Carboxypeptidase B-like Activity Research Articles

Structure and Function of REP34 Implicates Carboxypeptidase Activity in Francisella tularensis Host Cell Invasion

Francisella tularensis is the etiological agent of tularemia, or rabbit fever. Although F. tularensis is a recognized biothreat agent with broad and expanding geographical range, its mechanism of infection and environmental persistence remain poorly understood. Previously, we identified seven F. tularensis proteins that induce a rapid encystment phenotype (REP) in the free-living amoeba, Acanthamoeba castellanii. Encystment is essential to the pathogen's long term intracellular survival in the amoeba. Here, we characterize the cellular and molecular function of REP34, a REP protein with a mass of 34 kDa. A REP34 knock-out strain of F. tularensis has a reduced ability to both induce encystment in A. castellanii and invade human macrophages. We determined the crystal structure of REP34 to 2.05-Å resolution and demonstrate robust carboxypeptidase B-like activity for the enzyme. REP34 is a zinc-containing monomeric protein with close structural homology to the metallocarboxypeptidase family of peptidases. REP34 possesses a novel topology and substrate binding pocket that deviates from the canonical funnelin structure of carboxypeptidases, putatively resulting in a catalytic role for a conserved tyrosine and distinct S1' recognition site. Taken together, these results identify REP34 as an active carboxypeptidase, implicate the enzyme as a potential key F. tularensis effector protein, and may help elucidate a mechanistic understanding of F. tularensis infection of phagocytic cells.

Modified Human Beta 2‐Microglobulin (desLys58) Displays Decreased Affinity for the Heavy Chain of MHC Class I and Induces Nitric Oxide Production and Apoptosis

Beta2-microglobulin (beta2m) is the light chain of major histocompatibility complex class I (MHC-I) molecules, and is a prerequisite for the binding of peptides to the heavy chain and their presentation to CD8+ T cells. beta2m can be modified in vivo and in vitro by proteolytic cleavage by complement C1 and subsequent carboxypeptidase B-like activity--processes that lead to the generation of desLys(58) beta2m (dbeta2m). This work aims to study the effect of dbeta2m on peptide binding to MHC-I, the influence of dbeta2m on the binding of beta2m to the MHC-I heavy chain and the biological activity of dbeta2m. Both beta2m and dbeta2m are able to support the generation of MHC-I/peptide complexes at 18 degrees C, but complexes formed in the presence of dbeta2m destabilize at 37 degrees C. Moreover, a 250 times higher concentration of dbeta2m than of beta2m is needed to displace MHC-I associated beta2m from the cell surface. In addition, only beta2m is able to restore MHC-I/peptide complex formation on acid-treated cells whereas dbeta2m appears to bind preferentially to denatured MHC-I heavy chains. In cell cultures, exogenously added dbeta2m, but not beta2m, induces apoptotic cell death in monocytic leukaemic cell lines but spares other kinds of leukaemic cells. Additionally, the presence of dbeta2m, and to a lesser extent beta2m, enhances IFN-gamma-induced NO production by monocytic leukaemic cells. In conclusion, these data show that dbeta2m is not able to support the formation of a stable tri-molecular MHC-I complex at physiological temperature and that dbeta2m exerts other biological functions compared to beta2m when bound to cells.

Hepatitis Induced by an IgM Monoclonal Antibody against Procarboxypeptidase R

Procarboxypeptidase R (proCPR), also known as thrombin-activatable fibrinolysis inhibitor (TAFI), is present in plasma and can be activated to carboxypeptidase R (CPR) by trypsin-like enzymes such as thrombin and plasmin. CPR has the carboxypeptidase B-like activity that can inactivate the inflammatory peptides such as C5a by removing the C-terminal arginine and can interfere with fibrinolysis by removing C-terminal lysine residue of fibrin. In the present study, we conducted to produce monoclonal antibodies (mAbs) by using spleen cells from proCPR-deficient mice immunized by partially purified mouse proCPR. The mAbs obtained were IgM isotype and reacted with proCPR and interfered with activation of proCPR to CPR by thrombin-thrombomodulin complex. Some BALB/c mice implanted with the hybridoma died in 7 days, and intravenous injection of the mAb to BALB/c mice induced transient elevation of GOT and GPT in plasma although injection to the deficient mice did not. Furthermore, the histological features showed the focally lesions in liver tissue of BALB/c mice injected with the mAb. Since liver is the major site of proCPR synthesis, IgM mAb to proCPR should have induced local inflammation at the side resulting in induction of hepatitis.

Thrombin activatable fibrinolysis inhibitor: not just an inhibitor of fibrinolysis.

To review the activation of thrombin activatable fibrinolysis inhibitor (TAFI) and activity of activated TAFI (TAFIa) as it relates to the regulation of both fibrinolytic and proinflammatory substances. Published articles and reviews (from PubMed, published between 1962 and 2003) on experimental studies of coagulation, fibrinolysis, and inflammation. The principal physiologic role of TAFI is still a matter of debate. Although TAFI activation can result from proteolysis by a number of proteases, the most likely physiologic activators are thrombin (in complex with the cofactor thrombomodulin) and plasmin (in complex with polysaccharide cofactors). The activated enzyme, TAFIa, displays carboxypeptidase B-like activity and probably regulates both fibrinolysis and inflammation in response to injury and infection. At present, there is limited understanding of the role that TAFI plays in the interrelationships between coagulation, fibrinolysis, and inflammation. Although the potential therapeutic value of TAFIa inhibition/TAFI activation awaits further investigation, the data gathered to date suggest that, like activated protein C, TAFIa may play a pivotal role in regulating the crosstalk between coagulation, fibrinolysis, and inflammation.

DIGESTIVE ENZYME ACTIVITIES IN THE ALIMENTARY TRACT OF REDCLAW CRAYFISH, CHERAX QUADRICARINATUS (DECAPODA: PARASTACIDAE)

Abstract The redclaw crayfish, Cherax quadricarinatus, is of commercially important interest, but its digestive capabilities are not yet completely understood. The understanding of how its digestive enzymes operate can help in the formulation of better diets for this species. A range of digestive enzymes (proteases, carbohydrases, and lipase) was found in the midgut gland and gastric fluid, with activities generally higher in the gastric fluid. Total protease showed maximal activity at pH 7.0–7.5 in both midgut gland and gastric fluid and was inhibited by 1.5 mM CuCl2 and 10 mM phenylmethylsulfonyl fluoride (PMSF). On the other hand, protease was activated by 1.5 mM CaCl2. Trypsin-like activity showed pH optima at 7.0 in both the midgut gland and the gastric fluid and was activated by 1.5 mM CaCl2, FeSO4, and Na2SO4. Chymotrypsin-like activity and carboxypeptidase A-like activity showed higher activities in the gastric fluid. Carboxypeptidase B-like activity showed optima at pH 6.0 and 7.0 in both the mid...

Identification and Characterization of Two Thrombin-activatable Fibrinolysis Inhibitor Isoforms

Thrombin-activatable fibrinolysis inhibitor (TAFI) is synthesized by the liver and is thought to circulate in plasma as a plasminogen-bound zymogen. When it is activated by the thrombin/thrombomodulin complex, activated TAFI exhibits carboxypeptidase B-like activity. To study the structure-function relationship of TAFI, we expressed recombinant human TAFI in insect cells. During the cloning of TAFI cDNA from several human liver cDNA libraries, we identified a second TAFI cDNA which differed from the published sequence at 2 positions. One of these sequences resulted in a substitution of alanine for threonine at residue 147, the other was a silent mutation. These substitutions were found in several cDNA libraries from different sources. Using Southern blot analysis, we confirmed the existence of this TAFI polymorphism in the population. In order to compare the activation and activity of TAFI isoforms, we expressed both isoforms in the baculovirus expression system, and compared the enzyme kinetics of the purified proteins. The molecular weight of recombinant TAFI is lower than plasma TAFI due to differences in glycosylation. The two recombinant TAFI isoforms had similar activation kinetics and the activated enzymes had similar carboxypeptidase B-like activity towards small molecule substrates. Their ability to retard clot lysis was found to be similar in a plate clot lysis assay.

Both Cellular and Soluble Forms of Thrombomodulin Inhibit Fibrinolysis by Potentiating the Activation of Thrombin-activable Fibrinolysis Inhibitor

Thrombin-activable fibrinolysis inhibitor (TAFI) is a recently described plasma zymogen that can be activated by thrombin to an enzyme with carboxypeptidase B-like activity. The enzyme, TAFIa, potently attentuates fibrinolysis. TAFI activation, like protein C activation, is augmented about 1250-fold by thrombomodulin (TM). In this work, the effects of both soluble and cellular forms of TM on TAFI activation-dependent suppression of fibrinolysis were investigated. Soluble TM included in clots formed from purified components, barium citrate-adsorbed plasma, or normal human plasma maximally increased the tissue plasminogen activator-induced lysis time 2-3-fold, with saturation occurring at 5, 10, and 1 nM TM in the three respective systems. Soluble TM did not effect lysis in the system of purified components lacking TAFI or in plasmas immunodepleted of TAFI. In addition, the antifibrinolytic effect of TM was negated by monoclonal antibodies against either TAFI or TM. The inhibition of fibrinolysis by cellular TM was assessed by forming clots in dialyzed, barium citrate-adsorbed, or normal plasma over cultured human umbilical vein endothelial cells (HUVECs). Tissue plasminogen activator-induced lysis time was increased 2-fold, with both plasmas, in the presence of HUVECs. The antifibrinolytic effect of HUVECs was abolished 66% by specific anti-TAFI or anti-TM monoclonal antibodies. A newly developed functional assay demonstrated that HUVECs potentiate the thrombin-catalyzed, TM-dependent formation of activated TAFI. Thus, endothelial cell TM, in vitro at least, appears to participate in the regulation of not only coagulation but also fibrinolysis.

Tryptic Map Variation of Erythropoietin Resulting from Carboxypeptidase B-Like Activity

Abstract The recombinant human erythropoietin (r-HuEPO) is monitored by tryptic mapping for its purity and identity. Occasionally, these maps have produced an additional peak not evident in the reference standard. This peak was isolated, sequenced and characterized by mass spectrum. It is derived from a known r-HuEPO tryptic peptide 144–150 with the C-terminal arginine removed. Its formation can be reduced or totally eliminated by the addition of inhibitors of carboxypeptidase B activity. These results indicate the presence of residual carboxypeptidase B-like activity in some r-HuEPO lots. Similar enzymatic activity has been shown in varying amounts in the concentrated diafiltered media before purification. The peptide 144–150 is one of the first to be affected by the carboxypeptidase B-like activity. This peptide seems to locate in the most trypsin-sensitive part of r-HuEPO.

Expression, purification, and characterization of the yeast KEX1 gene product, a polypeptide precursor processing carboxypeptidase.

The Saccharomyces cerevisiae KEX1 gene encodes a protease with carboxypeptidase B-like activity involved in K1 and K2 killer toxins and alpha-factor (mating pheromone) precursors processing. The gene has been expressed using the baculovirus/insect cell system, and the KEX1 encoded protein (Kex1p) was purified to apparent homogeneity from detergent-solubilized membrane preparations of insect cells infected with the recombinant virus. The specific activity of the enzyme was enriched 126-fold as compared with the cell lysate, with a recovery of 29%. The NH2-terminal sequence of the purified active enzyme was identical to the predicted sequence after the removal of the signal peptide. This provides evidence that Kex1p, at least in insect cells, is not made as a proenzyme. The optimum pH for activity was 6.0, and the apparent pI value of the protein was below pH 3.0. The enzyme cleaves arginine or lysine from the COOH terminus of synthetic peptides: benzoyl-Phe-Ala-Arg (Km = 284 microM), furylacryloyl (fa)-Ala-Arg (Km = 516 microM), and fa-Ala-Lys (Km = 962 microM). The kinetic data obtained reveals that Kex1p preferentially cleaves the COOH-terminal arginine of peptides over the COOH-terminal lysine. Insect-derived Kex1p processes alpha-factor-Lys-Arg, its known natural substrate, to mature active alpha-factor, and this maturation event takes place in a sequential manner. Furthermore, the enzyme expresses very high affinity for the 15-amino acid-long peptide, alpha-factor-Lys-Arg (Ki = 22 microM), and somewhat lower affinity for the heptapeptides [Leu]enkephalin-Arg-Arg,-Arg-Lys, and [Met]enkephalin-Lys-Lys (Ki = 45, 57, and 81 microM, respectively). The data demonstrate that processing at the COOH terminus of the peptides tested stops after the cleavage of the Arg and/or Lys residues. The specificity of the enzyme for COOH-terminal basic amino acid residues of the peptides used in this study and its high affinity for alpha-factor-Lys-Arg confirms the role that Kex1p plays in polypeptide precursor processing in yeast.

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.

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 K cat values of 16 s −1 for AcYAR, 13 s −1 for dans-FAR, and 8.5 s −1 for hipp-R. The K m values for CPE with AcYAR (28 μ m) and dans-FAR (34 μ m) are similar, and are much lower than the K m with hipp-R (400 μ m). 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 × 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.

Serum acetylcholinesterase possesses trypsin-like and carboxypeptidase B-like activity

Acetylcholinesterase (AChE, EC 3.1.1.7) purified from the electric organ of eel possesses a protease activity resembling that of a neuropeptide processing enzyme. To examine whether any mammalian AChEs possess a similar protease activity, the enzyme was purified, 110,000-fold from foetal bovine serum. Purified serum AChE cleaved 2 synthetic peptide substrates in a manner resembling the combined actions of trypsin-like and carboxypeptidase B-like enzymes. A synthetic fragment of preproenkephalin A (residues 97–107) containing a complete methionine-enkephalin sequence was cleaved by serum AChE to yield free methionine-enkephalin. The carboxypeptidase action of AChE was weakly stimulated by the presence of 100 μM CoCl 2 suggesting the requirement of a metal ion for complete activity. The results support the hypothesis that in many tissues AChE may act as a neuropeptide processing enzyme.

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.

Determination of the carboxyl termini of the alpha and beta subunits of yeast K1 killer toxin. Requirement of a carboxypeptidase B-like activity for maturation.

The carboxyl-terminal sequences of the two polypeptide chains of the Saccharomyces cerevisiae K1 killer toxin were determined by protein sequencing and amino acid analysis of peptide fragments generated from the mature, secreted toxin. The COOH-terminal amino acid of the beta chain is histidine 316, the final residue encoded by the precursor gene. The COOH terminus of the alpha chain is at alanine 147 of the preprotoxin. Amino acid composition data for the purified toxin are consistent with that predicted from the gene sequence of the preprotoxin where the alpha and beta subunits consist of amino acid residues 45-147 and 234-316, respectively. The molecular weight of the mature alpha beta dimer is about 20,658. The COOH-terminal sequence determination completes the location of the toxin subunits in the precursor, and its configuration may be represented as prepropeptide-Pro-Arg-alpha-Arg-Arg-gamma-Lys-Arg-beta, where gamma represents the interstitial glycosylated peptide. The COOH terminal side of the paired basic residues (Arg-148 Arg-149 and Lys-232 Arg-233 of preprotoxin) are endoproteolytic processing sites for the product of the KEX2 gene (Julius, D., Brake, A., Blair, L., Kunisawa, R., and Thorner, J. (1984) Cell 37, 1075-1089), and thus maturation of the alpha subunit of killer toxin apparently requires a carboxypeptidase B-like activity. A possible candidate for this activity is the product of the KEX1 gene (Dmochowska, A., Dignard, D., Henning, D., Thomas, D.Y., and Bussey, H. (1987) Cell, in press).

An endopeptidase associated with bovine neurohypophysis secretory granules cleaves pro-ocytocin/neurophysin peptide at paired basic residues

The octacosapeptide sequence [Tyr 18] pro-ocytocin/neurophysin (1–18)NH 2 [ pro-OT Np(1–18)NH 2 ] was synthesized and used as substrate to detect endoprotease(s) possibly involved in the processing of this precursor in bovine hypothalamo-neurohypophyseal tract. An endopeptidase (58 Kda) was detected in lysates made from highly purified neurosecretory granules. This protease which cleaves the peptide bond on the carboxyl side of the Lys-Arg doublet, and no single basic residue, generates both OT-Gly 10-Lys 11-Arg 12+Ala 13-Val-Leu-Asp-Leu-Tyr 18(NH 2) from the octacosapeptide substrate. In addition, a carboxypeptidase B-like activity converting OT-Gly 10-Lys 11-Arg 12 into OT-Gly 10 was detected in the same granule lysates. It is hypothesized that a combination of these endoprotease and carboxypeptidase B-like activities together with the amidating enzyme of secretory granules might participate in the cleavage and processing of pro-OT Np in vivo .

Carboxypeptidase B-like converting enzyme activity in secretory granules of rat pituitary.

Recent amino acid sequence data suggest that trypsin-like and carboxypeptidase B-like activities are required for the processing of pituitary prohormones--e.g., pro-opiocortin (pro-adrenocorticotropin/lipotropin) and provasopressin in secretory granules. In this study the existence of a carboxypeptidase B activity in purified secretory granules from anterior, intermediate, and neural lobes of rat pituitary has been examined. A carboxypeptidase B activity that cleaved the COOH-terminal -Lys-Lys-Arg residues from the adrenocorticotropin fragment ACTH-(1-17) (a potential hormone product liberated from pro-opiocortin by a trypsin-like enzyme) was detected in anterior and intermediate lobe granules. A similar carboxypeptidase B activity was also present in purified secretory granules from rat pituitary neural lobes that cleaved the -Lys-Arg residues from [Arg8]vasopressin-Gly-Lys-Arg, a potential product cleaved from provasopressin. Secretory granule carboxypeptidase(s) from the three lobes of the pituitary was shown to cleave 125I-[Met]enkephalin-Arg6 to form 125I-[Met]enkephalin as well. 125I-[Met]Enkephalin was used as a model substrate for the quantitative assay of pituitary carboxypeptidase activity. The carboxypeptidase B in secretory granules from all three lobes was shown to be active at pH 5.5, but not at pH 7.4. Inhibition by the zinc metallocarboxypeptidase inhibitors guanidinopropylsuccinic acid, aminomercaptosuccinic acid, benzylsuccinic acid, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and the potato carboxypeptidase B inhibitor, and inhibition by the metal chelators EDTA and 1,10-phenanthroline demonstrate metal ion dependence of the pituitary granule carboxypeptidase activities. However, Co2+ stimulated the secretory granule carboxypeptidase B activities. Thiol protease inhibitors such as Cu2+ and p-chloromercuriphenylsulfonic acid also inhibited the activity. Thus, the secretory granule carboxypeptidase B-like activities in all three lobes of the pituitary appear to be similar thiol-metallopeptidases that differ from other carboxypeptidase activities previously described and may play an exclusive role in hormone biosynthesis in the pituitary.

Carboxypeptidase B-like activity for the processing of enkephalin precursors in the membrane component of bovine adrenomedullary chromaffin granules

Trypsin and carboxypeptidase B-like (CPB-like) peptidases should be involved in processing proenkephalin to form the small biologically active enkephalins. A carboxypeptidase B-like activity from the soluble fraction of bovine adrenomedullary chromaffin granules which converts 125I-(Met)-enkephalin-Arg 6 to 125I-(Met)enkephalin has previously been described and characterized (1,2). In this study, CPB-like activity in the membrane bound component of chromaffin granules is characterized and compared with that in the soluble fraction. Membrane and soluble CPB activities cleaved 125I-(Met)-enkephalin-Arg 6 or 125I-(Met)enkephalin-Lys 6 to form 125I-(Met)enkephalin. Like the soluble enzyme, the CPB-like activity in the membrane component had a pH optimum of 6.0, was inhibited by thiol agents (PCMPSA, CuCl 2) and metal ion chelators (EDTA, 1, 10-phenanthroline), and was stimulated by Co ++. The membrane CPB-like activity appeared to be an intrinsic membrane protein, since 80% of the activity remained with the membranes after washing with 1.0 M NaCl. Membrane and soluble CPB-like activities in chromaffin granules appear to be similar enzymes.

A sensitive radiometric assay for enkephalin convertase and other carboxypeptidase B-like enzymes

A sensitive radiometric assay for carboxypeptidase B-like enzymes has been developed using enkephalin convertase, an enkephalin synthesizing carboxypeptidase. The assay is based on the differential solubility of 3H-labeled substrate and product in chloroform. The substrates 3H-benzoyl-Phe-Ala-Arg or 3H-benzoyl-Phe-Leu-Arg are poorly soluble in chloroform due to the charged arginine. The products of carboxypeptidase B-like activity on these substrates, 3H-benzoyl-Phe-Ala or 3H-benzoyl Phe-Leu partition quantitatively into chloroform, allowing rapid separation of product from substrate. This assay is approximately 100 times more sensitive than a similar fluorometric assay utilizing dansyl-Phe-Ala-Arg as a substrate.

Processing of enkephalin-containing peptides in isolated bovine adrenal chromaffin granules.

Intact chromaffin granules isolated from bovine adrenal medulla were incubated at 37 degrees C for up to 22 hr. Processing of enkephalin-containing (EC) peptides in the granules was followed by the change in their size distribution as shown by chromatography on Sephadex G-75 columns. A gradual shift toward lower molecular weight EC peptides was observed during the incubation, indicating processing of the higher molecular weight to lower molecular weight EC peptides. The total amount of [Met]-enkephalin, free and in peptide linkage, remained constant indicating that little or no nonspecific degradation occurred during the experiment. HPLC resolution of the fraction containing the low molecular weight EC peptides showed that free enkephalins as well as [Met]enkephalin-Arg6-Phe7 and [Met]enkephalin-Arg6-Gly7-Leu8 accumulated while [Met]enkephalin-Arg6 and [Met]enkephalin-Lys6 disappeared. All the above data indicate the presence of an atypical trypsin activity and the presence of a carboxypeptidase B-like activity within the granules. From the rates of accumulation of the low molecular weight EC peptides and the disappearance of the higher molecular weight EC peptides, a processing rate of 65-70 pmol/g tissue per hr was estimated, which calculates to a lifetime of 6-8 days for EC peptides in the granules. Under steady-state conditions this rate of processing appears to be too low to produce significant amounts of free enkephalins from larger EC peptides. This is well in accord with previous observations that relatively small amounts of free enkephalins are found in bovine adrenal medulla.

Pituitary endopeptidases.

This review summarizes our knowledge of pituitary endopeptidases. Emphasis has been placed on well-characterized enzymes and their potential roles in proteolytic processes of the pituitary. Because of space limitations, degradation of biologically active peptide by crude preparations has generally not been discussed. Only a few proteolytic enzymes are at present adequately characterized, and knowledge of their physiological function in vivo is insufficient. Among the many functions of proteolytic enzymes, those that are specific for the pituitary as an endocrine gland are of primary interest. Such functions include inactivation of neuropeptides and factors that control the secretory function of the pituitary, processing of precursors destined for secretion, selective cleavage of prohormones into active fragments, and degradation of inactive fragments. While some of the enzymes described here, such as cathepsin D, could be expected to have primarily a degradative function, others could potentially be involved in hormonal metabolism, since they exhibit trypsin-like, chymotrypsin-like, and dipeptidyl carboxypeptidase-like activities, all potentially useful in hormonal conversions. Data suggestive of the presence in the pituitary of enzymes involved in removal of the 'signal sequence', and enzymes involved in hormone processing by cleavage of bonds after a pair of basic residues and in the subsequent removal of these residues by a carboxypeptidase B-like activity have been published. None of these enzymes, however, has been isolated or purified to a degree that would allow determination of its specificity, mechanisms of action, physicochemical properties, and susceptibility to specific inhibitors. Questions that remain unresolved ask whether differences in the processing pathways in various anatomical parts of the pituitary are due to the presence of proteases with different specificities, or to different disposition of these enzymes, and factors, such as conformation of the substrate and its secondary modification, for example by glycosylation or phosphorylation. Proof of a functional involvement of a protease in hormonal processing should include demonstration that inhibition of activity results in inhibition of processing in the intact cell. Specific inhibitors of processing enzymes could potentially be used to modulate pituitary function, and thus have pharmacological interest. Although there are few answers to the above problems at present, the questions are well defined, and it can be expected that the rapidly expanding research on pituitary proteases will soon provide some of the answers.