N-glycosylation of carnosinase influences protein secretion and enzyme activity: implications for hyperglycemia.

OBJECTIVE: The (CTG)(n) polymorphism in the serum carnosinase (CN-1) gene affects CN-1 secretion. Since CN-1 is heavily glycosylated and glycosylation might influence protein secretion as well, we tested the role of N-glycosylation for CN-1 secretion and enzyme activity. We also tested whether CN-1 secretion is changed under hyperglycemic conditions. RESULTS: N-glycosylation of CN-1 was either inhibited by tunicamycin in pCSII-CN-1-transfected Cos-7 cells or by stepwise deletion of its three putative N-glycosylation sites. CN-1 protein expression, N-glycosylation, and enzyme activity were assessed in cell extracts and supernatants. The influence of hyperglycemia on CN-1 enzyme activity in human serum was tested in homozygous (CTG)(5) diabetic patients and healthy control subjects. Tunicamycin completely inhibited CN-1 secretion. Deletion of all N-glycosylation sites was required to reduce CN-1 secretion efficiency. Enzyme activity was already diminished when two sites were deleted. In pCSII-CN-1-transfected Cos-7 cells cultured in medium containing 25 mmol/l d-glucose, the immature 61 kilodaltons (kDa) CN-1 immune reactive band was not detected. This was paralleled by an increased GlcNAc expression in cell lysates and CN-1 expression in the supernatants. Homozygous (CTG)(5) diabetic patients had significantly higher serum CN-1 activity compared with genotype-matched, healthy control subjects. CONCLUSIONS: We conclude that apart from the (CTG)(n) polymorphism in the signal peptide of CN-1, N-glycosylation is essential for appropriate secretion and enzyme activity. Since hyperglycemia enhances CN-1 secretion and enzyme activity, our data suggest that poor blood glucose control in diabetic patients might result in an increased CN-1 secretion even in the presence of the (CTG)(5) allele.

The mechanism of butyrate-induced collagen biosynthesis in cultured fibroblasts.

The data showing that butyrate may play an important role in cellular metabolism led us to study its effect on collagen biosynthesis in cultured fibroblasts. Since insulin-like growth factor-I (IGF-I) is the most potent stimulator of collagen biosynthesis in fibroblasts, the effect of butyrate on IGF-I receptor (IGF-IR) expression was evaluated. Confluent human dermal fibroblasts were treated with millimolar concentrations of sodium butyrate (NaB) for 48 hours. It was found that butyrate induced collagen biosynthesis and prolidase activity. It was found that the exposure of the cells to 4 mM butyrate contributed to a distinct increase in IGF-IR. It was accompanied by a parallel increase in the expression of Sos protein and MAP-kinases (ERK1, ERK2). It was found that the MEK inhibitor decreased collagen biosynthesis and expression of MAP-kinases (ERK1, ERK2), while NaB counteracted the process. The data suggests that butyrate-dependent stimulation of collagen biosynthesis in cultured human skin fibroblasts undergoes through IGF-IR signaling.

Pharmacological profile of ASP8497, a novel, selective, and competitive dipeptidyl peptidase-IV inhibitor, in vitro and in vivo.

Dipeptidyl peptidase (DPP)-IV is involved in the inactivation of glucagon-like peptide-1 (GLP-1), a potent insulinotropic peptide. Thus, DPP-IV inhibitors are expected to become a useful new class of antidiabetic agent. This report describes the pharmacological profile of the novel DPP-IV inhibitor, ASP8497 [(2S,4S)-4-fluoro-1-({[4-methyl-1-(methylsulfonyl)piperidin-4-yl]amino}acetyl)pyrrolidine-2-carbonitrile monofumarate], both in vitro and in vivo. ASP8497 inhibited DPP-IV in plasma from mice, dogs, and humans with median inhibition concentration (IC(50)) values of 2.6 nM, 7.3 nM, and 6.2 nM, respectively. In contrast, ASP8497 did not potently inhibit human DPP8 or DPP9 activity (IC(50)=1,700 nM and 100 nM, respectively) and exhibited selectivity against several proteases, including proline-specific proteases (IC(50)>10 microM). Kinetic analysis indicated that ASP8497 is a competitive DPP-IV inhibitor. In normal mice, ASP8497 inhibited plasma DPP-IV activity even 12 h after administration. ASP8497 significantly inhibited increases in the blood glucose level during the oral glucose tolerance test (OGTT) conducted 0.5 h after administration. This was accompanied by increases in the plasma active GLP-1 and insulin levels. In addition, ASP8497 significantly inhibited increases in the blood glucose level during the OGTT conducted 8 h after administration. Furthermore, in Zucker fatty rats, ASP8497 dose dependently improved glucose tolerance with significance at doses of 1 mg/kg or higher. In contrast, ASP8497 did not cause hypoglycemia in fasted normal mice. These results indicate that ASP8497 is a potent, competitive, and selective DPP-IV inhibitor with antihyperglycemic activity.

Mechanism of collagen biosynthesis up-regulation in cultured leiomyoma cells.

Uterine leiomyoma is the most common tumour in women with a reported incidence of 25-30%. The tumors are benign, composed of smooth muscle cells with variable amount of collagen - rich fibrous tissue. It is well established that accumulation of extracellular matrix in leiomyoma is key feature of tissue fibrosis. However, the pathogenesis of leiomyoma is still unclear. The aim of this study was to evaluate the metabolism of collagen in cultured leiomyoma cells and in control myometrium cells. The effect of estradiol, selective modulators of estrogen receptors (raloxifene, tamoxifen) and estrogen receptor down regulator (ICI 182.780) on collagen biosynthesis (measured by 5-[3H]-proline incorporation assay and measurement of prolidase activity) and collagen degradation (measured by metalloproteinase activity assay) was studied. It was found that collagen biosynthesis is strongly stimulated by low doses of estradiol (5 nM) in leiomyoma cells while it is not changed in control myometrium cells. An increase in estradiol concentration to 10 nM results in drastic decrease of this process both in leiomyoma as well as control cells. Although raloxifene and tamoxifen only slightly affected collagen biosynthesis in control myometrium cells, they significantly inhibited the process in leiomyoma cells. There was no coordinate correlation between collagen biosysignificantly inhibited the process in leiomyoma cells. There was no coordinate correlation between collagen biosynthesis and prolidase activity suggesting that regulation of this process may take place at transcriptional level. Both estrogen and SERMs were found to inhibit MMP-2 in leiomyoma as well as in control myometrium cells. The data suggest that stimulatory action of estrogen on collagen biosynthesis and inhibitory effect on MMP-2 activity in uterine leiomyoma may contribute to accumulation of this protein in ECM of this tissue.

Characteristics of prolinase against various iminodipeptides in erythrocyte lysates from a normal human and a patient with prolidase deficiency.

The effect of various amino acids and MnCl2 on prolinase activity in erythrocyte lysates from a healthy individual and a patient with prolidase deficiency was investigated. A concentration of 0.1 mM MnCl2 increased prolinase activity in normal erythrocytes against pro-gly, pro-glu, pro-leu, pro-ser and pro-phe, but inhibited that against pro-ala, pro-val, pro-met and pro-asp. However, prolinase activity against these iminodipeptides was enhanced by pre-incubation with glycine, independent of MnCl2. The same studies on erythrocytes from a prolidase-deficient patient showed almost the same results as the normal control, except that prolinase activity against pro-gly and pro-ser was slightly inhibited by adding 0.1 mM MnCl2. Some amino acids, glutamic acid and glutamine, slightly enhanced prolinase activity against pro-gly in erythrocytes from both the normal control and the prolidase-deficient patient, but N-acetyl-L-glutamic acid, gamma-aminobutyric acid (GABA) and beta-alanine showed no effect. Branched amino acids, L-valine, L-leucine and L-isoleucine strongly inhibited the prolinase activity against pro-gly. However, conversely, their isomers, D-valine, D-leucine and D-isoleucine, enhanced it. The kinetics of prolinase activity in the erythrocytes from both the normal individual and the prolidasedeficient patient were also studied. Their Km values were changed by adding glycine or 0.1 mM MnCl2, but Vmax values were almost the same.

New chromogenic dipeptide substrate for continuous assay of the D-alanyl-D-alanine dipeptidase VanX required for high-level vancomycin resistance.

A direct continuous UV-Vis spectrophotometric assay has been developed for VanX, a D-alanyl-D-alanine aminodipeptidase necessary for vancomycin resistance. This method is based on the hydrolysis of the alternative substrate D-alanyl-alpha-(R)-phenylthio-glycine D-Ala-D-Gly(S-Ph)-OH (H-DAla-DPsg-OH, 5a). Spontaneous decomposition of the released phenylthioglycine generates thiophenol, which is quantified using Ellman's reagent. The dipeptide behaved as an excellent substrate of VanX, exhibiting Michaelis-Menten kinetics with a kcat of 76 +/- 5/s and a KM of 0.83 +/- 0.08 mm (kcat = 46 +/- 3/s and KM = 0.11 +/- 0.01 mm for D-Ala-D-Ala). Determination of the kinetic parameters of the previously reported mechanism-based inhibitor D-Ala-D-Gly(SPhip-CHF2)-OH (H-D-Ala-DPfg-OH, 5c) [Araoz, R., Anhalt, E., René, L., Badet-Denisot, M.-A., Courvalin, P. & Badet, B. (2000) Biochemistry 39, 15971-15979] using the substrate reported in the present study yielded values of Kirr of 22 +/- 1 microM and kinact of 9.3 +/- 0.4/min in good agreement with values previously obtained in our laboratory (Kirr = 30 +/- 1 mm; kinact = 7.3 +/- 0.3/min). In addition, inhibition by the competing substrate D-Ala-D-Ala resulted in determination of a Ki = 70 +/- 6 microM close to the previously reported KM value. These results demonstrate that the present assay is a convenient, rapid and sensitive tool in the search for VanX inhibitors.

Piperine modulates permeability characteristics of intestine by inducing alterations in membrane dynamics: influence on brush border membrane fluidity, ultrastructure and enzyme kinetics.

Piperine (1-Piperoyl piperidine) is a major alkaloid of Piper nigrum Linn. and Piper longum Linn. It is shown to possess bioavailability-enhancing activity with various structurally and therapeutically diverse drugs. The mechanism of enhancing the bioavailability, is, however, not understood. We hypothesize that piperine's bioavailability-enhancing property may be attributed to increased absorption, which may be due to alteration in membrane lipid dynamics and change in the conformation of enzymes in the intestine. Results of membrane fluidity studies using an apolar fluorescent probe, pyrene (which measures the fluid properties of hydrocarbon core), showed an increase in intestinal brush border membrane (BBM) fluidity. Piperine also stimulated Leucine amino peptidase and Glycyl-glycine dipeptidase activity, due to the alteration in enzyme kinetics. This suggests that piperine could modulate the membrane dynamics due to its apolar nature by interacting with surrounding lipids and hydrophobic portions in the protein vicinity, which may decrease the tendency of membrane lipids to act as stearic constrains to enzyme proteins and thus modify enzyme conformation. Ultra structural studies with piperine showed an increase in microvilli length with a prominent increase in free ribosomes and ribosomes on the endoplasmic reticulum in enterocytes, suggesting that synthesis or turnover of cytoskeletal components or membrane proteins may be involved in the observed effect. In conclusion, it is suggested that piperine may be inducing alterations in membrane dynamics and permeation characteristics, along with induction in the synthesis of proteins associated with cytoskeletal function, resulting in an increase in the small intestine absorptive surface, thus assisting efficient permeation through the epithelial barrier.

Stability of new carbapenem DA-1131 to renal dipeptidase (dehydropeptidase I).

The stability of DA-1131 to renal dipeptidase (RDPase) (EC 3.4.13.19) was compared with that of imipenem and meropenem by V(max)/K(m) ratios as an index of the enzyme's preference for substrates. Our results showed a decreasing order of imipenem (6.24), meropenem (2.41), and DA-1131 (1.39). The biochemical evaluation of DA-1131 as the least preferred substrate of RDPase suggests its potential use as a novel beta-lactam antibiotic which may be usable without coadministration of RDPase inhibitors once its clinical suitability is proven.

The mechanism for anthracycline-induced inhibition of collagen biosynthesis.

One of the recognized side effects accompanying anti-neoplastic anthracyclines administration is poor wound healing resulting from impairment of collagen biosynthesis. However, the precise mechanism of anthracyclines-induced inhibition of collagen synthesis has not been established. We have suggested that prolidase, an enzyme involved in collagen metabolism, may be one of the targets for anthracyclines-induced inhibition of synthesis of this protein. Prolidase [EC 3.4.13.9] cleaves imidodipeptides containing C-terminal proline, providing large amount of proline for collagen synthesis. Therefore, we compared the effect of daunorubicin and doxorubicin on prolidase activity and collagen biosynthesis in confluent cultured human skin fibroblasts. We have found that daunorubicin and doxorubicin coordinately induced the inhibition of prolidase activity (IC(50)=0.3 and 10 microM, respectively) and collagen biosynthesis (IC(50)=1 and 15 microM, respectively) in cultured human skin fibroblasts. The inhibitory effect of daunorubicin or doxorubicin on prolidase activity and collagen biosynthesis was not due to anti-proliferative activity of these drugs as shown by cell viability tetrazoline test. The decrease in prolidase activity due to the treatment of confluent cells with the anthracyclines was not accompanied by any difference in the amount of enzyme protein recovered from these cells as shown by Western immunoblot analysis. It may be suggested that the inhibition is a post-translational event. Since prolidase is metalloprotease, requiring manganese for catalytic activity, and anthracyclines are known as chelators of divalent cations, we considered that the chelating ability of anthracyclines might be an underlying mechanism for the anthracyclines-induced inhibition of prolidase activity. In order to determine the ability of daunorubicin or doxorubicin to form complexes with manganese (II), potentiometric method was employed based on the measurement of protonation constant by pH-metric titrated assay. We have found that both anthracyclines form stable complexes with manganese (II). The composition of the daunorubicin-Mn(II) complex was calculated as 3:1 while that of doxorubicin-Mn(II) complex was 2:1. The constant stability value for the investigated complexes were calculated as beta(av)=(1.74+/-0.01)x10(23) for daunorubicin, and beta(av)=(1.99+/-0.025)x10(11) for doxorubicin. The higher ability of daunorubicin vs. doxorubicin to chelate manganese and inhibit prolidase activity may explain the potential mechanism for its greater potency to inhibit collagen biosynthesis.

Evaluation of the effect of low-dose oral theophylline therapy on some bone turnover markers and serum prolidase I activity in mild asthmatics.

Hypercalcemia and hypercalciuria observed both in humans and in animals who were on long-term theophylline therapy, prompted us to investigate whether oral theophylline treatment at optimal doses causes any adverse side effects on bone metabolism in mild asthmatics. Therefore, serum osteocalcin (BGP) and total alkaline phosphatase (TALP, EC 3.1.3.1) as bone formation markers, serum prolidase I (EC 3.4.13.9) activity as a marker for collagen metabolism, urinary deoxypyridinoline (Dpd), hydroxyproline (Hyp) and fasting urinary calcium as bone resorption markers, were measured in 18 mild asthmatics who had been treated with theophylline over 1-10 years. Among measured bone turnover markers, BGP, TALP, and Hyp levels were found to be increased in mild asthmatics; and BGP showed the greatest percent mean increase (98%) over the healthy subjects. However, these increments did not exceed the upper reference limits. Serum prolidase I activity was also increased in mild asthmatics receiving theophylline. Our results indicate that theophylline therapy at optimal doses may not exert adverse side effects on bone homeostasis, but patients receiving supratherapeutic doses of theophylline should be under close examination in order to predict future bone mass status.

Estrogen-dependent regulation of prolidase activity in breast cancer MCF-7 cells.

Prolidase [EC 3.4.13.9] plays an important role in the recycling of proline for collagen synthesis and cell growth. The increase in the enzyme activity is correlated with the increased intensity of collagen turnover, thus reflecting the intensity of collagen metabolism. Since estrogens alter collagen metabolism, it can be assumed that the changes may be reflected by prolidase activity. The effects of estrogen and antiestrogen (tamoxifen on the prolidase and collagenase activities and collagen biosynthesis) were measured in the estrogen-receptor (ER)-positive breast cancer cell line. Estradiol stimulated collagen biosynthesis and extracellular prolidase and collagenase activities in cultured MCF-7 cells without an effect on collagen accumulation in the extracellular matrix produced by these cells. On the other hand, tamoxifen inhibited the estrogen-dependent stimulatory effect on collagen biosynthesis but did not inhibit the stimulatory effect of estrogen on prolidase and collagenase activities. The inhibitory effect of tamoxifen on estrogen-dependent stimulation of collagen synthesis in MCF-7 cells and lack of its effect on estrogen-dependent stimulation of prolidase and collagenase activities suggest that both processes (collagen synthesis and degradation) are independently regulated in MCF-7 cells, possibly through antagonist, agonist and other estrogen receptor-independent actions of tamoxifen. Increased extracellular prolidase activity in estrogen-stimulated MCF-7 cells indicates potential diagnostic value of tissue prolidase in determining the ER status of breast cancer.

Insulin stimulates the release of the glycosyl phosphatidylinositol-anchored membrane dipeptidase from 3T3-L1 adipocytes through the action of a phospholipase C.

Membrane dipeptidase (MDP; EC 3.4.13.19) enzymic activity that was inhibited by cilastatin has been detected on the surface of 3T3-L1 cells. On differentiation of the cells from fibroblasts to adipocytes the activity of MDP increased 12-fold. Immunoelectrophoretic blot analysis indicated that on adipogenesis the increase in the amount of MDP preceded the appearance of GLUT-4. MDP on 3T3-L1 adipocytes was anchored in the bilayer by a glycosyl phosphatidylinositol (GPI) moiety as evidenced by its release into the medium in a hydrophilic form on treatment of the cells with bacterial phosphatidylinositol-specific phospholipase C and the appearance of the inositol 1,2-cyclic monophosphate cross-reacting determinant. Incubation of 3T3-L1 adipocytes with either insulin or the sulphonylurea glimepiride led to a rapid concentration- and time-dependent release of MDP from the cell surface. The hydrophilic form of MDP released from the cells on stimulation with insulin was recognized by antibodies against the inositol 1,2-cyclic monophosphate cross-reacting determinant, indicating that it had been generated by cleavage of its GPI anchor through the action of a phospholipase C.

Influence of 1,10-phenanthroline and its analogues, other chelators and transition metal ions on dipeptidase activity of the rumen bacterium, Prevotella ruminicola.

Prevotella ruminicola plays a prominent role in the breakdown of peptides in the rumen, a process which contributes to excessive ammonia production and inefficient nitrogen retention in ruminants. Various metal ions and chelators were examined to assess how the metal ion-dependent dipeptidase activity of P. ruminicola M384 might be inhibited. Using sonicated extracts, Cu2+, Cr2+ and Hg2+ were most inhibitory, decreasing Ala2 breakdown to 15, 15 and 5% of control activity, whereas Co2+, Mn2+ and Zn2+ stimulated activity by 189, 30 and 26%, respectively. The chelators, EDTA, EGTA, TPEN and 1,10-phenanthroline, were inhibitory, as were several phenanthroline analogues. Among the stereoisomers of 1,10-phenanthroline tested, derivatives methylated on C-2 and C-9 were less effective than the parent molecule, but 3,4,7,8-tetramethyl-1,10-phenanthroline (TMP) was more inhibitory. Titration of the most effective inhibitors showed that EDTA, TPEN and TMP had similar potency and were effective at 0.1 mmol l-1 and above. Thus some metal ions and chelators are potent inhibitors of P. ruminicola dipeptidase, although they are unlikely to be sufficiently specific to peptide metabolism to be useful in vivo.

Inhibition by 1,10-phenanthroline of the breakdown of peptides by rumen bacteria and protozoa.

The rate of peptide breakdown in the rumen frequently exceeds the rate at which the amino acids released can be used for microbial growth. The final step in this often wasteful process involves the cleavage of dipeptides. The main rumen bacterial species with high dipeptidase activity, Prevotella ruminicola, Fibrobacter succinogenes, Lachnospira multipara and Megasphaera elsdenii, had activities which were inhibited > 95% by 1,10-phenanthroline, a chelator of divalent metal ions and metalloprotease inhibitor. Dipeptidase activity in digesta taken from the rumen of sheep decreased by 33% in the presence of 1,10-phenanthroline, while mixed bacteria from the same samples were inhibited by 80% and the activity of mixed protozoa decreased by only 15%. Thus a substantial amount of dipeptide breakdown appears to be due to ciliate protozoa in the mixed population. Extensive washing of the protozoa increased the sensitivity of protozoal dipeptidase activity to 1,10-phenanthroline, suggesting that protozoa too have a metallo-dipeptidase activity but that it is normally protected from inhibition by 1,10-phenanthroline. Breakdown of the pentapeptide, Ala5, was also inhibited 27% by 1,10-phenanthroline in the mixed population, and when Trypticase, a pancreatic casein hydrolysate containing a mixture of oligopeptides, dipeptides and amino acids, was incubated with rumen fluid, the production of ammonia and free amino groups was inhibited 71% by 1,10-phenanthroline. It was concluded that metal ion chelation inhibits oligopeptidase and dipeptidase activities of rumen micro-organisms and may be a means of controlling ammonia production from peptides in the rumen.

Carbonate inhibition of leukotriene D4-dipeptidase in human serum.

Human serum contains an inhibitor of leukotriene D4 (LTD4) dipeptidase which was separated from the enzyme by ultrafiltration (Amicon, YM-10). Removal of the inhibitor resulted in a 3- to 5-fold increase in total LTD4-dipeptidase activity in the material retained by the filter. Inhibitor activity (which was assayed with a partially purified LTD4-dipeptidase) was recovered in the filtrate. Ultrafiltration of serum using YM-3, YM-1, and YC-05 membranes suggested an inhibitor molecular weight of less than 500. Elution of inhibitor activity from a Bio Gel P2 gel filtration column was identical to the elution pattern of pure carbonate. The inhibitor was heat stable (95 degrees C, 30 min), stable in 0.1 N NaOH, but rapidly inactivated by 0.1 N HCl at both 4 degrees C and 30 degrees C. Partially purified LTD4-dipeptidase was inhibited by carbonate and phosphate but not by nitrate, sulfate, or chloride. Based on these observations it was concluded that the inhibitor of LTD4-dipeptidase in human serum either was carbonate or required carbonate. The relative concentrations of LTC4, LTD4, and LTE4 appear to be important parameters in determining the duration and intensity of LT mediated reactions. The relative concentration of carbonate in serum or extracellular fluids might, therefore, be a factor in modulating localized LT mediated responses.

[Age-related changes in the dipeptide hydrolase activity of different sections of the small intestine in rats in response to hydrocortisone administration]. / Vozrastzavisimye izmeneniia dipeptidgidrolaznoi aktivnosti razlichnykh otdelov tonkoi kishki krys v otvet na vvedenie gidrokortizona.

The glycyl-L-leucinedipeptidehydrolase activity in different portions of the small intestine after hydrocortisone administration on the 1st, 5th, 10th, 16th, 30th day of life was studied in rats. The enzyme was mainly localized in distal portion of the small intestine for the first two weeks. The maximal reactivity of the enzyme-synthetizing system was shown after a single hydrocortisone injection during this period in proximal portion. The enzyme distribution along the small intestine became even in the end of the experimental period. The response to the hormone administration was practically absent from the 16th day of suckling life.