Abstract. Mutational studies were previously carried out at the co site in intact cells (Micanovic, R., L. Gerber, J. Berger, K. Kodukula, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA. 87:157-161; Micanovic R., K. Kodukula, L. Gerber, and S. Udenfriend. 1990. Proc. Natl. Acad. Sci. USA: 87:7939-7943) and at the co + 1 and co + 2 sites in a cell-free system (Gerber, L., K. Kodukula, and S. Udenfriend. 1992. J. Biol. Chem. 267:12168-12173) of nascent proteins destined to be processed to a glycosylphosphatidylinositol (GPI)-anchored form. We have now mutated the co + 1 and co + 2 sites in placental alkaline phosphatase (PLAP) cDNA and transfected the wild-type and mutant cDNAs into COS 7 cells. Only glycine at the co + 2 site yielded enzymatically active GPI membrane-anchored PLAP in amounts comparable to the wild type (alanine). Serine was less active and threonine and valine yielded very low but significant activity. By contrast the co + 1 site was promiscuous, with only prollne being inactive. These and the previous studies indicate that the co and ~0 + 2 sites of a nascent protein are key determinants for recognition by COOH-terminal signal transamidase. Comparisons have been made to specific requirements for substitution at the -1, -3 sites of amino terminal signal peptides for recognition by NH2-terminal signal peptidase and the mechanisms of NH2 and COOH-terminal signaling are compared.

Glycosylphosphatidylinositol (GPI) substitution is now recognized to be a ubiquitous method of anchoring a protein to membranes in eukaryotes. The structure of GPI and its biosynthetic pathways are known and the signals in a nascent protein for GPI addition have been elucidated. The enzyme(s) responsible for GPI addition with release of a COOH-terminal signal peptide has been considered to be a transamidase but has yet to be isolated, and evidence that it is a transamidase is indirect. The experiments reported here show that hydrazine and hydroxylamine, in the presence of rough microsomal membranes, catalyze the conversion of the pro form of the engineered protein miniplacental alkaline phosphatase (prominiPLAP) to mature forms from which the COOH-terminal signal peptide has been cleaved, apparently at the same site but without the addition of GPI. The products, presumable the hydrazide or hydroxamate of miniPLAP, have yet to be characterized definitively. However, our demonstration of enzyme-catalyzed cleavage of the signal peptide in the presence of the small nucleophiles, even in the absence of an energy source, is evidence of an activated carbonyl intermediate which is the hallmark of a transamidase.

Bovine adrenal chromaffin granules have been shown to contain, in addition to Met-enkephalin and Leu-enkephalin, at least three small peptides with opiate receptor activity. One of these adrenal peptides has been purified to homogeneity and its sequence was shown to be Met-enkephalin- [Arge, Phe7]. This heptapeptide was also found in beef striatal extracts in amounts comparable to those of Leu-enkephalin.The opioid pentapeptides Met-enkephalin and Leu-enkephalin were originally isolated from whole brain (1). When the Metenkephalin sequence was found at the NH2 terminus of the pituitary hormone (3-endorphin, it was assumed that Metenkephalin is derived from f3-endorphin. However, this relationship has never been established. Indeed, there have been studies that suggest a biosynthetic pathway for the brain enkephalins that does not include f3-endorphin as an intermediate (2)(3)(4).Recently, immunologic procedures have revealed the presence of opioid peptides in adrenal medulla, localized in chromaffin cells (5-9). We have not only corroborated these findings but also have found that the adrenal medulla contains more enkephalin-like material than does the brain. Furthermore, the medulla is also rich in several proteins and large peptides that yield opioid activity after treatment with trypsin (10). None of the proteins is related to f3-endorphin or its pituitary precursors.In the course of these studies we undertook the chemical characterization of those peptides in the medulla that are in the molecular weight range of the enkephalins and that show direct opioid activity. We found not only Met-and Leu-enkephalin, as expected, but also, at least three other small peptides with opioid activity. One of these unknown peptides has been purified to homogeneity and its sequence was shown to be Metenkephalin [Arg6, Phe7]. This peptide is found not only in the adrenal gland but also in the striatum in amounts comparable to those of Leu-enkephalin. Details of the isolation and characterization of this opioid heptapeptide are presented here. MATERIALS AND METHODSBovine adrenal glands were obtained from a local slaughterhouse and stored on ice until used (1-2 hr). The medullas were dissected out, and chromaffin granules were prepared by the procedure of Smith and Winkler (11). The isolated chromaffin granules were lysed in a solution (1:10, wt/vol) containing 1 M acetic acid, 20 mM HCI, 1 /Ag each of phenylmethylsulfonyl fluoride and pepstatin per ml, and 0.1% 2-mercaptoethanol. Membranes and cell debris were removed by centrifugation at 100,000 X g for 30 min, and proteins in the supernatant solution were precipitated by the addition of 50% (wt/vol) trichloroacetic acid to achieve a 10% final concentration. The precipitate was then removed by centrifugation at 26,000 X g for 15 min. After removal of trichloroacetic acid and lipids from the supernatant solution by ether extraction (three times with equal volumes), the sample was lyophilized. The residue was then dissolved in buffer and subjected to high-performance liq...

A carboxyl-terminal chymotryptic peptide from mature human placental alkaline phosphatase was purifled by HPLC and monitored by a specific RIA. Sequencing and amino acid assay showed that the carboxyl terminus of the peptide was aspartic acid, representing residue 484 of the proenzyme as deduced from the corresponding cDNA. Further analysis of the peptide showed it to be a peptidoglycan containing one residue of ethanolamine, one residue of glucosamine, and two residues of neutral hexose. The inositol glycan is apparently linked to the a carboxyl group of the aspartic acid through the ethanolamine. Location of the inositol glycan on Asp-484 of the proenzyme indicates that a 29-residue peptide is cleaved from the nascent protein during the posttranslational condensation with the phosphatidylinositolglycan.Membrane proteins vary greatly in the nature and extent of their interactions with the lipid bilayer. A number of diverse cell-surface proteins are anchored in plasma membranes by a phosphatidylinositol-glycan (PI-G) structure that is covalently attached to the carboxyl-terminal amino acid of the mature protein (1-3). Alkaline phosphatase [AP; nonspecific octophosphoric-monoester phosphohydrolase (alkaline optimum), EC 3.1.3.1] has been identified as a PI-G-tailed protein (4, 5). The mature enzyme, which is widely distributed in mammalian tissues, can be released from cellular membranes by phosphatidylinositol-specific phospholipase C (6, 7). Studies of AP, biosynthetically radiolabeled in cell culture with components of the putative PI-G moiety (8, 9), further support the initial classification of AP as a PI-Gtailed membrane protein. In higher primates and in man three isozymes of AP are present-namely, intestinal, placental, and the tissue-unspecific form present in liver, bone, kidney, and most other tissues (10,11). The AP isozymes are highly glycosylated homodimers that have subunit molecular masses ranging from 60 to 80 kDa. The cDNA sequences of all three major types of mammalian AP have been deduced (12-17), and they all indicate the presence of a stretch of -20 hydrophobic amino acid residues at the carboxyl terminus of the nascent protein. It is believed, by analogy to the two best understood PI-G-tailed proteins, variant surface glycoprotein of Trypanosoma brucei (18-22) and Thy-1 antigen (23)(24)(25)(26), that after synthesis on membrane-bound ribosomes the nascent form of AP is modified by (i) removal of an amino-terminal signal sequence, (it) addition of N-linked oligosaccharides, (ifi) replacement of a carboxyl-terminal hydrophobic peptide extension with a PI-G moiety that serves as a membrane anchor. The length of the carboxylterminal peptide that is removed from the nascent form of AP and the exact site of PI-G attachment, presumably via an amide bond between the amino group of ethanolamine and the carboxyl group of the carboxyl-terminal amino acid, are not known. To elucidate the post-translational modification of PLAP at its carboxyl terminus, we have purified the carboxyl-terminal chy...

A unique type of radioimmunoassay is described that does not require centrifugation or separation. Microbeads containing a fluorophor are covalently linked to antibody. When an 12SI-labeled antigen is added it binds to the beads and, by its proximity, the emitted short-range electrons of the 125I excite the fluorophor in the beads. The light emitted can be measured in a standard scintillation counter. Addition of unlabeled antigen from tissue extracts displaces the labeled ligand and diminishes the fluorescent signal. Application of scintillation proximity immunoassay to tissue enkephalins, serum thyroxin, and urinary morphine is described. Applications ofthe principle to study the kinetics of interaction between receptors and ligands are discussed.The term scintillation proximity assay was coined by Hart and Greenwald in 1979 (1) to describe a unique type of RIA. In their procedure they used two different types of polymer beads to which the same antigen (human serum albumin) had been linked covalently. One of the beads had a fluorophor immobilized in it, whereas the second bead was labeled with 3H. Since electrons emitted during 3H decay have a range of only a few micrometers in water, in dilute suspension few of the 3H beads would be close enough to excite the fluorophor beads so that only a low level of fluorescence would be observed. On addition of specific antibody to the suspended beads, agglutination occurs, bringing many of the 3H beads within sufficient proximity of the fluorophor beads, so as to excite them. The emitted fluorescence can be measured in a scintillation counter. The procedure described by Hart and Greenwald (1) MATERIALS AND METHODSThe fluorophor-containing polyvinyltoluene beads (NE-102A), 1-10 pum in diameter, were purchased from Nuclear Enterprises (Edinburgh, Scotland). The methyl groups on the beads were oxidized to carboxyl groups with potassium permanganate by the following procedure. Seven grams of beads was suspended in 70 ml ofa 3% solution ofTriton X-100 (Packard), shaken, and centrifuged. The bead pellet was washed six times by repeatedly suspending in 70 ml of water, shaking, and centrifuging. The washed beads were resuspended in 36 ml of water and stored in the refrigerator overnight to permit settling. The fine particles were removed the next morning by aspirating off the supernatant fluid and the beads were resuspended in water to a final volume of 70 ml.A 7-ml aliquot of the bead suspension (ca. 700 mg) was transferred to a 100-ml round-bottom flask to which 22 ml of water and 22 ml of a freshly prepared solution of 0.1 M KMnO4 were added. After layering the flask with argon, it was placed in a 60°C bath and incubated for 10 hr with stirring. The flask was then removed from the bath and incubated at room temperature for an additional 12 hr. After oxidation, 5 ml of a sodium bisulfite solution (40 g/100 ml) was added with stirring, followed by addition of 30 ml of 1 M HCl. The suspension was then transferred to a centrifuge tube and after centrifugation the sup...