Enzymes that process somatostatin precursors. A novel endoprotease that cleaves before the arginine-lysine doublet is involved in somatostatin-28 convertase activity of rat brain cortex

The selective processing activity which generates both the NH2- and COOH-terminal fragments of the octacosapeptide somatostatin-28 (S-28) was investigated. Separation into two distinct proteolytic activities was achieved by ion-exchange chromatography. An endoprotease cleaving either the substrate Pro-Arg-Glu-Arg-Lys-Ala-Gly-Ala-Lys-Asn-Tyr-NH2, i.e. [Ala17,Tyr20]S-28-(10-20)-NH2 (peptide I), or the octacosapeptide somatostatin-28, on the NH2 side of the Arg-Lys doublet was separated from an aminopeptidase B-like activity. Whereas the endoprotease cleaves a single peptide bond, between Glu12 and Arg13 of S-28, the aminopeptidase B-like enzyme removes both Arg13 and Lys14 stepwise from the NH2 terminus of the corresponding COOH-terminal fragment. This endoprotease activity peaks around pH 8.5, whereas the optimal aminopeptidase B-like activity is in the pH range 6.2-8.5. Combination of both enzymes resulted in the recovery of the overall S-28 convertase activity with an optimal pH at 7. In addition, this endoprotease appears to be very sensitive to divalent cations since it is strongly inhibited by chelating agents. The use of selectively modified undecapeptides derived from the reference substrate peptide I by a single modification of the amino acids Glu12, Arg13, and Lys14 at the cleavage locus showed that both basic residues are critically important, whereas Glu12 is not. It is proposed that S-28 processing involves a divalent cation-sensitive endoprotease that is sensitive to thiol reagents, which cleaves before the Arg-Lys doublet, which is not trypsin-like, and whose action is coupled to an aminopeptidase B-like enzyme.     

Partial purification and functional properties of an endoprotease from bovine neurosecretory granules cleaving proocytocin/neurophysin peptides at the basic amino acid doublet

An enriched preparation of neurosecretory granules from bovine pituitary neural lobes was used as a source of processing enzymes possibly involved in the cleavage of the proocytocin/neurophysin precursor. A synthetic eicosapeptide reproducing the entire (1-20) sequence of the NH2-terminal domain of the bovine ocytocin/neurophysin precursor was used as a substrate to monitor an endoprotease activity cleaving at the Lys11-Arg12 doublet. The 58-kDa endoprotease detected in the lysate of neurohypophyseal granules produced a single cleavage, after the doublet, at the Arg12-Ala13 peptide bond. This endoprotease with pHi 6.9 and 7.2 exhibits maximal activity at pH around neutrality (7.0) and was strongly inhibited by divalent cation chelating agents [ethylenediaminetetraacetic acid and ethylene glycol bis(beta-aminoethyl ether)-N,N,N',-N'-tetraacetic acid] and to some extent by p-(chloromercuri)benzoate and p-(chloromercuri)benzenesulfonic acid, while phenylmethanesulfonyl fluoride and pepstatin were not active. This endoprotease action was sensitive to any modification of the substrate at either basic amino acid of the doublet since replacement of either L-Lys11 or L-Arg12 by D-Lys or D-Arg and by L-Nle abolished the cleavage reaction. In contrast, reversal of the polarity of the doublet in [Arg11,Lys12]proocytocin/neurophysin(1-20) had no effect on the mode of endoproteolytic cleavage as well as modifications of Gly10 (replaced by Ala10). It is concluded that the selectivity of this endoprotease, which may be involved in the primary event occurring in proocytocin/neurophysin processing, is strictly dependent upon the integrity of the basic doublet but that other parameters determined by the amino acid sequence around this doublet may play an important role.     

Molecular analysis of dibasic endoproteolytic cleavage signals.

Biologically active peptide hormones are synthesized from larger precursor proteins by a variety of post-translational processing reactions. To characterize these processing reactions further we have expressed preprogastrin in two endocrine cell lines and examined the molecular determinants involved in endoproteolysis at dibasic cleavage sites. The Gly93-Arg94-Arg95 carboxyl-terminal processing site of progastrin must be processed sequentially by an endoprotease, a carboxypeptidase, and an amidating enzyme to produce bioactive gastrin. For these studies the dibasic Arg94-Arg95 residues that serve as signals for the initiation of this processing cascade were mutated to Lys94-Arg95, Arg94-Lys95, and Lys94-Lys95. In the GH3 cells the Lys94-Arg95 mutation slightly diminished synthesis of carboxyl-terminally amidated gastrin, whereas in the MTC 6-23 cells this mutation had no effect on amidated gastrin synthesis. In contrast, both Arg94-Lys95 and Lys94-Lys95 mutations resulted in significantly diminished production of amidated gastrin in both cell lines. A specific hierarchy of preferred cleavage signals at this progastrin processing site was demonstrated in both cell lines, indicating that cellular dibasic endoproteases have stringent substrate specificities. Progastrins with the Lys94-Arg95 mutation in GH3 cells also demonstrated diminished processing at the Lys74-Lys75 dibasic site, thus single amino acid changes at one processing site may alter cleavage at distant sites. These studies provide insight into the post-translational processing and biological activation of not only gastrin but other peptide hormones as well.     

Proocytocin/neurophysin convertase from bovine neurohypophysis and corpus luteum secretory granules: complete purification, structure-function relationships, and competitive inhibitor

Structure-function relationship studies were conducted on the proocytocin/neurophysin endoprotease previously characterized in both bovine neurohypophyseal and corpus luteum granules, using as a reference substrate a synthetic peptide reproducing the entire (1-20) NH2-terminal domain of the precursor. The [D-Arg12] derivative of proocytocin/neurophysin (1-20) was found to be a good competitive inhibitor of the enzyme (Ki = 30 microM), while the [D-Lys11] derivative was not. This allowed the complete purification of two isoforms of the endoprotease (Mr 58,000 and 52,000, respectively) by affinity chromatography using covalently immobilized [D-Arg12] proocytocin/neurophysin (1-20) as the affinity adsorbent. The use of selectively modified or truncated forms of the reference substrate or of the [D-Arg12] competitive inhibitor of the endoprotease established clearly that this basic pair specific convertase is sensitive to modification of the substrate structure either at the basic residues of the cleavage locus or at amino acids around this site (i.e., Pro7 and Gly9). It is concluded that longer distance interactions between amino acids situated on both the NH2 and COOH sides of the basic doublet Lys11Arg12 may contribute to the stabilization of a preferred substrate conformation allowing recognition by the enzyme subsites.     

Deficiencies in pro-thyrotropin-releasing hormone processing and abnormalities in thermoregulation in Cpefat/fat mice

Cpe(fat/fat) mice are obese, diabetic, and infertile. They have a mutation in carboxypeptidase E (CPE), an enzyme that converts prohormone intermediates to bioactive peptides. The Cpe(fat) mutation leads to rapid degradation of the enzyme. To test whether pro-thyrotropin-releasing hormone (TRH) conversion to TRH involves CPE, processing was examined in the Cpe(fat/fat) mouse. Hypothalamic TRH is depressed by at least 75% compared with wild-type controls. Concentrations of pro-TRH forms are increased in homozygotes. TRH-[Gly(4)-Lys(5)-Arg(6)] and TRH-[Gly(4)-Lys(5)] represent approximately 45% of the total TRH-like immunoreactivity in Cpe(fat/fat) mice; they constitute approximately 1% in controls. Levels of TRH-[Gly(4)] were depressed in homozygotes. Because the hypothalamus contains some TRH, another carboxypeptidase must be responsible for processing. Immunocytochemical studies indicate that TRH neurons contain CPE- and carboxypeptidase D-like immunoreactivity. Recombinant CPE or carboxypeptidase D can convert synthetic TRH-[Gly(4)-Lys(5)] and TRH-[Gly(4)-Lys(5)-Arg(6)] to TRH-[Gly(4)]. When Cpe(fat/fat) mice are exposed to cold, they cannot maintain their body temperatures, and this loss is associated with hypothalamic TRH depletion and reduction in thyroid hormone. These findings demonstrate that the Cpe(fat) mutation can affect not only carboxypeptidase activity but also endoproteolysis. Because Cpe(fat/fat) mice cannot sustain a cold challenge, and because alterations in the hypothalamic-pituitary-thyroid axis can affect metabolism, deficits in pro-TRH processing may contribute to the obese and diabetic phenotype in these mice.     

Catabolism of rat growth hormone-releasing factor(1-29) amide in rat serum and liver.

Clinical and veterinary uses of growth hormone-releasing factor [GRF(1- 29)NH2] require the design of analogs that are resistant to proteolysis by serum and liver degrading enzymes. This study investigated rat GRF(1-29)NH2 processing in serum and liver homogenate by means of high pressure liquid chromatography (HPLC). Synthetic rGRF(1-29)NH2 (30 microM) was incubated (0-120 min, 37 degrees C) in serum (49 +/- 8 mg prot./ml). The rGRF(1-29)NH2 (10 microM) was also incubated (0-120 min, 37 degrees C) with liver homogenate (200 +/- 6 micrograms prot./ml). Time course studies of rGRF(1-29)NH2 disappearance showed apparent half-lives of 18 +/- 4 min and 13 +/- 3 min in serum and liver homogenate, respectively. This was accompanied by the appearance of degradation products that were all less hydrophobic than the native peptide. In the serum, two major metabolites were detected and isolated by preparative HPLC. Combined results of amino acid analysis, sequencing, and chromatography with synthetic homologs revealed the presence of rGRF(1-20)OH and (3-20)OH. A small amount of rGRF(12-29)NH2, coeluting with rGRF(3-20)OH, was also found by sequencing. In the liver, rGRF(1-18)OH, (3-18)OH, and (1-10)OH were identified. The peptide bond Ala2-Asp3 (DPP IV cleavage site) was hydrolyzed in both serum and liver. Other tissue-specific cleavage sites were Arg11-Arg12 and Arg20-Lys21 (trypsin-like cleavage site) in the serum, and Tyr10-Arg11 and Tyr18-Ala19 (chymotrypsin-like cleavage site) in the liver.(ABSTRACT TRUNCATED AT 250 WORDS)     

Processing endoprotease recognizes a structural feature at the cleavage site of peptide prohormones. The pro-ocytocin/neurophysin model

Pro-ocytocin/neurophysin convertase is a divalent cation-dependent endoprotease isolated from both bovine corpus luteum and neurohypophyseal secretory granules. The putative pro-ocytocin/neurophysin converting enzyme cleaves the Arg12-Ala13 bonds of both pro-ocytocin/neurophysin (1----20) and pro-ocytocin/neurophysin obtained by hemisynthesis. The minimal efficient substrate structure allowing recognition by this processing endoprotease was defined by measuring its cleavage efficiency and the inhibitory properties of a set of 34 selectively modified derivatives of the (1----20) NH2-terminal domain of the ocytocin/neurophysin precursor. The data demonstrate that: (i) the basic Lys11-Arg12 doublet, although necessary, is not sufficient; (ii) a minimal substrate length of nine amino acids (residues 7-15 or 8-16) is essential; (iii) those amino acids around the Lys-Arg doublet which contribute to the formation of a possible beta-turn-alpha-helix secondary structure are critical; (iv) substrate recognition by the enzyme may involve several subsites in which structural determinants, situated on both sides of the basic doublet, participate; (v) the NH2-terminal sequence of neurophysin plays a critical role in the correct reading of the cleavage sequence by the processing endoprotease. It is proposed, first, that this type of structural feature may constitute the basis of a general coding system for endoproteases involved in the processing of polypeptide hormone precursors; second, that in addition to its role in the intragranular packaging of the nonapeptide hormone, neurophysin plays a key role in the correct processing of its common precursor with ocytocin.     

Human carboxypeptidase M. Purification and characterization of a membrane-bound carboxypeptidase that cleaves peptide hormones

A membrane-bound neutral carboxypeptidase B-like enzyme was solubilized from human placental microvilli with 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS) and purified to homogeneity by ion-exchange chromatography and affinity chromatography on arginine-Sepharose. It gave a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an apparent Mr of 62,000 with or without reduction. The enzyme is a glycoprotein as shown by its high affinity for concanavalin A-Sepharose and reduction in mass to 47,600 daltons after chemical deglycosylation. It has a neutral pH optimum, is activated by CoCl2, and inhibited by o-phenanthroline, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, or cadmium acetate, indicating it is a metallopeptidase. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides (e.g. Bz-Gly-Arg, Bz-Gly-Lys, Bz-Ala-Lys, dansyl-Ala-Arg, where Bz is benzoyl and dansyl is 5-dimethylaminonaphthalene-1-sulfonyl) as well as from several biologically active substrates: dynorphin A(1-13), Met5-Arg6-enkephalin (Km = 46 microM, kcat = 934 min-1), bradykinin (Km = 16 microM, kcat = 147 min-1), Met5-Lys6-enkephalin (Km = 375 microM, kcat = 663 min-1), and Leu5-Arg6-enkephalin (Km = 63 microM, kcat = 106 min-1). Although the enzyme shares some properties with other carboxypeptidase B-like enzymes, it is structurally, catalytically, and immunologically distinct from pancreatic carboxypeptidase A or B, human plasma carboxypeptidase N, and carboxypeptidase H ("enkephalin convertase"). To denote that the enzyme is membrane-bound, and to distinguish it from other known carboxypeptidases, we propose the name "carboxypeptidase M." Because of its localization on the plasma membrane and optimal activity at neutral pH, carboxypeptidase M could inactivate or modulate the activity of peptide hormones either before or after their interaction with plasma membrane receptors.     

Somatostatin-28 and pro-ocytocin/neurophysin convertases: basic pair selective endoproteases involved in pro-hormone processing in the rat brain cortex and bovine corpus luteum

Two neuropeptide precursor processing enzyme systems were characterized in the rat brain cortex and bovine neurohypophysis and corpus luteum. The first one combines the action of a 90 kDa endoprotease which cleaves somatostatin-28 before the Arg-Lys doublet and that of an aminopeptidase B-like enzyme. The second system associates the action of a 58 kDa endoprotease cleaving pro-ocytocin/neurophysin (1-20) after the Lys-Arg dibasic moiety and a carboxypeptidase B-like activity. Both systems appear to be located in membrane-limited secretory vesicles of the producing organs, and to exhibit the properties of metallo-enzymes sensitive to divalent cation chelators. In contrast, they do not show the characteristics of serine-proteases and of trypsin-like enzymes. Studies with substrate analogs selectively modified at the basic doublet indicated that the integrity of both basic amino acids is essential but that conformational parameters, probably governed by the amino acid sequences flanking the basic doublet, play an important role. These data will be discussed in relation to a hypothesis on the predicted preferred secondary structure of these restriction loci.     

Purification and Characterization of a Membrane-Bound Enkephalin-Forming Carboxypeptidase, "Enkephalin Convertase"

Enkephalin convertase, the enkephalin-synthesizing carboxypeptidase B-like enzyme, has been purified to apparent homogeneity from bovine pituitary and adrenal chromaffin granule membranes. The membrane-bound enkephalin convertase can be solubilized in high yield with 0.5% Triton X-100 in the presence of 1 M NaCl. Extensive purification is achieved by affinity chromatography with p-aminobenzoyl-L-arginine linked to Sepharose 6B. Enzyme purified from both pituitary and adrenal chromaffin granule membranes shows a single band by sodium dodecyl sulfate polyacrylamide gel electrophoresis with an apparent molecular weight of 52,500, whereas enkephalin convertase purified from soluble extracts of these tissues has an apparent molecular weight of 50,000. The regional distribution of the membrane-bound enzyme in the rat brain differs from that of the soluble enzyme. While the soluble enzyme shows 10-fold variations, resembling somewhat the enkephalin peptides, membrane-bound enkephalin convertase is more homogeneously distributed throughout the brain. In rat pituitary glands, membrane-bound enzyme activity is similar in the anterior and posterior lobes, whereas the soluble enzyme is enriched in the anterior lobe. Membrane-bound and soluble forms of enkephalin convertase isolated from either bovine pituitary glands or adrenal chromaffin granules show identical substrate and inhibitor specificities. As with the soluble enzyme, membrane-bound enkephalin convertase hydrolyzes [Met]- and [Leu]enkephalin-Arg6 and -Lys6 to enkephalin, with no further degradation of the pentapeptide.     

Carboxyl-terminal proteolytic processing of matrix Gla protein

The present study was undertaken to determine the extent of COOH-terminal proteolytic processing in matrix Gla protein (MGP), a 10-kDa protein which contains 5 residues of the vitamin K-dependent Ca2+ binding amino acid, gamma-carboxyglutamic acid (Gla). Two forms of MGP were isolated from demineralization and urea extracts of bovine cortical bone, one 79 residues in length with the COOH terminus Phe-Arg-Gln and the other 83 residues in length with the COOH terminus Phe-Arg-Gln-Arg-Arg-Gly-Ala. The 84-residue form of bovine MGP predicted from the message structure could not be detected in the bone extracellular matrix extracts, and it therefore seems probable that the lysine at position 84 was removed by the action of a carboxypeptidase B-like enzyme prior to secretion. A plausible sequence of proteolytic cleavages that could generate the 79-residue form of MGP would be a trypsin-like cleavage at Arg80-Arg81 or Arg81-Gly82 followed by carboxypeptidase B-like cleavage to remove COOH-terminal arginine(s). Since essentially equal amounts of the 79- and 83-residue forms of MGP were also detected in bovine articular cartilage and plasma, it seems likely that the COOH-terminal processing events identified in bone apply to many of the other tissues which synthesize this protein. Only one form of MGP was detected in human bone extracts, a 77-residue protein that lacks the COOH-terminal residues Arg-Lys-Arg-Arg-Gly-Thr-Lys. This shortened version of human MGP is consistent with the proposed model for COOH-terminal processing, since the amino acid substitution in the COOH terminus of the human protein, Lys79 for Gln79, would allow removal of the additional basic residues from the human MGP COOH terminus by the action of the carboxypeptidase B-like enzymic activity. Recent studies have shown that MGP is strongly induced by retinoic acid in fibroblasts, chondrocytes, and osteoblasts, a response which suggests that MGP mediates an action of retinoic acid on an aspect of cell growth or differentiation. If this hypothesis is true, the present evidence for complex COOH-terminal processing events could provide a means to regulate the as yet unknown activity of MGP in the extracellular environment in a mechanism similar to the activation of hormones such as anaphlotoxins and kinins.     

(Met)enkephalin and carboxypeptidase processing enzyme are co-released from chromaffin cells by cholinergic stimulation

A carboxypeptidase B-like enzyme is involved in processing of proenkephalin in adrenal medulla. Nicotine stimulated the co-release of this enzyme with (Met)enkephalin pentapeptide from bovine chromaffin cells in primary culture. The ratio of enzyme activity/immunoreactivity was determined for the released carboxypeptidase to provide an index of the level of enzyme activity per unit number of enzyme molecules. The ratio for the Co++-stimulated carboxypeptidase secreted into the cell culture medium upon nicotinic stimulation was 10.1 +/- 1.02 (pmol Met-enkephalin formed per ng carboxypeptidase immunoreactivity), while the Co++-stimulated carboxypeptidase in the soluble and membrane components of purified chromaffin granules had lower ratios of 5.46 +/- 0.70 and 1.07 +/- 0.13, respectively. Hexamethonium, a nicotinic receptor antagonist, blocked the nicotine-induced release of the carboxypeptidase processing enzyme and (Met)enkephalin. These data suggest that a pool of carboxypeptidase enzyme molecules at a high state of activation are present in functionally mature granules whose contents are released by nicotinic receptor stimulation.     

Reactivity of basic amino acid pairs in prohormone processing: model of pro-ocytocin/neurophysin processing domain

Statistical analysis of several potential dibasic cleavage sites reveals differences in the distribution of basic doublets when the in vivo cleaved sites were compared to those which are not cleaved. Analysis of the substrate specificity of protease Kex2 towards the pro-ocytocin/neurophysin processing domain (pro-OT/Np(7-15) with altered basic pairs shows a cleavage efficiency order in accord with the statistical data. Structural analysis of these substrates indicates that each basic pair is associated with a local and specific conformational change. Thus, the in vivo cleavage hierarchy of dibasic sites is encoded by both the nature of basic pairs and the plasticity of proteolytic processing domains.     

Effect of neurophysin on enzymatic maturation of oxytocin from its precursor

We examined the extent to which rates of enzymatic conversion of the oxytocin biosynthetic precursor to mature peptide are modulated by intramolecular and intermolecular assembly of precursor and polypeptide intermediates. The biosynthesized precursor contains hormone and neurophysin sequences linked by a Gly-Lys-Arg sequence and undergoes enzymatic processing reactions which include endoproteolytic cleavage at the Lys-Arg dibasic sequence, carboxypeptidase B-like exoproteolytic cleavage, and enzymatic amidation. We evaluated the effect of neurophysin on such processing reactions using semisynthetic precursors of oxytocin/bovine neurophysin I and synthetic oxytocinyl precursor intermediates as substrates. Neurophysin I at high concentration (0.7 mM) reduced the rates of carboxy-peptidase B-like conversion of oxytocinyl-Gly-Lys-Arg to oxytocinyl-Gly and the enzymatic amidation of oxytocinyl-Gly to mature (C-terminal amidated) oxytocin. The dependence of rate suppression on the concentrations of peptide substrate and neurophysin I suggested that suppression is due to intermolecular formation of hormone-neurophysin complexes which are aggregated at least to dimers. An analogous intramolecular neurophysin effect was found for endoproteolytic processing of semisynthetic precursors. Endoproteinase Lys-C cleaved the Lys11-Arg12 peptide bond in a native-like semisynthetic precursor at a significantly slower rate than it did an assembly-deficient precursor analogue. The difference in semisynthetic precursor endoproteolysis rates is most substantial at the high concentrations at which the native-like precursor would form dimers but the assembly-deficient analogue would not. The native-like semisynthetic precursor was more stable than the assembly-deficient precursor analogue to tryptic digestion. The concentration-dependent effects of neurophysin, both intramolecularly as a precursor domain and intermolecularly as an interacting protein, are likely to occur in the secretory granules in which the biosynthetic precursors are packaged. The molecular organization of both hormone/neurophysin precursors and the noncovalently complexed hormone-neurophysin intermediates can be expected to play a role in modulating enzymatic processing reactions that lead to mature neurohypophysial hormones.     

Design and synthesis of highly potent and selective melanotropin analogues of SHU9119 modified at position 6

The melanocortin receptors are involved in several important physiological functions. The potent and enzymatically stable analogues MT-II (Ac-Nle-c[Asp-His-DPhe-Arg-Trp-Lys]-NH(2)) and SHU9119 (Ac-Nle-c[Asp-His-DNal(2')-Arg-Trp-Lys]-NH(2)) are important ligands of these receptors but are relatively nonselective. To differentiate between the physiological functions of these receptors, agonists, and antagonists with improved receptor selectivities are needed. We report here analogues of the well-characterized antagonist SHU9119 in which we replaced His(6) with conformationally constrained amino acids. By this structure-activity study we discovered two important compounds, PG-901 (Ac-Nle(4)-c[Asp(5)-Pro(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)) and PG-911 (Ac-Nle(4)-c[Asp(5)-Hyp(6)-DNal(2')(7)-Arg(8)-Trp(9)-Lys(10)]-NH(2)), characterized to be full agonists at the hMC5R (EC(50) = 0.072 nM and 0.031 nM, respectively), but full antagonists at the hMC3R and the hMC4R. We also demonstrated that the relative stereochemistry of the amino acid at the 6-position is critical for activity, and could play an important role in potency as well as in selectivity for the melanocortin receptors.     

Comparison of a spectrophotometric, a fluorometric, and a novel radiometric assay for carboxypeptidase E (EC 3.4.17.10) and other carboxypeptidase B-like enzymes

Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme involved in the biosynthesis of numerous peptide hormones and neurotransmitters. A sensitive assay for CPE and other carboxypeptidase B-like enzymes has been developed using 125I-acetyl-Tyr-Ala-Arg (125I-AcYAR) as the substrate. This peptide is poorly soluble in ethyl acetate whereas the product of carboxypeptidase B-like enzymatic activity (125I-AcYA) can be quantitatively extracted with this solvent, allowing the rapid separation of product from substrate. This radiometric assay can detect less than 1 pg of either CPE or carboxypeptidase B. For CPE, the assay with 125I-AcYAR is approximately 1000 times more sensitive than a fluorescent assay using dansyl-Phe-Ala-Arg (dans-FAR), and 6000 times more sensitive than a spectrophotometric assay using hippuryl-Arg (hipp-R). CPE hydrolyzes the three substrates with Kcat values of 16 s-1 for AcYAR, 13 s-1 for dans-FAR, and 8.5 s-1 for hipp-R. The Km values for CPE with AcYAR (28 microM) and dans-FAR (34 microM) are similar, and are much lower than the Km with hipp-R (400 microM). Thus, the primary reason for the increased sensitivity of the 125I-AcYAR assay over the fluorescent assay is not a result of kinetic differences but is due to the detection limit of iodinated product (10(-15) mol), compared to the fluorescent product (5 x 10(-11) mol). Applications of this rapid and sensitive radiometric assay to detect CPE in cultured cells and in subcellular fractions of the pituitary are described.     

Two peptidases that convert 125I-Lys-Arg(Met)enkephalin and 125I-(Met)enkephalin-Arg6, respectively,to 125I-(Met)enkephalin in bovine adrenal medullary chromaffin granules

Two peptidases which convert ¹²⁵I-Lys-Arg-ME and ¹²⁵I-ME-Arg⁶, respectively, to ¹²⁵I-ME, have been identified and characterized in bovine adrenomedullary chromaffin granules. The former is referred to as a secretory granule peptidase (SGP) and the latter as a carboxypeptidase B-like enzyme (CPB-like) [7] which is here further characterized. SGP cleaved ¹²⁵I-Lys-Arg-ME to produce only ¹²⁵I-ME and was localized in chromaffin granules which contained co²⁺-stimulated CPB-like activity, ME, and catecholamines. Both the SGP and the CPB-like enzymes appear to be thiol-metalloproteases. While the CPB-like enzyme seems likely to be involved in processing the enkephalin precursors [7], SGP may function as a trypsin-like or aminopeptidase enzyme in secretory granules.     

Tryptase from rat skin: purification and properties

Tryptase was purified 13,000-fold to apparent homogeneity from rat skin. The two-step procedure involved ammonium sulfate fractionation of the initial extract followed by combined sequential affinity chromatography on agarose-glycyl-glycyl-p-aminobenzamidine and concanavalin A-agarose. The purified enzyme had a specific activity toward N-benzoylarginine ethyl ester (BzArgOEt) of 170 mumol/min mg-1 and was obtained in a yield of 28% as determined by the specific substrate, H-D-Ile-Pro-Arg-p-nitroanilide. Rat skin tryptase was thermal labile, losing 50% of its activity when preincubated for 30 min at 30 degrees C. The presence of NaCl (1 M) improved thermal stability and was necessary for long-term storage. Heparin did not stabilize the enzyme against thermal denaturation, and heparin-agarose failed to bind the enzyme. Rat skin tryptase was inhibited by diisopropylphosphofluoridate, antipain, leupeptin, and aprotinin but not by alpha 1-antitrypsin, ovomucoid, or soybean or lima bean trypsin inhibitors. Substrate specificity studies using a series of tri- and tetrapeptidyl-p-nitroanilide and peptidyl-7-amino-4-methylcoumarin substrates demonstrated the existence of an extended substrate binding site. Rat skin tryptase hydrolyzed [Arg8]vasopressin, neurotensin, and the oxidized B-chain of insulin at the -Arg8-Gly9-NH2, -Arg8-Arg9-, and -Arg22-Gly23-bonds, respectively. No general proteinase activity was observed toward casein, hemoglobin, or azocoll. Rat skin tryptase had a Mr of 145,000 by gel filtration. The subunit Mr was either 34,000 or 30,000 depending on the electrophoretic technique used. Treatment of the enzyme with peptide N-glycosidase F (N-glycanase) decreased the subunit Mr by 4000. The enzyme exhibited multiple isoelectric forms (pI's of 4.5-4.9). Rat skin tryptase was found to be related statistically to other tryptases on the basis of amino acid composition. The N-terminal amino acid sequence was Ile1-Val2-Gly3-Gly4-Gln5-Glu6-Ala7-+ ++Ser8-Gly9-Asn10-Lys11-Trp12-Pro13- Trp14- Gln15-Val16-Ser17-Leu18-Arg19-Val20- --21-Asp-22Thr23-Tyr24-Typ25-, with a putative glycosylation site at residue 21. This sequence was 72-80% homologous with the N-terminus of other tryptases but only 40% homologous with that of bovine trypsin.     

The insulin-secretory-granule carboxypeptidase H. Purification and demonstration of involvement in proinsulin processing.

A carboxypeptidase B-like enzyme was detected in the soluble fraction of purified insulin secretory granules, and implicated in insulin biosynthesis. To investigate the role of this activity further, we purified the enzyme from rat insulinoma tissue by gel-filtration chromatography and affinity elution from p-aminobenzoyl-arginine. A yield of 42%, with a purification factor of 674 over the homogenate, was achieved. Analysis of the purified carboxypeptidase by SDS/polyacrylamide-gel electrophoresis under either reducing or non-reducing conditions showed it to be a monomeric protein of apparent Mr 55,000. The preparation was also homogeneous by high-performance gel-filtration chromatography. The enzyme bound to concanavalin A, showing it to be a glycoprotein. Amino acid analysis or chemical deglycosylation and SDS/polyacrylamide-gel electrophoresis indicated a protein Mr of 50,000, suggesting a carbohydrate content of approx. 9% by weight. The purified enzyme was able to remove basic amino acids from the C-terminus of proinsulin tryptic peptides to generate insulin, but did not further degrade the mature hormone. It was inhibited by EDTA, 1,10-phenanthroline and guanidinoethylmercaptosuccinic acid, and stimulated 5-fold by CoCl2. The pH optimum of the conversion of diarginyl-insulin into insulin was in the range 5-6, with little activity above pH 6.5. Activity was also expressed towards a dansylated tripeptide substrate (dansyl-phenylalanyl-leucyl-arginine; Km = 17.5 microM), and had a pH optimum of 5.5. These properties are indistinguishable from those of the activity located in secretory granules, and are compatible with the intragranular environment. The insulin-secretory-granule carboxypeptidase shared several properties of carboxypeptidase H from bovine adrenal medulla and pituitary. We propose that the carboxypeptidase that we purified is the pancreatic isoenzyme of carboxypeptidase H (crino carboxypeptidase B; EC 3.4.17.10), and is involved in the biosynthesis of insulin in the pancreatic beta-cell.     

Structure-activity relationship of cyclic peptide penta-c[Asp-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys]-NH(2) at the human melanocortin-1 and -4 receptors: His(6) substitution

A series of MT-II related cyclic peptides, based on potent but non-selective hMC4R agonist (Penta-c[Asp-His(6)-DPhe(7)-Arg(8)-Trp(9)-Lys]-NH(2)) was prepared in which His(6) residue was systematically substituted. Two of the most interesting peptides identified in this study are Penta-c[Asp-5-ClAtc-DPhe-Arg-Trp-Lys]-NH(2) and Penta-c[Asp-5-ClAtc-DPhe-Cit-Trp-Lys]-NH(2) which are potent hMC4R agonists and are either inactive or weak partial agonists (not tested for their antagonist activities) in hMC1R, hMC3R and hMC5R agonist assays.