Arginine and lysine product inhibition of bovine adrenomedullary carboxypeptidase H, a prohormone processing enzyme

Carboxypeptidase H (CPH) is one of the later enzymes in the cascade of proteolytic steps required for the posttranslational processing of peptide hormone precursors, including processing of proenkephalin. In this study, CPH activity in the soluble and membrane fractions of enkephalin-containing bovine chromaffin granules was competitively inhibited by its products arginine and lysine. Ki values for arginine and lysine were 4.6 +/- 1.3 and 7.6 +/- 1.9 mM, respectively, indicating that arginine was a more effective inhibitor than lysine. Other amino acids (at 10 mM) had no effect. The in vivo intragranular concentrations of lysine and arginine are similar to the measured Ki values, indicating that product inhibition of CPH by basic amino acids may occur in vivo.     

The interaction of myelin basic protein with tubulin and the inhibition of tubulin carboxypeptidase activity

Tubulin carboxypeptidase was found to be inhibited by myelin basic protein in a concentration dependent manner. The inhibition was produced by the interaction between myelin basic protein with the substrate. As a consequence of this interaction, turbid insoluble aggregates were formed at either 5 degrees or 37 degrees C. The turbidity increased by increasing the myelin basic protein concentration and it reached a plateau at a molar ratio of myelin basic protein to tubulin dimer of about 6. At plateau, the molar ration in the insoluble aggregates was about 6. When tubulin was in excess, the formation of the insoluble aggregates was diminished. However, if the excess of tubulin was added after the formation of the aggregates, the turbidity was not significantly affected. Turbidity was diminished by increasing the ionic strength.     

Arginine and lysine residues as NADH-binding sites in NADH-nitrate reductase from spinach.

Chemical modifications of spinach leaf nitrate reductase, and its 28,000 M(r) fragment with phenylglyoxal, 2,3-butanedione and pyridoxal phosphate reduce the catalytic activity of the enzyme. The kinetics of the modification indicate a rapid inactivation followed by a slower rate of inactivation. NADH-nitrate reductase, NADH-cytochrome c reductase and NADH-ferricyanide reductase activities of the nitrate reductase complex are inactivated at a faster rate when compared to the loss of FMNH2-nitrate reductase and reduced methyl viologen (MVH)-nitrate reductase activities. NADH protects the inactivation of NADH-ferricyanide reductase activity of the 28,000 M(r) fragment of nitrate reductase. These data suggest that nitrate reductase contains active sites of arginine and lysine residues that are involved in the NADH binding site of the enzyme.     

Catalytic activity of carboxypeptidase B and of carboxypeptidase Y with anisylazoformyl substrates

Anisylazoformyllysine (CH3OC6H4-N = N-CO-Lys-OH) is rapidly hydrolyzed at the acyl-lysine linkage by the zinc-enzyme porcine carboxypeptidase B. The catalytic reaction is readily monitored spectrophotometrically by disappearance of the intense absorption (348.5 nm, epsilon 18400) of the azo chromophore, which chemically fragments after substrate cleavage. Carboxypeptidase Y has no activity toward this type of substrate.     

Arginine carboxypeptidase (CPR) in human plasma determined with sandwich ELISA.

There are two types of carboxypeptidases present in human blood, carboxypeptidase N (CPN) and arginine carboxypeptidase (CPR). CPR is generated during coagulation from a precursor (proCPR) which can be converted to the active form by trypsin in vitro. Since it is difficult to distinguish the two types of carboxypeptidases in human blood by the measurement of enzyme activity, we established a quantitative sandwich ELISA by which CPR can be quantitated. The amount of CPR in plasma, fresh serum and heated serum were essentially the same. Therefore the ELISA assay does not distinguish proCPR, activated CPR and inactivated CPR. With the ELISA method, CPR was quantitated in plasma from fifty patients with rheumatoid arthritis and eleven patients with severe hepatitis as well as healthy individuals. The amount of CPR in plasma obtained from patients with rheumatoid arthritis was not found to be lower than that of normal subjects. Furthermore, the patients who suffered severe hepatitis and had very low levels of CPR-total were fatal. This suggests that a decrease of CPR level might be a good indication of a patient's prognosis to death by hepatitis.     

Effect of chronic ethanol consumption on basic carboxypeptidase activity in regions of the rat brain

It is discovered that chronic consumption of ethanol induced decrease of carboxypeptidase H activity in striatum by 27%; increase of carboxypeptidase M activity in hippocampus by 67% and decrease in cerebral hemispheres by 34%; phenylmethylsulfonyl fluoride-inhibited carboxypeptidase activity increase in hypothalamus by 141%, in striatum by 60% and in optic and lamina quadrigemina by 34%. The role of basic carboxypeptidases in mechanisms of ethanol influence on the peptidergic systems are discussed.     

Substitution of lysine 213 with arginine in penicillin-binding protein 5 of Escherichia coli abolishes D-alanine carboxypeptidase activity without affecting penicillin binding.

All penicillin-binding proteins (PBPs) contain a conserved box of homology in the carboxyl-terminal half of their primary sequence that can be Lys-Thr-Gly, Lys-Ser-Gly, or His-Thr-Gly. Site-saturation mutagenesis was used to address the role of the lysine residue at this position (Lys213) in Escherichia coli PBP 5, a D-alanine carboxypeptidase enzyme. A soluble form of PBP 5 was used to replace Lys213 with 18 other amino acids, and the ability of these mutant proteins to bind [3H]penicillin G was assessed. Only the substitution of lysine with arginine resulted in a protein that was capable of forming a stable covalent complex with antibiotic. The affinity of [14C]penicillin G for the arginine mutant was 1.2-fold higher than for wild-type PBP 5 (4.4 versus 5.1 micrograms/ml for 20 min at 30 degrees C), and both proteins showed identical rates of hydrolysis of the [14C]penicilloyl-bound complex (t1/2 = 9.1 min). Surprisingly, the arginine-substituted protein was unable to catalyze D-alanine carboxypeptidase activity in vitro, which suggests that there is a substantial difference in the geometries of the peptide substrate and penicillin G within the active site of PBP 5.     

Zinc deficiency and dipeptidyl carboxypeptidase activity

Dipeptidyl carboxypeptidase (DC) is highly active in the testis and epididymis of rats and increases during pubertal development. Zinc deficiency during this period depresses the activity of DC in the testis. Experiments were conducted to determine the effects of zinc deficiency on epididymal DC activity. Comparisons were made between changes seen in this organ and those observed in testis. Three dietary treatments were used; zinc-deficient, fed ad libitum; zinc-adequate, pair-fed to the deficient group; and zinc-adequate, fed ad libitum. Results confirmed that testicular DC is affected negatively by zinc deficiency. DC activity was also lower in the epididymis of zinc-deficient rats than in control rats. These effects apparently were specific relative to changes in activity of other enzymes. Alkaline phosphatase activity in the epididymis was not affected by zinc deficiency and it was depressed in the testis. Gamma-glytamyl transferase activity in the epididymis was not affected by zinc deficiency but it was elevated in the testis. The results of this study suggest that part of the effect of zinc deficiency on sexual maturity in the male rat may be caused by reduced activity of DC. This enzyme is thought to be required for maturation and development of sperm cells. Presented in part at the 1988 Joint Meeting of the North Dakota and South Dakota Academies of Science, Bismarck, ND, April 30, 1988. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture, and does not imply its approval to the exclusion of other products that may also be suitable.     

Inhibitors of protein phosphatase 1 and 2A decrease the level of tubulin carboxypeptidase activity associated with microtubules.

The association of tubulin carboxypeptidase with microtubules may be involved in the determination of the tyrosination state of the microtubules, i.e. their proportion of tyrosinated vs. nontyrosinated tubulin. We investigated the role of protein phosphatases in the association of carboxypeptidase with microtubules in COS cells. Okadaic acid and other PP1/PP2A inhibitors, when added to culture medium before isolation of the cytoskeletal fraction, produced near depletion of the carboxypeptidase activity associated with microtubules. Isolation of the native assembled and nonassembled tubulin fractions from cells treated and not treated with okadaic acid, and subsequent in vitro assay of the carboxypeptidase activity, revealed that the enzyme was dissociated from microtubules by okadaic acid treatment and recovered in the soluble fraction. There was no effect by nor-okadaone (an inactive okadaic acid analogue) or inhibitors of PP2B and of tyrosine phosphatases which do not affect PP1/PP2A activity. When tested in an in vitro system, okadaic acid neither dissociated the enzyme from microtubules nor inactivated it. In living cells, prior stabilization of microtubules with taxol prevented the dissociation of carboxypeptidase by okadaic acid indicating that dynamic microtubules are needed for okadaic acid to exert its effect. On the other hand, stabilization of microtubules subsequent to okadaic acid treatment did not reverse the dissociating effect of okadaic acid. These results suggest that dephosphorylation (and presumably also phosphorylation) of the carboxypeptidase or an intermediate compound occurs while it is not associated with microtubules, and that the phosphate content determines whether or not the carboxypeptidase is able to associate with microtubules.     

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.     

Elucidation of cathepsin B-like activity associated with extracts of human myelin basic protein

Myelin basic protein (MBP) extracted from human delipidated white matter was found to be degraded at pH 3.0 by endogenous proteolytic activities of extracts. Electrophoretic peptide patterns were consistent with limited proteolysis of MBP. Based on pH, activation by EDTA and DTE, and inhibition by p-CMPS, E-64 and, in particular, by leupeptin, the protease involved was tentatively identified as cathepsin B or a cathepsin B-like enzyme. As pepstatin failed to inhibit acid proteolysis of MBP cathepsin D was ruled out.     

Kinetics of activated thrombin-activatable fibrinolysis inhibitor (TAFIa)-catalyzed cleavage of C-terminal lysine residues of fibrin degradation products and removal of plasminogen-binding sites

Partial digestion of fibrin by plasmin exposes C-terminal lysine residues, which comprise new binding sites for both plasminogen and tissue-type plasminogen activator (tPA). This binding increases the catalytic efficiency of plasminogen activation by 3000-fold compared with tPA alone. The activated thrombin-activatable fibrinolysis inhibitor (TAFIa) attenuates fibrinolysis by removing these residues, which causes a 97% reduction in tPA catalytic efficiency. The aim of this study was to determine the kinetics of TAFIa-catalyzed lysine cleavage from fibrin degradation products and the kinetics of loss of plasminogen-binding sites. We show that the k(cat) and K(m) of Glu(1)-plasminogen (Glu-Pg)-binding site removal are 2.34 s(-1) and 142.6 nm, respectively, implying a catalytic efficiency of 16.21 μm(-1) s(-1). The corresponding values of Lys(77)/Lys(78)-plasminogen (Lys-Pg)-binding site removal are 0.89 s(-1) and 96 nm implying a catalytic efficiency of 9.23 μm(-1) s(-1). These catalytic efficiencies of plasminogen-binding site removal by TAFIa are the highest of any TAFIa-catalyzed reaction with a biological substrate reported to date and suggest that plasmin-modified fibrin is a primary physiological substrate for TAFIa. We also show that the catalytic efficiency of cleavage of all C-terminal lysine residues, whether they are involved in plasminogen binding or not, is 1.10 μm(-1) s(-1). Interestingly, this value increases to 3.85 μm(-1) s(-1) in the presence of Glu-Pg. These changes are due to a decrease in K(m). This suggests that an interaction between TAFIa and plasminogen comprises a component of the reaction mechanism, the plausibility of which was established by showing that TAFIa binds both Glu-Pg and Lys-Pg.     

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.     

The emergence of transgenic potatoes as commercial products and tools for basic science

The potato has tremendous potential as a transgenic crop and is a good model system by which to analyse metabolic regulation and gene expression. The potato’s difficult genetics, but ease of genetic transformation and its clonal means of propagation make it ideal for applied agricultural molecular genetics. Thus, the next 4 years promise to put the potato (with a diversity of transgenic constructs expressed) in the limelight as many of the first transgenic agricultural products enter the marketplace.     

Tripeptides with C-Terminal Arginine as Potential Inhibitors of Urokinase

We synthesized and tested ten peptides with the molecular structure being H–d-Ser–AA–Arg–OH for their effect on the amidolytic activities against urokinase, thrombin, trypsin, plasmin, tissue plasminogen activator and kallikrein. The inserted amino acid in each peptide was either leucine, norleucine, izoleucine, valine, norvaline, α-metyloalanine, α-aminobutanoic acid, homoleucine, tert-leucine or neoglycine. H–d-Ser–NVal–Arg–OH (compound 4) was the most active inhibitor of urokinase plasminogen activator with a K_i value of 0.85 μM. Compound 4 showed cytotoxic effect against MDA-MB-231 and DLD cell lines, respectively, with IC₅₀ values of 25 and 19 μM. Synthesised compounds did not have activity against MCF-7 cancer cells. These peptides were nontoxic against pig’s erythrocytes in vitro.