Carboxypeptidase Y Research Articles

15] Deblocking in peptide synthesis with immobilized carboxypeptidase Y

This chapter describes carboxypeptidase Y (CPY) that involves the concept of a method for the synthesis of peptides in aqueous systems using very mild conditions for deblocking. Small protected peptides can be deblocked in alcohol/water mixtures. However, as the size of the peptide increases, the solubility decreases. To use the immobilized CPY in the presence of high levels of organic solvent is not possible. CPY is an exopeptidase found in yeast. The enzyme is unusual, because it has a broad specificity. It catalyzes the release of L-amino acids, including proline, from the C terminus of a polypeptide chain. Rates of amino acid release vary but not to the extent exhibited by other exopeptidases such as pancreatic enzymes. CPY also acts as an esterase. It rapidly cleaves the C-terminal alkyl ester group of large and small peptides. In conclusion, immobilized CPY is a versatile tool for the protein chemistry lab. It can be used for deblocking in peptide synthesis, the resolution of racemic mixtures of blocked amino acid esters or peptides, the sequencing of peptides, the total hydrolysis of peptides for analysis, and condensation reactions in peptide synthesis. The attachment of C-terminal amino acid amides may be accomplished using CPY to catalyze the peptide bond formation starting with the peptide ester and the amide. Also, the enzyme should be of interest in peptide modification and fragment condensation of peptides.

Gene dosage-dependent secretion of yeast vacuolar carboxypeptidase Y.

The structural gene for yeast vacuolar carboxypeptidase Y (PRC1) has been cloned by complementation of the prc1-1 mutation. As much as an eightfold elevation in the level of carboxypeptidase Y (CPY) results when a multiple-copy plasmid containing the PRC1 gene is introduced into yeast. Unlike the situation with a single copy of PRC1 in which newly synthesized CPY is efficiently localized to the vacuole, plasmid-directed overproduction results in secretion of greater than 50% of the protein as the precursor form. Secretion is blocked in a mutant that is defective at a late stage in the transport of periplasmic proteins. Unlike normal cell surface glycoproteins, secreted CPY precursor acquires no additional oligosaccharide modifications beyond those that accompany normal transport to the vacuole. In the periplasm, the CPY precursor is proteolytically activated to an enzymatically active form by an enzyme that is unrelated to the vacuolar processing enzyme. These findings suggest that proper sorting and transport of CPY is saturable. This may reflect limiting amounts of a CPY-sorting receptor, or of CPY-modifying machinery that is essential for recognition by such a receptor.

The interaction of platelet factor four and glycosaminoglycans

The interaction of platelet factor four (PF-4) with glycosaminoglycans (GAG) was evaluated using fluorescence spectroscopy, a radioligand binding assay, and a functional assay utilizing antithrombin III and factor X a. In these studies, we have (i) characterized the binding parameters for PF-4 to several forms of heparin and to dextran sulfate; (ii) examined the structural features of these glycosaminoglycans which support PF-4 binding; and (iii) examined the effects of selective digestion of the carboxy terminus of PF-4 on binding. The binding of PF-4 to unfractionated porcine intestinal mucosal heparin (< M r〉) = 11,000) was specific and saturable, with a molar stoichiometry of PF-4 to heparin of approximately 4:1 and an apparent estimated K d of 3 × 10 −8 m. Heparin fractions (< M r〉) = 6,000) with either low or high affinity for antithrombin III bound to PF-4 with a similar apparent K d . PF-4 also bound to dextran sulfate (< M r〉 = 22,500) with an estimated apparent K d of 6 × 10 −8 m and a molar stoichiometry of approximately 16:1. Carboxypeptidase Y (CP-Y) digestion of PF-4 progressively decreased GAG binding. After 30 min of digestion, by which time all of the carboxyterminal serine and glutamate, both of the two leucines, and approximately one-quarter of the four lysines were removed, the IC 50 for heparin binding shifted from 10 to 150 n m. These studies demonstrate the effect of GAG polymer size and degree of sulfation on the affinity and stoichiometry of PF-4 binding, and the critical importance of the carboxy-terminal amino acids of PF-4 for binding to natural and synthetic GAGS.

Isolation and partial characterization of five myotropic peptides present in head extracts of the cockroach Leucophaea maderae

1. 1. Five peptides were isolated by reverse-phase HPLC from head extracts of the cockroach Leurophaea muderae. 2. 2. Four of the peptides were inactivated by aminopeptidase M (APM). The inability of APM to digest the fifth peptide suggests a blocked NH 2-terminus. 3. 3. Four of the peptides were inactivated by carboxypeptidase Y (CPY). The activity of the fraction which would have contained proctolin was decreased by about 20%. The complete deactivation of proctolin by CPY indicated that a second peptide, co-eluting with proctolin but refractory to CPY digestion, was responsible for 80% of the biological activity in that fraction. 4. 4. Concentrations of the peptides necessary to produce a threshold response from the isolated cockroach hindgut ranged from 0.009 to 0.083 head equivalents/ml.

Protease-catalyzed synthesis of melanocyte-stimulating hormone (MSH) fragments

In the present study on enzymatic peptide bond formation the proteosynthetic potential of several proteases was explored. Trypsin, α-chymotrypsin, papain, carboxypeptidase Y (CPD-Y), and thermolysin served as catalysts for the protease-controlled synthesis of some fragments of melanocyte-stimulating hormones. To obviate possible proteolytic cleavage of preexisting peptide bonds—a drawback often encountered during enzymatic peptide syntheses—several expedients leading to the target peptides were developed. The enzymatic procedure enabled under mild conditions the preparation of the desired peptides whose amino acid composition may give rise to severe complications during conventional syntheses.

Early stages in the yeast secretory pathway are required for transport of carboxypeptidase Y to the vacuole.

Temperature-sensitive secretory mutants (sec) of S. cerevisiae have been used to evaluate the organelles and cellular functions involved in transport of the vacuolar glycoprotein, carboxypeptidase Y (CPY). Others have shown that CPY (61 kd) is synthesized as an inactive proenzyme (69 kd) that is matured by cleavage of an 8 kd amino-terminal propeptide. sec mutants that are blocked in either of two early stages in the secretory process and accumulate endoplasmic reticulum or Golgi bodies also accumulate precursor forms of CPY when cells are incubated at the nonpermissive temperature (37 degrees C). These forms are converted to a proper size when cells are returned to a permissive temperature (25 degrees C). Vacuoles isolated from sec mutant cells do not contain the proCPY produced at 37 degrees C. These results suggest that vacuolar and secretory glycoproteins require the same cellular functions for transport from the endoplasmic reticulum and from the Golgi body. The Golgi body represents a branch point in the pathway: from this organelle, vacuolar proenzymes are transported to the vacuole for proteolytic processing and secretory proteins are packaged into vesicles.

Topological studies of the membrane-binding segment of cytochrome b5 embedded in phosphatidylcholine vesicles.

Carboxypeptidase Y (CPY) digestion of cytochrome b5 incorporated into single-walled liposomes of egg phosphatidylcholine (PC) resulted in the release of 25 amino acid residues form each molecule of the cytochrome and concomitant detachment of the cytochrome from the vesicles. This finding suggests that the COOH-terminus of the incorporated cytochrome is exposed to the outer surface of the liposomal membrane, since the cytochrome-carrying vesicles were shown to be impermeable to macromolecules such as dextran T-40 by isopycnic centrifugation studies. The possibility that CPY had attacked a small amount of free cytochrome b5 which was in equilibrium with the liposome-bound cytochrome could be excluded by the finding that transfer of the bound cytochrome to the unbound state was a much slower process than CPY-induced detachment of cytochrome b5. CPY could also attack the COOH-terminus of cytochrome b5 embedded in dipalmitoyl-PC liposomes even below the phase transition temperature of the synthetic phospholipid. Moreover, the tyrosine residue(s) near the COOH-terminus of the PC vesicle-bound cytochrome could be readily iodinated by the action of lactoperoxidase added externally. These observations indicate that the COOH-terminus of the liposome-bound cytochrome, like its heme-containing, hydrophilic domain, is exposed to the outer surface of the vesicular membrane. The cytochrome detached from the vesicles by CPY digestion could neither rebind to the liposomes nor form an oligomeric aggregate, suggesting that CPY had removed all the amino acid residues which are required for the protein to bind to membranes. The mechanism of CPY-induced detachment of cytochrome b5 from liposomes and the size of the peptide segment which is directly involved in the interaction of the cytochrome with the lipid bilayer membranes are discussed.

Reduced nicotinamide adenine dinucleotide-cytochrome b5 reductase: location of the hydrophobic, membrane-binding region at the carboxyl-terminal end and the masked amino terminus

Microsomal NADH-cytochrome b5 reductase is an amphiphilic protein consisting of a hydrophilic (catalytic) region and a hydrophobic (membrane-binding) segment. Digestion of the reductase purified from rabbit liver microsomes with carboxypeptidase Y (CPY), but not with aminopeptidases, resulted in the abolishment of the capacities of the reductase to bind to phosphatidylcholine liposomes and to reconstitute an active NADH-cytochrome c reductase system upon mixing with cytochrome b5. The NADH-ferricyanide reductase activity of the flavoprotein was, however, inactivated only slightly by the CPY digestion. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid analyses indicated that the CPY treatment removed about 30 amino acid residues from the tcooh terminus of the reductase and that about 70% of the amino acids released were hydrophobic. It is concluded that the hydrophobic region of the reductase, responsible for both membrane binding and effective reconstitution of NADH-cytochrome c reductase activity, is located at the COOH-terminal portion of the molecule. No NH2-terminal residue could be detected in the intact and CPY-modified reductase preparations. The location of the hydrophobic, membrane-binding segment at the COOH-terminal end and the masked NH2 terminus have also been reported for cytochrome b5, another microsomal membrane protein.

固定化酵素(第2報)固定化カルボキシペプチダーゼC&lt;SUB&gt;N&lt;/SUB&gt;に対する阻害剤の効果

固定化酵素(第2報)固定化カルボキシペプチダーゼC&lt;SUB&gt;N&lt;/SUB&gt;に対する阻害剤の効果

Effects of cord factor on microsomal enzymes.

Injection of cord factor into mice induced marked suppression of pyrazinamide deamidase and aminopyrine demethylase activities and slight decreases of the contents of hemoproteins of liver microsomes. Pyrazinamide deamidase activity was most significantly and consistently affected, and might be considered a marker of microsomal damage induced by cord factor. Enzymologic properties of pyrazinamide deamidase of cord factor microsomes and control microsomes did not differ qualitatively. They were both similarly enhanced by bovine serum protein Fraction V, and had similar Km (substrate concentration at which the velocity of the reaction is half-maximal) values. In vitro treatment of microsomes with cord factor did not affect microsomal enzymes.

The presence of zinc in carboxypeptidase C and coupling of the enzyme to sepharose and sephadex

The presence of zinc in carboxypeptidase C and coupling of the enzyme to sepharose and sephadex

Effects of cord factor on pyrazinamide deamidase.

Effects of cord factor on liver pyrazinamide deamidase were studied in mice. Suppression of enzyme activity was first noticed 24 hours after injection of cord factor. Effects of cord factor were dose-dependent to approximately 50 μg, at which the maximal effect was observed. Repeated injections of small amounts of cord factor caused the same degree of suppression as a single larger dose. In vitro treatment of liver homogenate had no effect. Cord factor did not influence intracellular distribution, affinity for substrate, or other enzymologic properties of the enzyme.