Rank-order of potencies for inhibition of the secretion of abeta40 and abeta42 suggests that both are generated by a single gamma-secretase.

The Alzheimer's disease amyloid peptide Abeta has a heterogeneous COOH terminus, as variants 40 and 42 residues long are found in neuritic plaques and are secreted constitutively by cultured cells. The proteolytic activity that liberates the Abeta COOH terminus from the beta-amyloid precursor protein is called gamma-secretase. It could be one protease with dual specificity or two distinct enzymes. By using enzyme-linked immunosorbent assays selective for Abeta40 or Abeta42, we have measured Abeta secretion by a HeLa cell line, and we have examined the dose responses for a panel of five structurally diverse gamma-secretase inhibitors. The inhibitors lowered Abeta and p3 secretion and increased levels of the COOH-terminal 99-residue beta-amyloid precursor protein derivative that is the precursor for Abeta but did not alter secretion of beta-amyloid precursor protein derivatives generated by other secretases, indicating that the inhibitors blocked the gamma-secretase processing step. The dose-dependent inhibition of Abeta42 was unusual, as the compounds elevated Abeta42 secretion at sub-inhibitory doses and then inhibited secretion at higher doses. A compound was identified that elevated Abeta42 secretion at a low concentration without inhibiting Abeta42 or Abeta40 at high concentrations, demonstrating that these phenomena are separable pharmacologically. Using either of two methods, IC50 values for inhibition of Abeta42 and Abeta40 were found to have the same rank-order and fall on a trend line with near-unit slope. These results favor the hypothesis that Abeta variants ending at residue 40 or 42 are generated by a single gamma-secretase.

Purification and characterization of PAM-1, an integral membrane protein involved in peptide processing.

Peptidylglycine alpha-amidating monooxygenase (PAM) catalyzes the two-step alpha-amidation of peptidylglycine intermediates. PAM-1, a Type I integral membrane protein, was solubilized from the membranes of stably transfected hEK-293 cells and purified to homogeneity by antibody affinity chromatography. Purified PAM-1 exhibits an acidic pH optimum and a lower maximal velocity than soluble bifunctional PAM. Limited tryptic digestion of this integral membrane protein releases monofunctional peptidylglycine alpha-hydroxylating monooxygenase, increasing its specific activity almost fourfold and shifting its pH optimum to coincide with the pH optimum of peptidyl-alpha-hydroxyglycine alpha-amidating lyase.

Peptide alpha-amidation and peptidylglycine alpha-hydroxylating monooxygenase: control by disulfiram.

The final two steps in the biosynthesis of alpha-amidated bioactive peptides are catalyzed by peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5). These enzymes are derived from the bifunctional precursor protein, peptidylglycine alpha-amidating monooxygenase. Because PHM is rate-limiting in peptide amidation and is copper-dependent, we examined the consequences of in vivo treatments with the copper-chelating drug disulfiram (Antabuse) on levels of alpha-amidated peptides and expression of PHM and PAL. Decreases in two amidated peptides (alpha-melanotropin and cholecystokinin) after disulfiram treatment were extremely pronounced outside the blood-brain barrier, with moderate decreases in the central nervous system. Unexpectedly, when assayed under optimal conditions in vitro, PHM activity was increased by disulfiram treatment, whereas PAL activity was unaltered. The increase in PHM activity in pituitary and atrium occurred within a few hours after the start of disulfiram treatment and was sustained up to 2 weeks after the cessation of treatment, whereas levels of alpha-amidated peptides remained low. Northern and Western blot analyses demonstrated that disulfiram had no influence on levels of peptidylglycine alpha-amidating monooxygenase mRNA or protein. Thus, inhibition of alpha-amidation by disulfiram in vivo occurs despite an increased Vmax of PHM assayed in vitro. The increase in PHM activity may result from induction of a physiologic mechanism that normally regulates this rate-limiting enzyme.

Use of endoproteases to identify catalytic domains, linker regions, and functional interactions in soluble peptidylglycine alpha-amidating monooxygenase.

The production of alpha-amidated peptides is accomplished through the sequential action of two enzymes, peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL), that are contained within the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) protein. Tissue-specific alternative splicing and endoproteolysis are known to generate both soluble and integral membrane mono- and bifunctional PAM proteins. In order to investigate the functional consequences of these differences we purified PAM-3, a soluble 95-kDa bifunctional form of the enzyme, from the spent medium of stably transfected hEK-293 cells. Using NH2-terminal sequence analysis of products of limited endoproteolysis and antibody cross-reactivity we identified protease-sensitive regions at the NH2 terminus, between the 35-kDa PHM and 42-kDa PAL domains and at the COOH terminus of the protein. Endoproteolytic removal of the COOH-terminal region from the bifunctional PAM-3 protein shifted the pH optimum of PHM to a more alkaline pH, increased the turnover number (kappa(cat)) of PHM and decreased its KM for alpha-N-acetyl-Tyr-Val-Gly; the catalytic properties of PAL were not altered. Since peptide amidation can be a rate-limiting step in the biosynthesis of neuropeptides, similar increases in PHM activity in vivo may play an important role in regulating the extent of peptide alpha-amidation.

Peptidylglycine alpha-amidating monooxygenase and other processing enzymes in the neurointermediate pituitary.

Studies on the mRNAs encoding PAM and on the various PAM proteins have begun to reveal some of the intricate mechanisms used to optimize the ability of this enzyme to carry out the alpha-amidation of peptides. Comparison of the regulatory elements governing expression of the various enzymes involved in peptide processing should reveal common elements. Knowledge of the processing enzymes themselves should help us to understand how these enzymes function in the secretory granule environment. In addition to their catalytic domains, other processing enzymes, like PAM, may well have processing domains and routing domains designed to optimize their ability to function in secretory granules.

Peptidylglycine alpha-amidating monooxygenase: a multifunctional protein with catalytic, processing, and routing domains.

Peptide alpha-amidation is a widespread, often essential posttranslational modification shared by many bioactive peptides and accomplished by the products of a single gene encoding a multifunctional protein, peptidylglycine alpha-amidating monooxygenase (PAM). PAM has two catalytic domains that work sequentially to produce the final alpha-amidated product peptide. Tissue-specific alternative splicing can generate forms of PAM retaining or lacking a domain required for the posttranslational separation of the two catalytic activities by endoproteases found in neuroendocrine tissue. Tissue-specific alternative splicing also governs the presence of a transmembrane domain and generation of integral membrane or soluble forms of PAM. The COOH-terminal domain of the integral membrane PAM proteins contains routing information essential for the retrieval of PAM from the surface of endocrine and nonendocrine cells. Tissue-specific endoproteolytic processing can generate soluble PAM proteins from integral membrane precursors. Soluble PAM proteins are rapidly secreted from stably transfected nonneuroendocrine cells but are stored in the regulated secretory granules characteristic of neurons and endocrine cells.

The membrane-bound bifunctional peptidylglycine alpha-amidating monooxygenase protein. Exploration of its domain structure through limited proteolysis.

The biosynthesis of alpha-amidated peptides from their glycine-extended precursors is catalyzed by the sequential action of peptidylglycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The two enzymes are part of a bifunctional, integral membrane protein precursor, peptidylglycine alpha-amidating monooxygenase (PAM). The major forms of PAM mRNA in the adult rat atrium differ by the presence or absence of optional exon A, a 315-nucleotide segment separating the PHM and PAL domains. Using antipeptide antibodies specific to the PHM, exon A, PAL, and cytoplasmic domains of rat PAM, carbonate-washed atrial membranes were found to contain proteins corresponding to rPAM-1 and rPAM-2. Digestion of atrial membranes with a variety of endoproteinases released PHM and PAL catalytic activities. Dose-response curves indicated that both catalytic activities were extremely resistant to inactivation by trypsin. Endoproteolytic digestion of atrial membranes with trypsin, chymotrypsin, elastase, thermolysin, or endoproteinase Lys-C generated a 35-kDa PHM fragment. Digestion with trypsin, elastase, thermolysin, or endoproteinase Lys-C generated a 42-kDa PAL fragment. In contrast to the stability exhibited by the PHM and PAL domains, the cytoplasmic domain of PAM was destroyed by most of the enzymes; only digestion with endoproteinase Lys-C generated a stable fragment. Digestion with endoproteinase Arg-C removed the carboxyl-terminal tail from PAM but failed to release the PHM or PAL domains from the membranes. The PHM fragments generated by some of the endoproteinases showed a tendency to adhere to the membranes. Thus the bifunctional PAM protein consists of independent catalytic domains separated from each other and from the putative transmembrane domain by flexible regions accessible to attack by a wide variety of endoproteinases.

Peptidyl-alpha-hydroxyglycine alpha-amidating lyase. Purification, characterization, and expression.

The production of alpha-amidated peptides from their glycine-extended precursors is a two-step process involving the sequential action of two catalytic domains encoded by the bifunctional peptidylglycine alpha-amidating monooxygenase (PAM) precursor. The NH2-terminal third of the PAM precursor contains the first enzyme, peptidylglycine alpha-hydroxylating monooxygenase (PHM), a copper, molecular oxygen, and ascorbate-dependent enzyme. The middle third of the PAM precursor contains the second enzyme, peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). The COOH-terminal third of the PAM precursor encodes a transmembrane domain and a hydrophilic domain that may form a cytoplasmic tail. Antisera to a peptide within the PAL domain were used to identify a 50-kDa protein as the major form of PAL in bovine neurointermediate pituitary granules. This 50-kDa PAL protein was purified and found to begin at Asp434 of bPAM, indicating that it could arise through endoproteolytic cleavage of the bPAM precursor at Lys432-Lys433. With alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine as the substrate, PAL exhibits a pH optimum of 5.0; enzymatic activity is inhibited by high concentrations of salt but is relatively resistant to thiol reagents and urea. PAL activity is inhibited by EDTA and restored by a number of divalent metals, including Cd2+, Cu2+, Zn2+, and Ca2+. Kinetic studies using alpha-N-acetyl-Tyr-Val-alpha-hydroxyglycine indicate that PAL has a Km of 38 microM and a turnover number of 220/s. Expression vectors encoding only the soluble PHM domain or the PAM precursor from which the PHM domain had been deleted were constructed. hEK293 cells transfected with the PHM vector exhibited a 10-fold increase in secretion of PHM activity with no PHM activity detectable in control or transfected cells. hEK293 cells transfected with the PAL vector exhibited a 2-fold increase in secretion of PAL activity and a 15-fold increase in cellular PAL activity. Most of the PAL activity produced by the transfected cells remained membrane-associated.

pH-dependent stimulation of peptidylglycine alpha-amidating monooxygenase activity by a granule-associated factor.

Peptidylglycine alpha-amidating monooxygenase (PAM; EC 1.14.17.3) is a granule-associated enzyme that catalyzes the production of alpha-amidated peptides from their glycine-extended precursors, a posttranslational modification often required for full biological activity. PAM activity in crude homogenates of bovine neurointermediate pituitary has an acidic pH optimum for the peptide substrate alpha-N-Ac-Tyr-Val-Gly. During purification, the pH optimum shifts, so that purified bovine (b)PAM exhibits an alkaline pH optimum for this substrate with virtually no activity below pH 6.5. A factor that restores the ability of purified bPAM to produce alpha-amidated products at pH 6 was identified. In rat anterior pituitary this factor (denoted SPAM for stimulator of PAM activity) was a soluble protein with a mol wt of 44 K by gel filtration; its stimulatory activity could be reduced or eliminated by trypsin digestion or boiling. SPAM stimulated PAM activity at acidic pH by increasing the apparent Vmax and decreasing the apparent Michaelis-Menten constant (Km) for the peptide substrate. Like PAM, SPAM activity is localized to the secretory granule. Levels of SPAM activity in various rat tissues correlated closely with levels of PAM activity, with the greatest amount of SPAM activity in atrium, anterior pituitary, and neurointermediate pituitary. The distribution of PAM and SPAM between soluble and membrane fractions also correlated closely. In AtT-20 cell lines transfected with a complementary DNA (cDNA) encoding the full-length bPAM precursor, both SPAM and PAM activities were increased compared to wild type cells; both activities were decreased in a cell line expressing an antisense rat (r)PAM mRNA. In marked contrast, an AtT-20 cell line transfected with a cDNA encoding a truncated, soluble form of bPAM had elevated levels of PAM activity, but levels of SPAM activity were not increased compared to wild-type cells. These results suggest that SPAM activity is closely linked to the expression of full-length PAM. The interaction of PAM and SPAM may represent a site for regulation of the synthesis of bioactive peptides, particularly at low intragranular pH.

The 108-kDA peptidylglycine alpha-amidating monooxygenase precursor contains two separable enzymatic activities involved in peptide amidation.

A 43-kDa protein factor that increases the ability of purified bovine peptidylglycine alpha-amidating monooxygenase (PAM)-A and -B to produce alpha-amidated peptides at physiological pH was purified to homogeneity from bovine neurointermediate pituitary. At each step of the purification, the amount of activity correlated with the amount of protein detected on Western blots by antibody to bovine PAM(561-579). In the bovine neurointermediate pituitary the 108-kDa PAM precursor protein is cleaved to form a peptidylglycine alpha-hydroxylating monooxygenase and a peptidyl-alpha-hydroxyglycine alpha-amidating lyase, which function sequentially in the 2-step formation of alpha-amidated peptides.

The active site of membrane-bound meizothrombin. A fluorescence determination of its distance from the phospholipid surface and its conformational sensitivity to calcium and factor Va.

The distance between the phospholipid surface and the active site of membrane-bound meizothrombin, a derivative of prothrombin, was determined directly using fluorescence energy transfer. The active site of prothrombin was exposed after a single cleavage by Echis carinatus protease in the presence of [5-(dimethylamino)-1-naphthalenesulfonyl]glutamylglycylarginyl+ ++ (DEGR) chloromethyl ketone to yield DEGR-meizothrombin and thereby minimize secondary proteolysis. When DEGR-meizothrombin was titrated with 80% phosphatidylcholine, 20% phosphatidylserine vesicles containing octadecylrhodamine, singlet-singlet energy transfer was observed between the donor dyes in the active sites of the membrane-bound proteins and the acceptor dyes at the outer surface of the phospholipid bilayer. This energy transfer required both Ca2+ and phosphatidylserine. Assuming k2 = 2/3, the dependence of the efficiency of energy transfer upon the acceptor density showed that the distance of closest approach between the active site probe and the bilayer surface was 71 +/- 2 A. In the presence of factor Va, the distance was 67 +/- 3 A. These direct measurements show that the active site of meizothrombin is located far above the membrane surface. Also, association of factor Va with meizothrombin on the phospholipid surface appears to cause a slight movement of the meizothrombin protease domain toward the membrane surface. The environment of the dansyl dye covalently attached to the active site of meizothrombin was particularly sensitive to the presence of calcium: addition of Ca2+ ions to metal-free DEGR-meizothrombin reduced the dansyl fluorescence lifetime from 11.7 to 9.0 ns and the dansyl emission intensity by 24%. Hence, the conformation of the active site changed when Ca2+ ions bound to meizothrombin. Since the intensity change was half-maximal at 0.2 mM and was also elicited by the binding of Mg2+ ions, this spectral change correlates with the calcium-dependent conformational change previously observed in fragment 1. We conclude, therefore, that the binding of Ca2+ ions to meizothrombin and, by extension, perhaps to prothrombin, elicits a conformational change that extends beyond the fragment 1 domains into the distant (cf. above) active site or protease domain. The association of factor Va with membrane-bound DEGR-meizothrombin increased both the dansyl emission intensity (by 7%) and polarization. This intensity change and the factor-Va dependent change in energy transfer indicate that the cofactor of the prothrombinase complex functions to modulate the conformation and orientation of both the substrate and the enzyme of the complex.

The active site of blood coagulation factor Xa. Its distance from the phospholipid surface and its conformational sensitivity to components of the prothrombinase complex.

The location of the active site of membrane-bound factor Xa relative to the phospholipid surface was determined both in the presence and absence of factor Va using fluorescence energy transfer. Factor Xa was reacted with 5-(dimethylamino)-1-naphthalenesulfonyl- glutamylglycylarginyl(DEGR) chloromethyl ketone to yield DEGR-Xa, an analogue of factor Xa with a fluorescent dye attached covalently to the active site. When DEGR-Xa was titrated with phosphatidylcholine/phosphatidylserine vesicles containing octadecylrhodamine, fluorescence energy transfer was observed between the donor dyes in the active sites of the membrane-bound enzymes and the acceptor dyes at the outer surface of the phospholipid bilayer. Based on the dependence of the efficiency of singlet-singlet energy transfer upon the acceptor density and assuming kappa 2 = 2/3, the distance of closest approach between the active site probe and the surface of the phospholipid bilayer averaged 61 A in the absence of factor Va and 69 A in the presence of factor Va. These direct measurements show that the active site of factor Xa is located far above the membrane surface. Also, association of factor Xa with factor Va on the membrane surface to form the prothrombinase complex results in a substantial movement of the active site of the enzyme relative to the membrane surface. The 5-(dimethylamino)-1-naphthalenesulfonyl emission in the complete prothrombinase complex was distinct from that in any other combination of components. It therefore appears that the optimum conformation of the prothrombinase active site is achieved only when factor Va, Ca2+, and a membrane surface interact simultaneously with factor Xa. Thus, in addition to its previously demonstrated ability to stimulate factor Xa binding to membranes, factor Va, upon association with factor Xa on a phospholipid surface, allosterically induces a particular active site conformation in factor Xa and also positions the active site at the correct distance above the membrane for prothrombin activation.

A domain of membrane-bound blood coagulation factor Va is located far from the phospholipid surface. A fluorescence energy transfer measurement.

The larger subunit of blood coagulation factor Va was covalently labeled with iodoacetamido derivatives of fluorescein and rhodamine without loss of functional activity, as measured by either the one-stage clotting assay or the ability to accelerate prothrombin activation in a purified system. The spectral properties of the dyes were not altered by the presence or absence of the smaller subunit of factor Va, Ca2+, prothrombin, factor Xa, or phosphatidylcholine/phosphatidylserine (PC/PS, 4:1) vesicles. When fluorescein-labeled protein (factor VaF) was titrated with PC/PS vesicles containing either octadecylrhodamine or 5-(N-hexadecanoylamino)eosin, fluorescence energy transfer was observed between the protein-bound donor dyes and the acceptor dyes at the outer surface of the phospholipid bilayer. The extent of energy transfer correlated directly with the extent of protein binding to the vesicles monitored by light scattering. The distance of closest approach between the fluorescein on factor Va and the bilayer surface averaged 90 A for the two different acceptors. Association of factor VaF with factor Xa on the phospholipid surface reduced this separation by 7 A, but association with prothrombin did not alter the distance between the labeled domain on factor VaF and the surface. The efficiency of diffusion-enhanced energy transfer between rhodamine-labeled factor Va and terbium dipicolinate entrapped inside PC/PS vesicles was less than 0.01, consistent with the location of the dye far above the inner surface of the vesicle. Thus, a domain of membrane-bound factor Va is located a minimum of 90 A above the phospholipid surface.(ABSTRACT TRUNCATED AT 250 WORDS)