitor D-serine to pyruvate and ammonia. Potassium ion was shown to promote the appearance of a second upfield shifted cofactor 31P resonance at 4.0 ppm (pH 7.8, 25 degrees C), that increased in area at the expense of the resonance at 4.4 ppm observed in the absence of K+. Na+ antagonized the K+ promoted appearance of the second resonance. These observations suggest that K+ and Na+ stabilize conformational states that differ with respect to O-P-O bond angle, conformation, and/or hydrogen bonding of the phosphate group. An analysis of the dependence of the relative intensities of the two resonances on the K+ concentration yielded a value of ca. 10 mM for the equilibrium constant for dissociation of K+ from D-serine dehydratase. The chemical shift difference between the two resonances indicated that the K+-stabilized and Na+-stabilized forms of the enzyme interconvert at a frequency less than 16 s-1 at pH 7.8, 25 degrees C.

The interaction of the stinging nettle rhizome lectin (UDA) with carbohydrates was studied by using the techniques of quantitative precipitation, hapten inhibition, equilibrium dialysis, and uv difference spectroscopy. The Carbohydrate binding site of UDA was determined to be complementary to an N,N',N"-triacetylchitotriose unit and proposed to consist of three subsites, each of which has a slightly different binding specificity. UDA also has a hydrophobic interacting region adjacent to the carbohydrate binding site. Equilibrium dialysis and uv difference spectroscopy revealed that UDA has two carbohydrate binding sites per molecule consisting of a single polypeptide chain. These binding sites either have intrinsically different affinities for ligand molecules, or they may display negative cooperativity toward ligand binding.

The catalytically active, tyrosyl-phosphorylated form of insulin receptor kinase was isolated from human placenta by a procedure which exploits the propensity for the intact alpha 2 beta 2 form of insulin receptor to undergo insulin-promoted autophosphorylation at tyrosyl residues and concomitant activation as a tyrosyl kinase. Purification of tyrosyl-phosphorylated insulin receptor was effected by adsorption on and elution (with a hapten) from a column of O-phosphotyrosyl-binding antibody immobilized on protein A-Sepharose (Ab-protein A). The starting material for the purification process was protein which had been solubilized from placental membranes and purified by chromatography on immobilized wheat germ agglutinin. After chromatography on Ab-protein A to remove preexisting O-phosphotyrosyl-containing proteins, the fraction which did not adsorb to the Ab-protein A column was incubated with insulin and briefly treated with ATP so as to maximize selective autophosphorylation of insulin receptor. This material was then subjected to chromatography on Ab-protein A. Although the amount of the intact alpha 2 beta 2 form of insulin receptor present in the starting material was only a small fraction of the protein (approximately 0.2%) and only approximately 20% of the insulin-binding forms of the receptor present, it was eluted (with 10 mM p-nitrophenyl phosphate) from the column in greater than or equal to 80% purity. Chromatography on Ab-protein A appears to have an advantage over the alternative affinity chromatographic procedures which utilize immobilized insulin or antiinsulin receptor antibody to adsorb insulin receptor, since these procedures do not resolve the intact alpha 2 beta 2 form of insulin receptor from the nicked insulin-binding forms of the receptor which do not undergo insulin promoted autophosphorylation.

Insulin receptor partially purified from human placenta by chromatography on immobilized wheat germ agglutinin was subjected to affinity cross linking to determine the relationship between the subunit structure of the multiple forms of the insulin receptor and their competence to bind insulin and undergo autophosphorylation. It was demonstrated that, whereas the 340-kDa intact receptor undergoes autophosphorylation, the 290- and 320-kDa insulin binding forms of the receptor do not. Phosphorylation at tyrosyl residues in the intact receptor was verified using a new facile method for determination of phosphorylated amino acids. The competence of the phosphorylated 340-kDa protein to bind insulin was demonstrated using a double-probe labeling protocol wherein receptor phosphorylated with [gamma-32P]ATP was cross-linked with disuccinimidyl suberate (DSS) in the presence of N epsilon B29-biotinylinsulin. The observation that succinylavidin, by virtue of its interaction with biotinyl residues, decreased the electrophoretic mobility of receptor radiochemically labeled with 32P indicated that the phosphorylated 340-kDa protein was competent to bind insulin. This result is compelling evidence that the 340-kDa phosphorylated species is insulin receptor itself, rather than a closely associated contaminant. Treatment of the receptor with the crosslinking agent DSS produced (after reduction and denaturation) alpha-dimer, beta-dimer, and a smaller amount of tetramer. This observation is consistent with a symmetrical, tetrameric, alpha 2 beta 2 structure for insulin receptor from human placenta, and excludes previously proposed alternative structures containing one alpha and one beta chain.

The present work discloses a procedure for preparation of human liver plasma membranes containing catalytically competent insulin receptor kinase. In addition to insulin promoted phosphorylation of the beta-subunit of insulin receptor kinase, insulin promoted phosphorylation of pp 120 and two other new proteins was demonstrated. The new proteins with molecular weights of 50,000 and 120,000 do not bind to WGA, pp 120 antibody or insulin receptor antibody, but bind to the antiphosphotyrosyl antibody. The identity and physiological significance of these putative substrates for insulin receptor kinase remains to be established.