Isolation, molecular cloning, and partial characterization of a novel carboxypeptidase B from human plasma

A novel plasminogen-binding protein has been isolated from human plasma utilizing plasminogen-Sepharose affinity chromatography. This protein copurified with alpha 2 antiplasmin when the plasminogen affinity column was eluted with high concentrations of epsilon-aminocaproic acid (greater than 20 mM). Analysis by sodium dodecyl sulfate suggests this protein has an apparent Mr of 60,000. The amino-terminal amino acid sequence showed no similarity to other protein sequences. Based on the amino-terminal amino acid sequence, oligonucleotide probes were designed for polymerase chain reaction primers, and an approximately 1,800 base pair cDNA was isolated that encodes this Mr 60,000 protein. The deduced amino acid sequence reveals a primary translation product of 423 amino acids that is very similar to carboxypeptidase A and B and consists of a 22-amino acid signal peptide, a 92-amino acid activation peptide, and a 309-amino acid catalytic domain. This protein shows 44 and 40% similarity to rat procarboxypeptidase B and human mast cell procarboxypeptidase A, respectively. The residues critical for catalysis and zinc and substrate binding of carboxypeptidase A and B are conserved in the Mr 60,000 plasminogen-binding protein. The presence of aspartic acid at position 257 of the catalytic domain suggests that this protein is a basic carboxypeptidase. When activated by trypsin, it hydrolyzes carboxypeptidase B substrates, hippuryl-Arg and hippuryl-Lys, but not carboxypeptidase A substrates, and it is inhibited by the specific carboxypeptidase B inhibitor (DL-5-guanidinoethyl)mercaptosuccinic acid. We propose that the Mr 60,000 plasminogen-binding protein isolated here is a novel human plasma carboxypeptidase B and that it be designated pCPB.     

Inhibition of the development of the cellular slime mould Dictyostelium discoideum by omega-aminocarboxylic acids.

Four omega-aminocarboxylic acids - epsilon-aminocaproic acid (EACA), trans-4-aminomethylcyclohexane-1-carboxylic acid (t-AMCHA), p-aminomethylbenzoic acid (PAMBA) and omega-aminocaprylic acid (OACA) -- prevented fruiting body formation of the cellular slime mould Dictyostelium discoideum. At concentrations of 40 mM, 75 mM, 10 mM and 5 mM, respectively, they allowed aggregation but prevented all further development at 24 degrees C. At lower concentrations, EACA allowed fruiting body formation but with a reduced number of spores per fruiting body. Only t-AMCHA had a significant inhibitory effect on the growth of myxamoebae. EACA affected development only if it was present between 8 and 16 h after the cells were deposited on the filters. Its effect was enhanced by high salt concentrations and by higher temperature, and was also dependent on the manner in which the cells were grown. Only strains capable of axenic growth displayed this sensitivity to EACA, although strains carrying only one of the genetic markers for axenic growth (axe A) were partially sensitive.     

Actions of two serine proteases from Trimeresurus jerdonii venom on chromogenic substrates and fibrinogen

Jerdonobin and jerdofibrase are two serine proteases purified from the venom of Trimeresurus jerdonii. The Michaelis constant K(m) and the catalytic rate constant K(cat) of jerdonobin or jerdofibrase on three chromogenic substrates, H-D-Pro-Phe-Arg-pNA (S2302), H-D-Phe-pipecolyl-Arg-pNA (S2238), and H-D-Val-Leu-Lys-pNA (S2251) were obtained from lineweaver-Burk plots. Jerdofibrase could hydrolyze all three substrates, but jerdonobin had no detectable activity on S2251, suggesting a relatively broader substrate specificity for jerdofibrase than jerdonobin. By SDS-PAGE, jerdofibrase preferentially degraded Bbeta-chain of fibrinogen. It also degraded Aalpha-chain of fibrinogen with relatively slow activity, but did not act on the gamma-chain. In contrast, jerdonobin did not degrade fibrinogen within 12 h. Fibrinopeptides liberation test, identified by HPLC, showed jerdonobin released fibrinopeptide A and a small amount of fibrinopeptide B. Unlike jerdonobin, jerdofibrase mainly released fibrinopeptide B. These results indicate that the two enzymes differ in their ability to hydrolyze chromogenic substrates and in their actions on fibrinogen.     

Carboxypeptidase T--intracellular carboxypeptidase of Thermoactinomycetes--a distant analog of animal carboxypeptidase

Carboxypeptidase T, an extracellular carboxypeptidase from Thermoactinomyces sp. was isolated and purified by affinity chromatography on bacitracin adsorbents. The enzyme homogeneity was established by SDS electrophoresis (Mr = 38 000) and isoelectrofocusing in PAAG (pI 5.3). Carboxypeptidase T reveals a mixed specificity in comparison with pancreatic carboxypeptidases A and B and cleaves with nearly the same efficiency the peptide bonds formed by the C-terminal residues of basic and neutral hydrophobic amino acids. The enzyme is insensitive to serine and thiol proteinase inhibitors but is completely inhibited by EDTA and o-phenanthroline. The maximal enzyme activity is observed at pH 7-8. With an increase of temperature from 20 to 70 degrees C the enzyme activity is enhanced approximately 10-fold. In the presence of 1 mM Ca2+ the enzyme thermostability is also increased. In terms of some properties, e.g. substrate specificity carboxypeptidase T is similar to metallocarboxypeptidase secreted by Streptomyces griseus. The N-terminal sequence of carboxypeptidase T: Asp-Phe-Pro-Ser-Tyr-Asp-Ser-Gly- Tyr-His-Asn-Tyr-Asn-Glu-Met-Val-Asn-Lys-Ile-Asn-Thr-Val-Ala-Ser-Asn-Tyr- Pro-Asn - Ile-Val-Lys-Thr-Phe-Ser-Ile-Gly-Lys-Val-Tyr-Glu-Gly-Xaa-Gly-Leu- coincides by 21% with that of pancreatic carboxypeptidases A and B. Thus, it may be concluded that these enzymes originate from a common precursor.     

Decreased levels of alpha 1(I) procollagen mRNA in dermal fibroblasts grown on fibrin gels and in response to fibrinopeptide B

We have investigated human neonatal fibroblast synthetic activity in response to fibrin substrates and components of fibrin formation and degradation. Greater than threefold downregulation of procollagen mRNA levels was seen 24 hours after fibroblasts were grown on fibrin gels as compared to tissue culture plastic. This downregulation occurred in both reptilase-generated fibrin (retention of fibrinopeptide B) and thrombin-generated fibrin (loss of both fibrinopeptide A and B). However, fibroblasts grown on fibrin retained their capacity to respond to the stimulatory action of transforming growth factor (TGF)-beta 1. Fibroblasts seeded on reptilase-generated fibrin displayed an abnormal morphology manifested by dendritic appearance and cell rounding, while fibroblast attachment was enhanced by 30% on thrombin-generated fibrin substrate (P < 0.02). Fibrinopeptides A and B, which are generated during fibrin formation, increased and decreased procollagen mRNA levels, respectively. Tissue plasminogen activator (t-PA) increased procollagen mRNA and TGF-beta 1 levels as early as 6 hours after cells were grown on tissue culture plastic, but this stimulation did not occur in cells cultured on a fibrin substrate. We conclude that alpha 1(I) procollagen mRNA levels in cultures of human dermal fibroblasts are consistently down-regulated by a fibrin substrate and are directly and profoundly influenced by complex interactions between components involved in the formation and removal of fibrin. © 1995 Wiley-Liss, Inc.     

Molecular cloning and sequencing of the cDNA for human membrane-bound carboxypeptidase M. Comparison with carboxypeptidases A, B, H, and N

Carboxypeptidase M, a widely distributed membrane-bound carboxypeptidase that can regulate peptide hormone activity, was purified to homogeneity from human placenta (Skidgel, R. A., Davis, R. M., and Tan, F. (1989) J. Biol. Chem. 264, 2236-2241). The NH2-terminal 31 amino acids were sequenced, and two complementary oligonucleotide probes were synthesized and used to isolate a carboxypeptidase M clone from a human placental cDNA library. Sequencing of the cDNA insert (2009 base pairs) revealed an open reading frame of 1317 base pairs coding for a protein of 439 residues. The NH2-terminal protein sequence matched the deduced amino acid sequence starting with residue 14. Hydropathic analysis revealed hydrophobic regions at the NH2 and COOH termini. The NH2-terminal 13 amino acids probably represent part of the signal peptide, and the COOH-terminal hydrophobic region may act either as a transmembrane anchor or as a signal for attachment to a phosphatidylinositol glycan moiety. The carboxypeptidase M sequence contains six potential Asn-linked glycosylation sites, consistent with its glycoprotein nature. The sequence of carboxypeptidase M was 41% identical with that of the active subunit of human plasma carboxypeptidase N, 41% identical with bovine carboxypeptidase H (carboxypeptidase E, enkephalin convertase), and 15% with either bovine pancreatic carboxypeptidase A or B. Many of the active site residues identified in carboxypeptidases A and B, including all of the zinc-binding residues (2 histidines and a glutamic acid), are conserved in carboxypeptidase M. These data indicate that all of the metallocarboxypeptidases are related, but the nondigestive carboxypeptidases with more specialized functions, present in cell membranes, blood plasma, or secretory granules (i.e., carboxypeptidase M, carboxypeptidase N and carboxypeptidase H), are more closely related to each other (41-49% identity) than they are to carboxypeptidase A or B (15-20% identity).     

Rat mast cell carboxypeptidase: amino acid sequence and evidence of enzyme activity within mast cell granules

The amino acid sequence of rat mast cell carboxypeptidase has been determined. The major form has 308 residues; a minor form has an additional (glutamyl) residue at the amino terminus that may indicate an alternate cleavage site during zymogen activation. The enzyme is homologous to pancreatic carboxypeptidases A and B, with conservation of the functional amino acid residues of the active site. The putative substrate binding site resembles that of carboxypeptidase A, although other structural features bear more similarity to carboxypeptidase B. Mast cell carboxypeptidase retains enzymatic activity toward a peptide substrate (angiotensin I) while bound within the granular matrix of the rat connective tissue mast cells. Evidence is presented to suggest that a cluster of positively charged lysyl and arginyl residues binds the enzyme to the negatively charged heparin of the granular matrix but leaves the active site exposed to bind and cleave peptide substrates.     

Cultured Astrocytes and Neurons Synthesize and Secrete Carboxypeptidase E, a Neuropeptide-Processing Enzyme

Carboxypeptidase E (EC 3.4.17.10) is a carboxypeptidase B-like enzyme associated with the biosynthesis of many peptide hormones and neurotransmitters. Media collected from cultured astrocytes contain a carboxypeptidase E-like activity. Cultured astrocytes secrete approximately 73% of their cellular level of carboxypeptidase E per hour, and secretion is not substantially influenced by 35 mM KCl. In contrast, neurons secrete only 29% of their cellular carboxypeptidase E per hour, but secretion increases to 86% on stimulation with 35 mM KCl. Secretion of carboxypeptidase E activity from both neuronal and astrocyte cultures is relatively selective; neither acid phosphatase or acetylglucosaminidase is secreted in appreciable amounts. Cultured neurons and astrocytes express a carboxypeptidase E mRNA of a similar size. The levels of this mRNA differ in astrocytes cultured from different brain regions, with high levels in striatal, cortical, hippocampal, and hypothalamic astrocytes and low levels in cerebellar astrocytes. The level of carboxypeptidase E mRNA in hypothalamic astrocyte cultures is four- to fivefold higher than the level in hypothalamic neuronal cultures. These results indicate that cultured astrocytes express carboxypeptidase E mRNA and enzymatic activity and thus contain one of the enzymes required in the biosynthesis of many peptide hormones and neurotransmitters.     

Reversible inactivation of pancreatic deoxyribonuclease A by sodium dodecyl sulfate. Removal of COOH-terminal residues from the denatured protein by carboxypeptidase A.

In the course of experiments on the role of the COOH-terminal residues in pancreatic deoxyribonuclease, we undertook to ascertain whether the presence of sodium dodecyl sulfate would render the normally unavailable terminus susceptible to hydrolysis by carboxypeptidase A. When DNase A is dissolved in 0.005% sodium dodecyl sulfate the protein becomes enzymically inactive when assayed against DNA in the same sodium dodecyl sulfate concentration. The loss of activity caused by treatment with sodium dodecyl sulfate for 1 hour at 45 degrees can be fully restored if the detergent-containing solution is diluted 10-fold into 6 M guanidinium chloride and then 10-fold into a pH 7.0 buffer, 10 mM in CaCl2, prior to a 100-fold dilution for assay. The presence of Ca2+ is essential for the refolding process. If the same degree of dilution is made into sodium dodecyl sulfate-free buffer without the guanidinium chloride step, there is very little reversal of the inactivation. An almost complete loss of regenerable activity is caused by 1 hour of digestion by carboxypeptidase at 45 degrees in the presence of 0.03% sodium dodecyl sulfate. Although up to 6 amino acid residues can be removed from the COOH terminus, the loss of activity can be correlated with the removal of either 1 or 2 amino acid residues (-Leu-Thr) from the COOH-terminal sequence. Thus, DNase A is one of the several enzymes in which residues at the COOH terminus are essential to the active conformation. If the enzyme minus 2 to 6 terminal residues was mixed with a 15-residue COOH-terminal peptide (obtained by cyanogen bromide cleavage), only about 2% activity could be regenerated.     

Purification and characterization of a new arginine carboxypeptidase in human serum

A carboxypeptidase capable of cleaving basic amino acids from synthetic peptide substrates is present in fresh human serum, and not in human heparinized plasma. Its activity is generated during the process of coagulation. Because of its unstability at room temperature and at 37 degrees C, we named it unstable carboxypeptidase (carboxypeptidase U). Carboxypeptidase U was partially purified from fresh human serum by chromatography on DEAE-cellulose and Mono-Q sepharose and was found to be a 435 kDa protein. We compared this enzyme with carboxypeptidase N, purified from human serum by a two-step affinity chromatography on arginine-Sepharose 4B, followed by ion-exchange chromatography on Mono-Q sepharose. Carboxypeptidase U cleaves hippuryl-L-arginine and hippuryl-L-lysine, but at a different relative rate than carboxypeptidase N, and has no esterase activity on hippuryl-L-argininic acid. Its activity was inhibited by o-phenanthroline, DL-2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, CoCl2, 2-mercaptoethanol, dithiothreitol and 4-chloromercuribenzoic acid. These characteristics differentiate carboxypeptidase U from carboxypeptidase N and other known carboxypeptidases.     

Isolation and characterization of a basic carboxypeptidase from human seminal plasma

A carboxypeptidase which cleaves the C-terminal arginine or lysine from peptides was purified by a two-step procedure; gel filtration on Sephacryl S-300 and affinity chromatography on arginine-Sepharose. The activity increased 280% after the first step, indicating the removal of an inhibitor from the crude starting material. The activity in the crude seminal plasma eluted from the Sephacryl S-300 column with an apparent Mr 98,000 and after purification with an Mr 67,000, indicating that it binds to another protein in the crude seminal plasma. When analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, a single band at Mr 53,000 was seen which was converted to two smaller bands (Mr 32,000 and/or 26,000) after reduction. The seminal plasma carboxypeptidase has a neutral pH optimum, is inhibited by o-phenanthroline and by the inhibitor of carboxypeptidase B-type enzymes, 2-mercaptomethyl-3-guanidinoethylthiopropanoic acid, and can be activated by cobalt. The purified enzyme has a high specific activity (67.8 mumol/min/mg) with the ester substrate benzoyl (Bz)-Gly-argininic acid and readily cleaves Bz-Ala-Lys, Bz-Gly-Arg, and Bz-Gly-Lys. It also hydrolyzes biologically active peptides such as bradykinin (Km = 6 microM, kcat = 43 min-1), Arg6-Met5-enkephalin (Km = 103 microM, kcat = 438 min-1), and Lys6-Met5-enkephalin (Km = 848 microM, kcat = 449 min-1). The seminal plasma carboxypeptidase did not cross-react with antiserum to human plasma carboxypeptidase N; other properties distinguish it from the blood plasma enzyme as well as from pancreatic carboxypeptidase B and granular, acid carboxypeptidase H (enkephalin convertase). The carboxypeptidase could be involved in the control of fertility by activating or inactivating peptide hormones in the seminal plasma. In addition it could contribute to the degradation of basic proteins during semen liquefaction.     

Excess zinc ions are a competitive inhibitor for carboxypeptidase A

The mechanism for inhibition of enzyme activity by excess zinc ions has been studied by kinetic and equilibrium dialysis methods at pH 8.2, I = 0.5 M. With carboxypeptidase A (bovine pancreas), peptide (carbobenzoxyglycyl-L-phenylalanine and hippuryl-L-phenylalanine) and ester (hippuryl-L-phenyl lactate) substrates were inhibited competitively by excess zinc ions. The Ki values for excess zinc ions with carboxypeptidase A at pH 8.2 are all similar [Ki = (5.2-2.6) X 10(-5) M]. The apparent constant for dissociation of excess zinc ions from carboxypeptidase A was also obtained by equilibrium dialysis at pH 8.2 and was 2.4 X 10(-5) M, very close to the Ki values above. With arsanilazotyrosine-248 carboxypeptidase A ([(Azo-CPD)Zn]), hippuryl-L-phenylalanine, carbobenzoxyglycyl-L-phenylalanine, and hippuryl-L-phenyl lactate were also inhibited with a competitive pattern by excess zinc ions, and the Ki values were (3.0-3.5) X 10(-5) M. The apparent constant for dissociation of excess zinc ions from arsanilazotyrosine-248 carboxypeptidase A, which was obtained from absorption changes at 510 nm, was 3.2 X 10(-5) M and is similar to the Ki values for [(Azo-CPD)Zn]. The apparent dissociation and inhibition constants, which were obtained by inhibition of enzyme activity and spectrophotometric and equilibrium dialysis methods with native carboxypeptidase A and arsanilazotyrosine-248 carboxypeptidase A, were almost the same. This agreement between the apparent dissociation and inhibition constants indicates that the zinc binding to the enzymes directly relates to the inhibition of enzyme activity by excess zinc ions. Excess zinc ions were competitive inhibitors for both peptide and ester substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

Electrochemiluminescence assay for basic carboxypeptidases: inhibition of basic carboxypeptidases and activation of thrombin-activatable fibrinolysis inhibitor

Carboxypeptidases catalyze the removal of the C-terminal amino acid residues in peptides and proteins and exert important biological functions. Assays for carboxypeptidase activity that rely on change of absorbance generally suffer from low sensitivity and are difficult to adapt to high-throughput screening. We have developed a sensitive, robust assay for basic carboxypeptidase activity that makes use of electrochemiluminescent (ECL) detection of reaction product. In this assay, a peptide substrate contains the epitope for antibody (G2-10) binding which is masked by a C-terminal arginine. Carboxypeptidase activity exposes the epitope, allowing the binding of ruthenylated G2-10 which is then detected using ECL. High sensitivity allowed detection limits of 1-2 pM enzyme for carboxypeptidase B and activated thrombin-activatable fibrinolysis inhibitor (TAFIa). The inhibition of several basic carboxypeptidases by commercially available inhibitors was studied. This antibody-based method can be extended to other sensitive detection techniques such as amplified luminescent proximity homogeneous assay. The high sensitivity of the assay allowed the determination of the activatable levels of TAFI in human and other animal plasma in the presence of epsilon -aminocaproic acid, an active-site inhibitor that stabilizes TAFIa. A method to isolate in situ activated TAFIa from human serum in the presence of epsilon -aminocaproic acid was also developed.     

Peptides with 6-Aminohexanoic Acid: Synthesis and Evaluation as Plasmin Inhibitors

Fifteen new peptide derivatives of ?-aminocaproic acid (EACA) containing the known fragment –Ala–Phe–Lys– with an affinity for plasmin were synthesised in the present study. The synthesis was carried out a solid phase. The following compounds were synthesised: H–Phe–Lys–EACA–X, H–d-Ala–Phe–Lys–EACA–X, H–Ala–Phe–Lys–EACA–X, H–d-Ala–Phe–EACA–X and H–Ala–Phe–EACA–X, where X = OH, NH₂ and NH–(CH₂)₅–NH₂. All peptides, except for those containing the sequence H–Ala–Phe–EACA–X, displayed higher inhibitory activity against plasmin than EACA. The most active and selective inhibitor of plasmin was the compound H–d-Ala–Phe–Lys–EACA–NH₂ which inhibited the amidolytic activity of plasmin (IC₅₀ = 0.02 mM), with the antifibrinolytic activity weaker than EACA. The resulting peptides did not affect the viability of fibroblast cells, colon cancer cell line DLD-1, breast MCF-7 and MDA-MB-231 cell lines.     

The kininases of Bothrops jararaca plasma

Evidence is presented to suggest that kininase activity of Bothrops jararaca plasma is due to the presence of at least three distinct enzymes: a carboxypeptidase B type enzyme, similar to that found in human plasma in that its activity is enhanced by Co2+ (1 X 10(-4) M); a carboxypeptidase B type enzyme whose activity is unaffected by Co2+, and an enzyme which cleaves bradykinin to liberate Phe-Arg as the major peptide fragment formed. The latter enzyme is responsible for the major kininase activity of this snake plasma and is identified as a dipeptide hydrolase.     

On the substrate specificity of cathepsins B1 and B2 including a new fluorogenic substrate for cathepsin B1.

Cathepsin B1 from bovine spleen exhibited its greatest rates of hydrolysis on peptide β-naphthylamide (βNA) derivatives containing paired basic residues, i.e., Cbz-Arg-Arg-βNA, t-Boc-Lys-Lys-βNA, and t-Boc-Lys-Arg-βNA. Internal peptide bonds were not attacked. At its pH 6.5 optimum, cathepsin B1 hydrolyzed Cbz-Arg-Arg-βNA (K_m 0.18 mM) 64 times faster than Bz-DL-Arg-βNA (K_m 3.3 mM or 1.6 mM for the L isomer) and was therefore chosen to replace the latter as a more soluble and sensitive substrate for the assay of cathepsin B1. Although cathepsin B2 had no action on the β-naphthylamide substrates, it did manifest carboxypeptidase activity by attacking COOH-terminal residues exposed by the action of cathepsin B1. At its pH 5.0 optimum, cathepsin B2 behaved as a SH-dependent, non-specific carboxypeptidase by releasing COOH-terminal amino acids from a variety of Cbz-Gly-X substrates and polypeptides such as glucagon, Val-Leu-Ser-Glu-Gly, and penta-lysine.     

Carboxypeptidase A in mouse mast cells. Identification, characterization, and use as a differentiation marker

By using a conventional spectrophotometric assay with hippuryl-L-phenylalanine as the substrate, 10(6) BALB/c mouse serosal mast cells possessed 1.5 +/- 0.43 U (mean +/- SE, n = 5, range = 0.48 to 2.5) of carboxypeptidase A activity, while T cell factor-dependent, mouse bone marrow-derived mast cells (BMMC) had barely detectable levels of 0.01 +/- 0.001 U/10(6) cells (mean +/- SE, n = 3). In order to characterize the carboxypeptidase A present in the BMMC, a sensitive assay was developed that used angiotensin I as the substrate and reverse phase-high performance liquid chromatography to separate and quantify production of the cleavage product des-leu-angiotensin I. Using this assay, mouse BMMC carboxypeptidase A had a neutral to basic pH optimum and hydrolyzed angiotensin I with a Km of 0.78 mM. The antigen-induced net percent release of carboxypeptidase A from IgE-sensitized BMMC was proportional to that of the secretory granule component beta-hexosaminidase which indicates a secretory granule location for the exopeptidase. As defined by exclusion during Sepharose CL-2B chromatography, carboxypeptidase A was exocytosed as a greater than 1 X 10(7) m.w. complex bound to proteoglycans. Because BMMC cocultured with mouse skin-derived 3T3 fibroblasts are known to undergo an increase in histamine content and biosynthesis of 35S-labeled heparin proteoglycans, carboxypeptidase A activity was measured during BMMC/fibroblast coculture for 0 to 28 days. The carboxypeptidase A activity increased progressively during 28 days of co-culture from 0.004 +/- 0.002 U/10(6) starting BMMC (mean +/- SE, n = 3) to 0.36 +/- 0.10 U/10(6) co-cultured mast cells. These findings indicate that carboxypeptidase A, a neutral protease, is exocytosed from the secretory granules of mouse mast cells bound to proteoglycan and is increased during the in vitro differentiation of mouse BMMC from mucosal-like mast cells to serosal-like mast cells.     

Appearance of gamma-D-glutamyl-(L) meso-diaminopimealate peptidoglycan hydrolase during sporulation in Bacillus sphaericus

Particulate preparations from sporulating cells of Bacillus sphaericus 9602 contained an endopeptidase activity that hydrolyzed the gamma-d-glutamyl-(l)meso-diaminopimelic acid linkages found in the spore cortical peptidoglycan of this organism. Diaminopimelic acid did not occur in the vegetative cell wall peptidoglycan, and the gamma-d-glutamyl-l-lysine linkages found in this polymer were not hydrolyzed by the endopeptidase. The endopeptidase hydrolyzed (X)-l-alanyl-gamma-d-glutamyl-(l)meso-diaminopimelyl(l)-d-alanyl-d-alanine only after removal of the terminal d-alanine residue. The preparations contained an acyl-d-alanyl-d-alanine carboxypeptidase I activity which converted such pentapeptides into substrates for the endopeptidase and which was inhibited 50% by 4 x 10(-7) M benzylpenicillin. This activity also hydrolyzed the analogous pentapeptide substrates containing l-lysine. The preparations also contained an acyl-l-lysyl-d-alanine carboxypeptidase II activity that was not active on the meso-diaminopimelic acid-containing analogue. Neither this activity nor the endopeptidase was inhibited by 10(-3) M benzylpenicillin. The specificities of the carboxypeptidases were consistent with the exclusive presence of l-lysine C-termini in the vegetative peptidoglycan and of meso-diaminopimelyl-d-alanine C-termini in the spore cortical peptidoglycan of B. sphaericus 9602.     

Hydrophobic-ionic chromatography. Its application to purification of porcine pancreas enzymes.

1. All the porcine pancreas enzymes tested, regardless of their pI's were adsorbed on Amberlite CG-50 (a weakly acidic cation exchange resin) at pH 4, where the ion-exchange group (carboxyl group) is not dissociated. The adsorption is hardly influenced by ionic strength. 2. At pH 4, the adsorbed enzymes were partially eluted by organic solvents such as 50% propanol. 3. The adsorbed enzymes were effectively eluted by increasing the pH from 4 to 6. Trypsin (pI 10.5) was eluted before carboxypeptidase A (pI 4.5 AND 5.3) WITH 0.5 M acetate buffer, whereas the former enzyme was eluted after the latter enzyme with 0.2 M 3,3-dimethyl glutarate buffer. However, with either buffer, the elution order of enzymes was not always the same as the order of the pI's. 4. By a single Amberlite CG-50 column chromatography of porcine pancreas extracts, kallikrein, carboxypeptidase B, deoxyribonuclease, carboxypeptidase A, and trypsin were purified 100-fold, 16-fmately 13%. The purification procedures included treatment with protamine, ammonium sulfate fractionation, treatment with acid, DE-32 cellulose column chromatography, gel filtration on Sephadex G-100, preparative polyacrylamide gel electrophoresis, and affinity chromatography on 5' AMP-Sepharose 4B. The last procedure, affinity chromatography on 5' AMP-Sepharose 4B, was useful for the removal of other dehydrogenases. The enzyme which was homogeneous, as shown by polyacrylamide gel electrophoresis, had a molecular weight of about 92,000. The optimum pH was at 10.0 and isoelectric point at 5.2. The enzyme accepted both L-fucose and D-arabinose as substrate, but was specific for NAD+ as coenzyme. Km values were 0.15 mM, 1.4 mM, and 0.07 mM for L-fucose, D-arabinose, and NAD+, respectively. A single enzyme catalyzed the oxidation of L-fucose and D-arabinose, which had the same configurations of hydroxyl groups from C-2 to C-4. The reaction products obtained with L-fucose as substrate were L-fucono-lactone and L-fuconic acid. The L-fucono-lactone was an immediate product of oxidation and was hydrolyzed to L-fuconic acid spontaneously. This reaction was irreversible. Therefore, it is likely that L-fucose dehydrogenase is involved in the initial step of the catabolic pathway of L-fucose in rabbit liver.