Dissection of glutathione conjugate turnover in yeast

Xenobiotics are widely used as pesticides. The detoxification of xenobiotics frequently involves conjugation to glutathione prior to compartmentalization and catabolism. In plants, degradation of glutathione-S-conjugates is initiated either by aminoterminal or carboxyterminal amino acid cleavage catalyzed by a γ-glutamyl transpeptidase and phytochelatin synthase, respectively. In order to establish yeast as a model system for the analysis of the plant pathway, we used monochlorobimane as a model xenobiotic in Saccharomyces cerevisiae and mutants thereof. The catabolism of monochlorobimane is initiated by conjugation to form glutathione-S-bimane, which is then turned over into a γ-GluCys-bimane conjugate by the vacuolar serine carboxypeptidases CPC and CPY. Alternatively, the glutathione-S-bimane conjugate is catabolized by the action of the γ-glutamyl transpeptidase Cis2p to a CysGly-conjugate. The turnover of glutathione-S-bimane was impaired in yeast cells deficient in Cis2p and completely abolished by the additional inactivation of CPC and CPY in the corresponding triple knockout. Inducible expression of the Arabidopsis phytochelatin synthase AtPCS1 in the triple knockout resulted in the turnover of glutathione-S-bimane to the γ-GluCys-bimane conjugate as observed in plants. Challenge of AtPCS1-expressing yeast cells with zinc, cadmium, and copper ions, which are known to activate AtPCS1, enhanced γ-GluCys-bimane accumulation. Thus, initial catabolism of glutathione-S-conjugates is similar in plants and yeast, and yeast is a suitable system for a study of enzymes of the plant pathway.     

Occurrence of acid and neutral carboxypeptidases in germinating cereals

High neutral metallocarboxypeptidase activity (EC 3.4.17) has earlier been detected in young seedlings of rice ( Oryza sativa L.) using benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala) as substrate at pH 7. This finding was confirmed, and it was observed that the activity could be assayed with higher specificity and sensitivity by using Z-Gly-Ala or Z-Gly-Phe as substrate at pH 6.5–7. No corresponding activity was detected in seedlings of barley ( Hordeum vulgare L. cv. Himalaya), oats ( Avena sativa L.) or maize ( Zea mays L.). The seedlings of the four cereals possessed similar activities of acid carboxypeptidases (EC 3.4.16; hydrolysis of Z-Phe-Ala and Z-Ala-Phe at pH 5.2 and of Z-Ala-Arg at pH 5.7). However, in endosperms of germinating rice and maize these activities were only about 1–5% of those in barley and oats. A corresponding, although less pronounced, difference was evident between the scutella of the two pairs of cereals. The possible relationship between neutral carboxypeptidase activity and ability to grow in anaerobic conditions is discussed.     

Precursors of the Cocoa-Specific Aroma Components are Derived from the Vicilin-Class (7S) Globulin of the Cocoa Seeds by Proteolytic Processing

Essential precursors of the cocoa-specific aroma components are formed during fermentation of cocoa seeds in the tropics. During the past decades, indications have accumulated that these aroma precursors are derived from seed proteins by acid-induced proteolysis. This has been recently corroborated by in vitro studies on the formation of the aroma-related components. It has been shown that the essential proteolysis products are derived from the vicilin-class (7S) globulins of the cocoa seeds by the cooperative action of an endogenous aspartic endoprotease and a carboxypeptidase.     

The association of tubulin carboxypeptidase activity with microtubules in brain extracts is modulated by phosphorylation/dephosphorylation processes

Tubulin carboxypeptidase, the enzyme which releases the COOH terminal tyrosine from the a-chain of tubulin, remains associated with microtubules through several cycles of assembly/disassembly (Arce CA, Barra HS: FEBS Lett 157: 75–78, 1983). Here, we present evidence indicating that in rat brain extract the carboxypeptidase/microtubules association is regulated by the relative activities of endogenous protein kinase(s) and phosphatase(s) which seem to determine the phosphorylation state of the enzyme (or another entity) and in some way the affinity of the enzyme for microtubules. The presence of 2.5 mM ATP during the in vitro microtubule formation resulted in a low recovery of carboxypeptidase activity in the microtubule fraction. This ATP-induced effect was not due to alteration of the enzyme activity or to inhibition of microtubule assembly but to a decrease of the association of the enzyme with microtubules. We found that the ATP-induced effect was not mediated by modifications on the microtubules but, presumably, on the enzyme molecule. The non-hydrolyzable ATP analogue, AMP-PCP, did not reproduce the effect of ATP. The inclusion of phosphatase inhibitors in the homogenization buffer also led to a decrease in the amount of tubulin carboxypeptidase associated with microtubules. Finally, we found that, in concordance with the mechanism hypothesized, the magnitude of the carboxypeptidase/microtubule association correlated well with the different incubation conditions created to favor maximal, minimal or intermediate protein phosphorylation states.     

Activity cycles and the control of four digestive proteinases in the posterior midgut of Rhodnius prolixus Stål (Hemiptera: Reduviidae)

Proteinase levels in the posterior midgut of fifth-instar and adult Rhodnius prolixus follow a cyclic pattern after ingestion of the bloodmeal. In the fifth instar, cathepsin B showed two peaks: the first occurred 6 days after ingestion and the second at the time of ecdysis. Cathepsin D, cathepsin B and lysosomal carboxypeptidase B reached maximal levels 6 days after ingestion. At this time the highest levels of these proteinases were found in mated females, the lowest in males and intermediate levels in virgin females. Maximal levels of aminopeptidase occurred later than catheptic enzymes, and the decline, after maximal levels were achieved, was much more gradual.Catheptic-proteinase levels within the posterior midgut in fifth-instar larvae and adults correlated positively with the amount of protein contained in this gut region. This indicates that production of these proteinases is controlled by a secretagogue mechanism. Aminopeptidase levels were controlled in a different manner. The mated state or sex of adults altered the proteinase levels by changing the amount of protein that was passed into the digestive midgut from the crop.     

Action on peptides by wheat carboxypeptidase

A kinetic analysis has been performed with purified wheat carboxypeptidase by the use of N-acyl dipeptides, Z-Gly-Pro-Leu-Gly (Z = benzyloxycarbonyl), angiotensin II and bradykinin. The values of k_cat were dramatically influenced by amino acid residues occupying the penultimate position from the carboxyl terminus of substrates. The structure of the substrate did not appreciably affect the K_m values.     

Acid carboxypeptidase from a wood-deteriorating basidiomycete, Pycnoporus Sanguineus

An acid carboxypeptidase (EC 3.4.16.1) has been isolated from the culture filtrate of a wood-degrading Basidiomycete, Pycnoporus sanguineus and the molecular and enzymatic properties of the enzyme were determined. The extracellular acid carboxypeptidase was homogeneous on polyacrylamide gel electrophoresis at pH 9.4 and SDS-disc gel electrophoresis. The MWs as determined by gel filtration and SDS-gel electrophoresis were 50 000 and 54 000, respectively. The isoelectric point was pH 4.78 using electrofocusing. The purified enzyme had a pH optimum of 3.4, a K_m of 0.74 mM and a k_cat of 16/sec with benzyloxycarbonyl-l-glutamyl-l-tyrosine. The K_m and k_cat values for bradykinin at pH 3.4 and 30° were 2.0 mM and 25/sec. Values for angiotensin at pH 3.4 and 30° were 0.76 mM and 2.4/sec, respectively.     

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.     

Subunit structure and immunological properties of wheat carboxypeptidase

Wheat carboxypeptidases I, II, III and IV from wheat seeds with isoelectric points of 4.8, 5.6, 6.0 and 6.5, respectively, were found to be homogeneous by the Ouchterlony double immunodiffusion technique using an antiserum of the enzyme III. In a previous paper [1], the native enzyme III (MW = 118 k) was separated into two 58 k subunits (MW = 58 k) and further divided into the 35 k and 25 k fragments (MW = 35 k and 25 k, respectively). The native enzyme III and the 58 k subunit produced a single precipitin line against the antiserum. The 35 k and 25 k fragments did not cross-react with the antiserum. The amino acid compositions of the 35 k and 25 k fragments were similar to each other. Amino-terminal amino acids of the 35 k and 25 k fragments were both glutamic acid. Carboxy-terminal groups of the 35k and 25k fragments were determined to be -(Gly, Ser)-Glu-OH and -Thr-Pro-Glu-OH, respectively. The Ouchterlony double immunodiffusion technique revealed the presence of a common antigen in a carboxypeptidase from wheat seeds and one from germinated wheat. Comparison of both enzymes is discussed.     

Activation and membrane binding of carboxypeptidase E

Carboxypeptidase E (CPE) is a carboxypeptidase B-like enzyme that is thought to be involved in the processing of peptide hormones and neurotransmitters. Soluble and membrane-associated forms of CPE have been observed in purified secretory granules from various hormone-producing tissues. In this report, the influence of membrane association on CPE activity has been examined. A substantial amount of the membrane-associated CPE activity is solubilized upon extraction of bovine pituitary membranes with either 100 mM sodium acetate buffer (pH 5.6) containing 0.5% Triton X-100 and 1 M NaCl, or by extraction with high pH buffers (pH greater than 8). These treatments also lead to a two- to threefold increase in CPE activity. CPE extracted from membranes with either NaCl/Triton X-100 or high pH buffers hydrolyzes the dansyl-Phe-Ala-Arg substrate with a lower Km than the membrane-associated CPE. The Vmax of CPE present in extracts and membrane fractions after the NaCl/Triton X-100 treatment is twofold higher than in untreated membranes. Treatment of membranes with high pH buffers does not affect the Vmax of CPE in the soluble and particulate fractions. Pretreatment of membranes with bromoacetyl-D-arginine, an active site-directed irreversible inhibitor of CPE, blocks the activation by NaCl/Triton X-100 treatment. Thus the increase in CPE activity upon extraction from membranes is probably not because of the conversion of an inactive form to an active one, but is the result of changes in the conformation of the enzyme that effect the catalytic activity.