A sensitive and specific assay for dipeptidyl-aminopeptidase II in serum and tissues by liquid chromatography-fluorometry

A highly sensitive and specific method for the assay of dipeptidyl-aminopeptidase II (DAP II) in crude enzyme preparations such as serum and tissue homogenates has been established by using a newly synthesized fluorogenic substrate, 7-Lys-Ala-4-methylcoumarinamide. The enzymatically formed 7-amino-4-methylcoumarin was determined by high-performance liquid chromatography with fluorescence detection. The activities of other aminopeptidases in human serum and rat brain homogenates were completely inhibited by o-phenanthroline without any effect on DAP II activity to permit specific determination of DAP II. The limit of sensitivity for DAP II activity was about 300 fmol/30 min. DAP II activity was found to be increased in sera from cancer patients, in contrast to the decrease in serum DAP IV activity. DAP II activity was found to be unequally distributed in rat brain regions, and the highest activity was found in the hypothalamus.     

Quantitative histochemistry of the lysosomal dipeptidyl aminopeptidase II in the proximal tubule of the rat kidney

The activity of the lysosomal dipeptidyl aminopeptidase II (DAP II) was measured by quantitative histochemical methods in the S1/S2 segments of the proximal tubule using freeze dried and celloidin mounted cryostat sections (FDC sections) of rat kidney. The methodological studies show that there is a linear relationship between the amount of reaction product and reaction time for the first 5 min, as well as section thickness between 4 and 10 microns. Maximal DAP II activities were demonstrated at pH 5.5. The Km of DAP II was about 2.3 mM. In addition to the methodological studies, DAP II activity was also measured in the proximal tubule (S1/S2 segments) of experimental animals (sham-operated and castrated male and female rats). Sham-operated females showed significantly higher DAP II activities than males. DAP II activity increased significantly in castrated males so that there were no significant differences between castrated males, sham-operated and castrated females. The quantitative histochemical results are largely in agreement with biochemical data published earlier.     

Serum activities of dipeptidyl-aminopeptidase II and dipeptidyl-aminopeptidase IV in tumor-bearing animals and in cancer patients

Serum activities of dipeptidyl-aminopeptidases (DAP) II and IV were measured in tumor-bearing animals and in patients with blood and solid cancers using highly sensitive and specific fluorometric methods. In mice with intraperitoneal or subcutaneous implantation of Ehrlich ascites tumor cells, serum DAP II activity was increased and serum DAP IV activity was decreased, resulting in a significant increase in the ratio of serum DAP II and DAP IV activities. The increase in the ratio of these two activities paralleled the size of the subcutaneous tumors. However, both serum DAP II and DAP IV activities were increased in rats with experimental hepatic cancer induced by 3'-methyl-4-dimethylaminoazobenzene, and the increase in the ratio of the two activities was not significant. In cancer patients, as compared with healthy subjects, serum DAP II activity was increased and serum DAP IV activity was decreased, the ratio of serum DAP II and DAP IV activities being markedly increased in cancer patients. Both serum DAP II and DAP IV activities were increased in patients with hepatic cancer as were those in rats with hepatic cancer, but the increase in DAP II was greater than that of DAP IV; thus the ratio of the two activities increased significantly. These data suggest that the increase of the serum DAP II/DAP IV ratio could be a biochemical index of cancer.     

Dipeptidyl-aminopeptidase II in human cerebrospinal fluid: changes in patients with Parkinson's disease

By using a sensitive and specific method, DAP II activity was found in CSF. DAP II activity in CSF of control patients without neurological diseases was 0.416 +/- 0.141 (mean +/- SD) nmole/min/ml and was higher than DAP IV activity in CSF, 0.221 +/- 0.062 (mean +/- SD) nmole/min/ml. In contrast, DAP II activity in serum was 1.16 +/- 0.16 (mean +/- SD) nmole/min/ml and was lower than serum DAP IV activity [41.85 +/- 3.36 (mean +/- SD) nmole/min/ml]. This relatively high activity of DAP II in CSF compared with the activity of DAP IV in CSF together with recent histochemical evidence on the localization of DAP II in some neurons (7) suggests that CSF DAP II may be derived from the brain and may be a marker of some peptidergic neurons. DAP II activity in CSF of patients with Parkinson's disease was significantly increased, whereas DAP IV activity in CSF did not change significantly.     

Quantitative histochemistry of the lysosomal dipeptidyl aminopeptidase II in the proximal tubule of the rat kidney

Summary The activity of the lysosomal dipeptidyl aminopeptidase II (DAP II) was measured by quantitative histochemical methods in the S₁/S₂ segments of the proximal tubule using freeze dried and celloidin mounted cryostat sections (FDC sections) of rat kidney. The methodological studies show that there is a linear relationship between the amount of reaction product and reaction time for the first 5 min, as well as section thickness between 4 and 10 m. Maximal DAP II activities were demonstrated at pH 5.5. The K _m of DAP II was about 2.3 mM. — In addition to the methodological studies, DAP II activity was also measured in the proximal tubule (S₁/S₂ segments) of experimental animals (sham-operated and castrated male and female rats). Sham-operated females showed significantly higher DAP II activities than males. DAP II activity increased significantly in castrated males so that there were no significant differences between castrated males, sham-operated and castrated females. The quantitative histochemical results are largely in agreement with biochemical data published earlier.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)Dedicated to Prof. Dr. T.H. Schiebler, Chairman of the Institute of Anatomy of the University of Würzburg, on the occasion of his 60th birthday     

Cytochemistry of dipeptidylaminopeptidase IV and II in normal and neoplastic lymphoid cells

Cytochemical methods were used to determine the distribution of dipeptidylaminopeptidase IV (DAP IV) and II (DAP II) in lymphoid cell populations from patients with lymphoproliferative diseases. Special attention was paid to unusual intracellular distribution patterns which might correlate with the presence of various membrane markers. In healthy patients, about 50% of the circulating lymphocytes were found to be positive to both reactions, the intracellular distribution patterns being variable. The DAP IV reaction was negative in all B-CLL cases. In 2 cases of T-CLL with phenotype E+ OKT3+T4-T8+ one was negative and one was weakly positive, while two cases of T-CLL with phenotype E+ OKT3+T4+T8- were both strongly positive. The other non-T lymphoproliferative diseases studied were negative for DAP IV, while one T-ALL and three T-lymphoma cases showed a strong granular or diffuse distribution. The DAP II reaction was strongly positive in all the T lymphoproliferative diseases studied, irrespective of their immunological phenotype. This reaction was also weakly positive in some cases of plasmocytoma and lymphoplasmacytoid lymphoma.     

Detection of a lysosomal carboxypeptidase and a lysosomal dipeptidase in highly-purified dipeptidyl aminopeptidase I (cathepsin C) and the elimination of their activities from preparations used to sequence peptides

The best preparations of dipeptidyl aminopeptidase I (DAP I) from beef spleen and rat liver were found to contain a carboxypeptidase (“catheptic carboxypeptidase C”) and a dipeptidase (“Ser-Met dipeptidase”). Each had a pH optimum near 5.5, a resistance to sulfhydryl inhibitors, and a lysosomal origin. The carboxypeptidase, which was inhibited by diisopropylphosphorofluoridate (DFP), preferentially cleaved COOH-terminal residues adjacent to proline, as in angiotensin II and Z-Pro-Phe. No action was detected on Z-Pro-Phe-NH₂. The dipeptidase, which was separable by electrofocusing, was most active on Ser-Met, and showed no action on Z-Ser-Met, Ser-Met-NH₂, Ser-Met-Glu, Gly-Gly or Gly-Leu. Ser-Met dipeptidase was unaffected by DFP, but was strongly inhibited by EDTA. A metal requirement was not apparent, however. A simplified method is described for preparing DAP I as a sequencing reagent free of these contaminating activities.     

Renal cathepsin-B activities in rats after castration and treatment with sex hormones

The activities of the lysosomal endopeptidase cathepsin B (cath B; CZB-Ala-Arg-Arg-MNA as substrate) and the lysosomal exopeptidase dipeptidylpeptidase II (DAP II; Lys-Ala-2NA as substrate) were fluorometrically determined in the renal homogenate of normal and experimental (castration followed by a 14-day treatment with estradiol and testosterone) rats of both sexes. In addition, methodological investigations of the renal homogenate were performed in order to differentiate cath B from other proteinases. These showed that cath-B activity was highest at around pH 6, was strongly inhibited by 4-hydroxymercuribenzoate and leupeptin, and was activated by dithiothreitol. Trypsin-like activities were not demonstrable under the used incubation conditions. The animal experiments showed that renal cath-B activities (1) were significantly higher in females than in males, (2) increased significantly in males and decreased significantly in females after castration (no significant difference between both sexes), (3) decreased in female and male castrates after treatment with testosterone and increased strongly after treatment with estradiol, and (4) showed an activity pattern similar to that of DAP II. The results are discussed in relation to the sex-dependent and sex-hormone-dependent proteinuria of rats. It is suggested that there is a correlation between protein catabolism in the kidney and proteinuria, i.e. high lysosomal proteinase activities correspond with low proteinuria.     

Renal cathepsin-B activities in rats after castration and treatment with sex hormones

Summary The activities of the lysosomal endopeptidase cathepsin B (cath B; CZB-Ala-Arg-Arg-MNA as substrate) and the lysosomal exopeptidase dipeptidylpeptidase II (DAP II; Lys-Ala-2NA as substrate) were fluorometrically determined in the renal homogenate of normal and experimental (castration followed by a 14-day treatment with estradiol and testosterone) rats of both sexes. In addition, methodological investigations of the renal homogenate were performed in order to differentiate cath B from other protenases. These showed that cath-B activity was highest at around pH 6, was strongly inhibited by 4-hydroxymercuribenzoate and leupeptin, and was activated by dithiothreitol. Trypsin-like activities were not demonstrable under the used incubation conditions. The animal experiments showed that renal cath-B activities (1) were significantly higher in females than in males, (2) increased significantly in males and decreased significantly in females after castration (no significant difference between both sexes), (3) decreased in female and male castrates after treatment with testosterone and increased strongly after treatment with estradiol, and (4) showed an activity pattern similar to that of DAP II. The results are discussed in relation to the sexdependent and sex-hormone-dependent proteinuria of rats. It is suggested that there is correlation between protein catabolism in the kidney and proteinuria, i.e. high lysosomal proteinase activities correspond with low proteinuria.Supported by the Deutsche Forschungsgemeinschaft (SFB 105)     

Ca2+-dependent phosphorylation of syntaxin-1A by the death-associated protein (DAP) kinase regulates its interaction with Munc18

Syntaxin-1 is a key component of the synaptic vesicle docking/fusion machinery that binds with VAMP/synaptobrevin and SNAP-25 to form the SNARE complex. Modulation of syntaxin binding properties by protein kinases could be critical to control of neurotransmitter release. Using yeast two-hybrid selection with syntaxin-1A as bait, we have isolated a cDNA encoding the C-terminal domain of death-associated protein (DAP) kinase, a calcium/calmodulin-dependent serine/threonine protein kinase. Expression of DAP kinase in adult rat brain is restricted to particular neuronal subpopulations, including the hippocampus and cerebral cortex. Biochemical studies demonstrate that DAP kinase binds to and phosphorylates syntaxin-1 at serine 188. This phosphorylation event occurs both in vitro and in vivo in a Ca2+-dependent manner. Syntaxin-1A phosphorylation by DAP kinase or its S188D mutant, which mimics a state of complete phosphorylation, significantly decreases syntaxin binding to Munc18-1, a syntaxin-binding protein that regulates SNARE complex formation and is required for synaptic vesicle docking. Our results suggest that syntaxin is a DAP kinase substrate and provide a novel signal transduction pathway by which syntaxin function could be regulated in response to intracellular [Ca2+] and synaptic activity.     

Dipeptidyl aminopeptidase activities of guinea-pig brain

• 1.1. The subcellular distribution ofdipeptidyl aminopeptidase activities in guinea-pig brain was investigated. Our studies show that DAP I (Gly-Arg-NH-Mec hydrolase) type activity was found to have an acidic optimum and was associated with the nuclear pellet.
• 2.2. No DAP II (Lys-Ala-NH-Mec hydrolase) type activity could be detected. Apparant hydrolysis was mainly due to aminopeptidase activity.
• 3.3. DAP III (Arg-Arg-NH-Mec hydrolase) type activity is largely cytoplasmic, but there was evidence of a membrane form associated with the synaptosomes.
• 4.4. DAP IV (Gly-Pro-NH-Mec hydrolase) type activity is present on the synaptosomal membrane, and also enriched in the microsomes. A soluble form of Gly-Pro-NH-Mec hydrolase activity is also present in the cytoplasm. Whether this activity is a DAP II or IV type activity is still yet to be determined.

     

The usefulness of the analytical electrofocusing in a thin-layer polyacrylamide gel (PAG) in the histochemistry of enzymes cleaving peptide bonds

Summary The usefulness of the analytical electrofocusing in a thin-layer polyacrylamide (PAG) plate is shown on the basis of experiments with 10%–20% homogenates of various rat, rabbit and human organs as well as in lysates of isolated human lymphocytes and leucocytes in 2% Triton X100. 0.1–0.3 l of 12000 g supernatants were applied on LKB Ampholine PAG plates pH range 3.5–9.5 and subjected to electrofocusing. Afterwards portions of PAG plates were processed in optimized histochemical media for the demonstration of enzymes cleaving peptide bonds using various substrates. The same media were used in the histochemical detection of enzymes in sections on slides or semipermeable membranes. Electrofocused zymograms display species and organ differences. Ala-MNA, Leu-MNA and Met-MNA furnish similar zymograms. Bands obtained with Ala-MNA are most intense. Zymograms with Gly-Pro-MNA and Lys-Pro-MNA at pH 7.2 are not entirely identical. The majority of bands is more intense when Gly-Pro-MNA is used as the substrate and is due to the activity of DAP IV. The anodal band(s) focusing around pH 4.9 (rat) or 5.5 (man) is (are) much stronger with Lys-Pro-MNA and DAP II is responsible for it (them). Zymograms with His-Ser-MNA and Lys-Ser-MNA are similar. However, they differ form those revealed with Gly-Pro-MNA and Lys-Pro-MNA. Zymograms of lysates of leucocytes obtained with naphthol AS-D-chloroacetate and Ac-Ala-l-naphthyl ester are not identical showing that more than one enzyme is responsible for the bands. Results obtained with closely related substrates such as Ac-Ala-l-naphthyl ester and Ac-Met-l-naphtyl ester are not identical either. Zymograms of lysates of human lymphocytes revealed with Gly-Pro-MNA at pH 7.2 and Lys-Ala-MNA or Lys-Pro-MNA at pH 5.5 or 5.3 respectively show clearly the presence of DAP IV and DAP II in these cells. The analytical electrofocusing in PAG plates is a very useful tool in the histochemistry (and biochemistry) of enzymes cleaving peptide bonds. It helps very much in the evaluation of the substrate specificity, choosing of the discriminating substrate and enables a quick and reliable testing of the quality of various batches of commercially supplied substrates, diazonium salts and other reagents. The correlation of zymograms with the in situ pattern helps in the elucidation of the origin of individual bands in zymograms and suggests different molecular forms of peptidases in different localizations.     

Mammalian chymotrypsin-like enzymes. Comparative reactivities of rat mast cell proteases, human and dog skin chymases, and human cathepsin G with peptide 4-nitroanilide substrates and with peptide chloromethyl ketone and sulfonyl fluoride inhibitors

The extended substrate binding sites of several chymotrypsin-like serine proteases, including rat mast cell proteases I and II (RMCP I and II, respectively) and human and dog skin chymases, have been investigated by using peptide 4-nitroanilide substrates. In general, these enzymes preferred a P1 Phe residue and hydrophobic amino acid residues in P2 and P3. A P2 Pro residue was also found to be quite acceptable. The S4 subsites of these enzymes are less restrictive than the other subsites investigated. The substrate specificity of these enzymes was also investigated by using substrates which contain model desmosine residues and peptides with amino acid sequences of the physiologically important substrates angiotensin I and angiotensinogen and alpha 1-antichymotrypsin, the major plasma inhibitor for chymotrypsin-like enzymes. These substrates were less reactive than the most reactive tripeptide reported here, Suc-Val-Pro-Phe-NA. The thiobenzyl ester Suc-Val-Pro-Phe-SBzl was found to be an extremely reactive substrate for the enzymes tested and was 6-171-fold more reactive than the 4-nitroanilide substrate. The four chymotrypsin-like enzymes were inhibited by chymostatin and N-substituted saccharin derivatives which had KI values in the micromolar range. In addition, several potent peptide chloromethyl ketone and substituted benzenesulfonyl fluoride irreversible inhibitors for these enzymes were discovered. The most potent sulfonyl fluoride inhibitor for RMCP I, RMCP II, and human skin chymase, 2-(Z-NHCH2CONH)C6H4SO2F, had kobsd/[I] values of 2500, 270, and 1800 M-1 s-1, respectively. The substrates and inhibitors reported here should be extremely useful in elucidating the physiological roles of these proteases.     

Rat and mouse CD94 associate directly with the activating transmembrane adaptor proteins DAP12 and DAP10 and activate NK cell cytotoxicity

Signaling by the CD94/NKG2 heterodimeric NK cell receptor family has been well characterized in the human but has remained unclear in the mouse and rat. In the human, the activating receptor CD94/NKG2C associates with DAP12 by an ionic bond between oppositely charged residues within the transmembrane regions of NKG2C and DAP12. The lysine residue responsible for DAP12 association is absent in rat and mouse NKG2C and -E, raising questions about signaling mechanisms in these species. As a possible substitute, rat and mouse NKG2C and -E contain an arginine residue in the transition between the transmembrane and stalk regions. In this article, we demonstrate that, similar to their human orthologs, NKG2A inhibits, whereas NKG2C activates, rat NK cells. Redirected lysis assays using NK cells transfected with a mutated NKG2C construct indicated that the activating function of CD94/NKG2C did not depend on the transmembrane/stalk region arginine residue. Flow cytometry and biochemical analysis demonstrated that both DAP12 and DAP10 can associate with rat CD94/NKG2C. Surprisingly, DAP12 and DAP10 did not associate with NKG2C but instead with CD94. These associations depended on a transmembrane lysine residue in CD94 that is unique to rodents. Thus, in the mouse and rat, the ability to bind activating adaptor proteins has been transferred from NKG2C/E to the CD94 chain as a result of mutation events in both chains. Remarkable from a phylogenetic perspective, this sheds new light on the evolution and function of the CD94/NKG2 receptor family.     

A high-performance liquid chromatography method for the simultaneous assay of diaminopimelate epimerase and decarboxylase

A sensitive and comparatively simple method for the assay of diaminopimelate (DAP) decarboxylase, which simultaneously monitors DAP epimerase activity, in the reverse of the biosynthetic direction, is described. The substrate, meso-DAP and products LL-DAP and L-lysine are derivatized with o-phthaldialdehyde and resolved by reversed-phase high-performance liquid chromatography. Separation is achieved on a Spherisorb C18 column using a gradient elution system. This technique offers a high degree of sensitivity as the detection method described can measure picomole quantities of substrate and products.     

Peptidases in the kidney of male rats following castration and testosterone substitution

The localization of aminopeptidase M (APM), dipeptidyl peptidase I (DAP I), II (DAP II) and IV (DAP IV) in the renal section was investigated histochemically, and their activities were determined fluorometrically in renal homogenate of normal, castrated and testosteron treated male rats.--After castration the activities of the lysosomal DAP II (pars convoluta of the proximal tubule), DAP I (distal and proximal tubule) and of the mainly membrane-bound DAP IV (glomeruli, brush border of the proximal tubule) increase in comparison to normal males, whereas the activities of the brush border-bound APM decrease. After testosteron treatment of castrated animals (0.1, 0.5 and 1.0 mg testosterone proprionate/100 g BW and day; 5-day treatment) the activities of DAP I, II and IV decrease again, so that after treatment with 0.1 mg testosterone proprionate, the activities of DAP I and II approach those in normal males.--The additionally determined urinary protein excretion shows that there is a significant decrease in proteinuria after castration, whereas testosterone treatment of castrated animals is accompanied by an increase of proteinuria.--Our results would suggest that the protein catabolism in the proximal tubule and the proteinuria are interrelated, and that testosterone influences (decreases) the protein catabolism in the proximal tubule. This means that high activities of lysosomal proteinases in the proximal tubule (castrates) are accompanied by a low proteinuria, and low activities of those proteinases (testosterone treated castrated or normal males) by a high proteinuria.     

Phosphodiesterase II in epithelial cells from guinea-pig and rat small intestine

Phosphodiesterase II activity was determined by using a synthetic substrate, the 2,4-dinitrophenyl ester of thymidine 3'-phosphate. The enzyme activity was determined in fractions obtained by differential centrifugation of homogenates of epithelial cells from the small intestinal mucosa of guinea pigs and rats. In guinea-pig preparations phosphodiesterase II occurred with highest specific activity in those fractions rich in succinate dehydrogenase and acid phosphatase. A lysosomal location for the guinea-pig enzyme was indicated by its structure-linked latency and by its association with particles that under-went a characteristic decrease in equilibrium density when Triton WR-1339 was injected into the animals. With rat preparations a much greater proportion of the phosphodiesterase II activity was found in the soluble fraction after ultracentrifugation. The rat enzyme exhibited a lower degree of latency and administration of Triton WR-1339 had no effect. The rat enzyme further differed from that of the guinea pig in other respects; it was more labile at 60 degrees C, it exhibited a lower pH optimum and it had a higher molecular weight as determined by gel-filtration chromatography.     

Studies on the ferrochelatase activity of isolated rat liver mitochondria with special reference to the effect of oxidizable substrates and oxygen concentration.

The mitochondrial ferrochelatase activity has been studied in coupled rat liver mitochondria using deuteroporphyrin IX (incorporated into liposomes of lecithin) and Fe(III) or Co(II) as the substrates. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value deltae (mM-1 - cm-1) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM - min-1 - mg-1 of protein at 37 degrees C, PH 7.4 and optimal substrate concentrations, i.e. 75 muM Fe(III) and 50 muM deuteroporphyrin. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.     

Efficient production and partial characterization of aspartyl aminopeptidase from Aspergillus oryzae

Aims: Aspartyl aminopeptidase (DAP) has a high degree of substrate specificity, degrading only amino-terminal acidic amino acids from peptides. Therefore, attention is focused here on the efficient production of this enzyme by a recombinant Aspergillus oryzae and characterization of its biochemical properties. Methods and Results: The gene encoding DAP was overexpressed under a taka-amylase gene promoter, with His-tag linker in A. oryzae, during cultivation in a Co²⁺-containing medium. The enzyme was extracted from the mycelia and purified with immobilized nickel ion absorption chromatography using a buffer containing cobalt ion and imidazole. The active fraction was further purified with gel filtration chromatography. The resultant, electrophoretically pure enzyme displayed a molecular mass of 520 kDa. This enzyme displayed high reactivity towards peptide substrate rather than synthetic substrates. Conclusions: Recombinant A. oryzae DAP was purified to homogeneity with an increased specific activity, when cultivated in a Co²⁺-rich medium. Moreover, the use of suitable metal ions in microbial cultivation and purification processes may help in increasing the specific activity of other metalloproteases and their functional analysis. Significance and Impact of the Study: Recombinant DAP produced using a cobalt ion in culture media of A. oryzae and purification process allow high yield of the enzyme activity.