Presence Of Bestatin Research Articles

Oxytocin is hydrolyzed by an enzyme in human placenta that is identical to the oxytocinase of pregnancy serum.

The hydrolysis of oxytocin (OT) by human placental subcellular fractions and pregnant sera was studied in the presence of bestatin, a potent inhibitor of aminopeptidases, and the antibody against pregnant serum oxyotocinase (P-LAP)(EC 3.4 11.3) by measuring liberated amino acids by high performance liquid chromatography (HPLC). Our immunotitration study and the effect of bastatin on the oxytocin-degrading protease showed that the initiating and responsible protease in oxyotocin degradation in human placenta and pregnant serum is P-LAP.

Inactivation of interleukin-8 by aminopeptidase N (CD13).

Aminopeptidase (APN) was found to degrade interleukin‐8 (IL‐8) and inactivate its chemotactic activity. The chemotactic activity of IL‐8 was decreased by APN or neutrophil plasma membranes dose‐ and time‐dependently. The chemotactic activity was not inactivated in the presence of bestatin or WM15 monoclonal antibody. The expression of IL‐8 was measured by flow cytometry. On lipopolysaccharide (LPS) stimulation, IL‐8 expression increased for 60 min and then decreased markedly. In contrast, on treatment with LPS and bestatin, the expression of IL‐8 increased continuously for at least 120 min. These results suggest that the expression and release of IL‐8 from phagocytic cells are regulated by the proteolytic effect of APN on IL‐8. J. Leukoc. Biol. 57: 129–134; 1995.

Inhibition of tumor invasion and extracellular matrix degradation by ubenimex (bestatin)

We have investigated the effect of the immunomodulator ubenimex (hereafter referred to as bestatin) on the enzymatic degradation of the extracellular matrix by human renal cell carcinoma SN12M cells during the invasive process. The invasion of SN12M cells into reconstituted basement membrane (Matrigel) was inhibited by the presence of bestatin in a concentration-dependent manner. However, bestatin did not have any effect on tumor cell adhesion and migration to the extracellular matrices which may be involved in tumor cell invasion. Bestatin inhibited the degradation of type IV collagen by tumor cells, but not by tumor-conditioned medium (TCM), in a concentration-dependent manner. We also found that bestatin inhibited hydrolysing activities towards substrates of aminopeptidases in SN12M cells. Since bestatin was found to inhibit aminopeptidase activity, the inhibition of tumor invasion by bestatin is likely to be associated with its action as an enzyme inhibitor. Bestatin only slightly inhibited tumor cell plasmin activity, which can lead to the conversion of the latent collagenase to the active form, but this slight effect was not significant. The zymography of TCM from SN12M cells showed that the treatment of tumor cells with bestatin resulted in the disappearance of the 68 kDa type IV collagenase-enzyme level (active form) and slight reduction of the 72 kDa type IV collagenase-enzyme level (latent form). These results indicated that bestatin may inhibit tumor cell invasion through a mechanism involving its inhibitory action on aminopeptidases in tumor cells, suggesting that the aminopeptidase may partly be associated with the conversion of a latent form of type IV procollagenase to an active form or the secretion of the collagenases from tumor cells.

Evidence that the agonist action of dynorphin A(1–8) in the guinea‐pig myenteric‐plexus may be mediated partly through conversion to [Leu5]enkephalin

1. The agonist action of the opioid peptide dynorphin A(1-8) on the myenteric plexus-longitudinal muscle of the guinea-pig ileum has been characterized. 2. The endogenous opioid peptide dynorphin A(1-8) was rapidly degraded by slices of myenteric plexus-longitudinal muscle of the guinea-pig ileum. 3. A product of the degradation was the delta-receptor preferring [Leu5]enkephalin. Levels of [Leu5]enkephalin were markedly increased in the presence of the peptidase inhibitors bestatin, thiorphan and captopril. 4. In the myenteric plexus dynorphin A(1-8) acted as a kappa-receptor agonist. In the presence of bestatin, thiorphan and captopril a mu-receptor agonist effect was observed. This mu-agonist action was lost in the presence of N-[1-(RS)-carboxy-2-phenylethyl]Ala-Ala-Phe-p-aminobenzoate, an inhibitor of the endopeptidase enzyme EC 3.4.24.15. 5. The results suggest that formation of [Leu5]enkephalin from dynorphin A(1-8) may be an important conversion process. The enzyme responsible may be the Zn2(+)-metalloendopeptidase, EC 3.4.24.15.

Inhibition of Tumor Cell Invasion by Ubenimex (Bestatin) in vitro

We have investigated the effect of the immunomodulator ubenimex (bestatin) on tumor cell invasion of reconstituted basement membrane (Matrigel). The invasion of B16‐BL6 melanoma cells and Lewis lung carcinoma (3LL) cells into Matrigel‐coated filters was inhibited by the presence of bestatin in a concentration‐dependent manner. The prctrcatment of either tumor cells or Matrigel with bestatin, however, had little effect on the invasion of tumor cells. Since bestatin was found to inhibit aminopeptidase in addition to its immunomodulating activities, the inhibition of tumor invasion by bestatin is likely to be associated with the action as an enzyme inhibitor. Other aminopeptidase inhibitors, arphamcnine B and amastatin A, could also inhibit tumor cell invasion into Matrigel. Bestatin inhibited hydrolyzing activities towards substrates of aminopeptidases in B16‐BL6 melanoma cells. However, bestatin did not have any effect on the haptotactic migration and adhesion of tumor cells to the substrates. These results indicated that bestatin may inhibit tumor cell invasion through a mechanism involving its inhibitory action on aminopeptidases in tumor cells.

Bestatin potentiates the antinociception but not the motor dysfunction induced by intracerebrally administered dynorphin-B in mice

The intracerebrally administered dynorphin-B produced not only an antinociceptive response but a motor dysfunction such as “barrel-rolling”, circling, jumping or ataxia in mice. These two effects were observed in a same dose range. Both responses were also produced by a nonopioid fragment, des-Tyr-dynorphin-B. Bestatin, an aminopeptidase inhibitor, markedly potentiated the antinociceptive response induced by dynorphin-B but not the motor dysfunction. Bestatin did not affect the responses produced by des-Tyr-dynorphin-B. In the presence of bestatin, low doses of dynorphin-B produced an antinociceptive response without the motor dysfunction. Naloxone antagonized the potentiated antinocicpetion but had no effect on the motor dysfunction. These results suggest that dynorphin-B produced an analgesia through opioid receptors and that this peptide also induced a motor dysfunction through a nonopioid receptor.

A novel dipeptidyl aminopeptidase in rat brain membranes. Its isolation, purification, and characterization.

A new type of dipeptidyl aminopeptidase, which releases basic aminoacyl dipeptides from the NH2-terminal end of oligopeptides, was purified about 2100-fold with 6.8% recovery from rat brain membranes by column chromatography on Cellex D, Arg-Tyr-AH-Sepharose 4B, hydroxylapatite, and Sephadex G-75, after the membranes were solubilized with Nonidet P-40. Activity was assayed by high performance liquid chromatography (HPLC) using Arg0-Met5-enkephalin (Arg0-enk)* as substrate in the presence of bestatin, thiorphan, and captopril. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the purified enzyme is apparently homogeneous with a mass of 64,000 daltons. This thiol enzyme is optimally active at pH 7 and is selectively activated by Mn(II). It loses 94% of its activity after EDTA treatment and can be reactivated by Mn(II), Co(II), and Zn(II). It splits Arg0-enk into equimolar amounts of Arg-Tyr and Gly-Gly-Phe-Met with a Km of 100 microM, and Vmax of 3.8 mumol/mg of protein/min. Dipeptidyl aminopeptidase does not hydrolyze model substrates for dipeptidyl aminopeptidases I, II, III, and IV, aminoacyl beta-naphthylamides, actin, desmin, tubulin, glial fibrillary acidic protein, and cytoskeletal neurofilament proteins. The enzyme is insensitive to puromycin, but is inhibited by several neuropeptides. Angiotensin III is the most potent with a Ki of 0.3 microM. Substrate specificity, pH optimum, molecular weight, activators, and catalytic site demonstrate that this enzyme is distinct from dipeptidyl aminopeptidases previously described.

Intermediate peptides of insulin degradation in liver and cultured hepatocytes of rats

1. 1. Bestatin, a microbial aminopeptidase inhibitor, induced accumulation of low-molecular weight intermediate peptides of insulin degradation in liver of rats in vivo and in primary cultured rat hepatocytes. However, bestatin did not affect the association and internalization of the hormone into hepatic cells. 2. 2. Results of the HPLC analyses showed that the intermediate peptides of insulin degradation are small ones and specifically accumulate only in the presence of bestatin. 3. 3. The above results, together with those employing other protease inhibitors, show that cytosolic bestatin-sensitive protease(s), trypsin-like protease(s) and thiol protease(s) play an important role in the intracellular degradation process of insulin.

Angiotensin II inactivation process in cultured mouse spinal cord cells.

The pattern of hydrolysis of [3H]angiotensin II ( [3H]AII; 20 nM) by intact cells was studied on cultured mouse spinal cord cells. Degradation products were identified by HPLC analysis after incubation for 2 h at 37 degrees C. In the absence of peptidase inhibitors, 70% of [3H]AII was degraded, and the main labeled metabolite was [3H]tyrosine (40% of total radioactivity). Minor quantities of [3H]AII1-5 and [3H]AII4-8 were formed. Results obtained in the presence of various inhibitors indicate that several enzymes were involved in the AII-hydrolyzing process. Dipeptidyl aminopeptidase III (EC 3.4.14.4) could play a critical role, as suggested by the formation of [3H]Val3-Tyr4 and [3H]-Tyr4-Ile5 in the presence of bestatin (2 X 10(-5) M). This hypothesis was confirmed by the potency of dipeptidyl amino-peptidase III inhibitors to inhibit both [3H]AII hydrolysis and formation of these 3H-labeled dipeptides. An arylamidase-like activity could also be participating in [3H]AII hydrolysis, because higher concentrations of bestatin (10(-4) M) in association with dipeptidyl aminopeptidase III inhibitors totally inhibited [3H]tyrosine formation, increased protection of [3H]AII and [3H]AII1-7 formed, and provoked a slight accumulation of [3H]AII2-8. These results suggest that the formation of [3H]AII2-8 is due to the action of a bestatin-insensitive acidic aminopeptidase and that the Pro7-Phe8 cleavage is also a step of AII hydrolysis, resulting from the action of an unidentified peptidase different from prolyl endopeptidase.(ABSTRACT TRUNCATED AT 250 WORDS)

Purification and characterization of two Cl- -activated aminopeptidases hydrolysing basic termini from human skeletal muscle.

Two aminopeptidases (I and II), hydrolysing basic termini, were purified to homogeneity (as judged by polyacrylamide gel electrophoresis) from human quadriceps muscle by anion-exchange chromatography and preparative electrophoresis. The electrophoretic migration rate of II was approximately 80% of that of I. Both enzymes had the following properties: optimum activity was at pH 6.5; addition of 0.15 M Cl- or Br- anions resulted in a 20-fold or 10-fold increase in activity respectively. There was little or no increase in activity on the addition of other anions, or divalent cations (0.05-5mM). Approximately 50% inhibition of activity was obtained in the presence of bestatin (0.1 microM), rho-hydroxymercuriphenylsulphonic acid (0.1 microM), EDTA (10 mM), 1,10-phenanthroline (100 microM), N-ethylmaleimide (1 mM) and But-Thr-Phe-Pro (0.5 mM). The molecular mass was 72 000 Da (gel filtration). Only the arginyl and lysyl 7-amino-4-methylcoumarin (Amc) derivatives were appreciably hydrolysed; approximate Km values for the reaction of I and II with these substrates (10-250 microM) were estimated as follows: Arg-Amc, KmI = 70 microM, KmII = 270 microM; Lys-Amc KmI = 280 microM, KmII = 400 microM. Both enzymes hydrolysed dipeptides with Arg or Lys as the NH2-terminal amino acid, however this was not an absolute requirement for dipeptide hydrolysis. The action of I and II on physiologically active oligopeptides was very restricted, with only bradykinin, proangiotensin and neurotensin being appreciably degraded. The breakdown of these peptides did not occur by classical aminopeptidase action (i.e. hydrolysis of the NH2-terminal residues), but via cleavage of internal peptide bonds. These results suggest that I and II may be isoenzymes of a Cl- -requiring, thiol-type aminopeptidase, which hydrolyses basic termini. These enzymes may act primarily as dipeptidases, with a very restricted mode of action in the degradation of naturally occurring oligopeptides.

Participation of both ‘enkephalinase’ and aminopeptidase activities in the metabolism of endogenous enkephalins

The pattern of hydrolysis of [ 3H]Met 5-enkephalin by slices from rat striatum has been determined by Chromatographic isolation of [ 3H]peptide fragments and by assessment of the effects of various selective peptidase inhibitors. It consists in the formation of [ 3H]Tyr, [ 3H]Tyr-Gly and [ 3H]Tyr-Gly-Gly which represent 80%, 2% and 18%, respectively, of total 3H-metabolite formation. The formation of [ 3H]Tyr, presumably occurring under the action of membrane-bound aminopeptidases, is reduced in the presence of either 10 −4 M puromycin or 2.10 −5 M bestatin by about 40% and 75%, respectively. Both aminopeptidase inhibitors display significantly higher potency on a crude particulate fraction from rat striatum. The formation of [ 3H]Tyr-Gly-Gly in the slice preparation is strongly reduced by addition of 10 −7 M thiorphan, an enkephalin dipeptidylcar☐ypeptidase (‘enkephalinase’) inhibitor, but only slightly in the presence of 10 −6 M captopril, an angiotensin-converting enzyme inhibitor. When the aminopeptidase pathway is inhibited by bestatin, [ 3H]Tyr-Gly-Gly represents more than 60% of total hydrolysis products, suggesting that the tripeptide is partly hydrolysed after being formed under the action of ‘enkephalinase’. In the presence of both bestatin and thiorphan, the total hydrolysing activity of the slice preparation is reduced by 75% while [ 3H]Tyr-Gly level is slightly increased. These data indicate that the hydrolysis of exogenous Met 5-enkephalin in the extracellular space of the slice preparation primarily involves the ‘enkephalinase’ as well as the aminopeptidase pathways. The participation of both pathways in the inactivation of endogenous Met 5-enkephalin has also been evidenced by evaluating the recovery of the opioid peptide released by K +-induced depolarisation of striatal slices in the presence of bestatin and thiorphan. While the presence of either inhibitor allows a partial protection of the released peptide, their co-presence results in total recovery of the enkephalin in intact form in the medium. Finally, administration of either the aminopeptidase or the ‘enkephalinase’ inhibitor in mice results in antinociceptive effects as evaluated in the hot-plate jump test. These effects are additive and prevented by naloxone, an opiate receptor antagonist. Thus, the various experimental approaches used indicate that both (but only) the ‘enkephalinase’ and the aminopeptidase pathways play a critical role in the inactivation of endogenous enkephalins.

Circadian rhythmicity of glycyl-l-leucine absorption mechanism in rat small intestine.

Adult rats fed ad libitum and on restricted-feeding (food available between 10:00 and 16:00) regimen showed feeding-cued circadian rhythms in the activity patterns of glycyld-L-leucine and L-leucine absorption by the proximal small intestine. Absorption of the two compounds by the distal small intestine followed these similar, but less overt, rhythmic patterns. The glycyl-L-leucine hydrolase activity of cytosolic fraction of the mucosal homogenates from the proximal intestine also exhibited a similar daily rhythm in the rats on restricted-feeding regimen, but no discernible rhythm in the rats fed ad libitum. Rhythmicity of the hydrolase activity was hardly noticeable in the membrane fraction on either feeding regimen. Restricted-feeding resulted in an enhanced daily average level for those intestinal activities. In the presence of bestatin, rhythmicity of glycyl-L-leucine absorption by the proximal intestine was unimpaired while cytosolic and membrane hydrolysis of the dipeptide was severely inhibited. Th...

Perturbation by bestatin of glycyl-L-leucine absorption in isolated epithelial cells from rat intestine.

Glycyl-L-leucine is one of the best substrates for peptide hydrolases in the intestinal mucosa. Its absorption and hydrolysis were investigated in epithelial cells isolated from the rat intestine in the presence of bestatin, a specific inhibitor of certain peptide hydrolases. Bestatin competi-tively inhibited dipeptide hydrolase activities in isolated cells with a Ki value of 10-8M, but noncompetitively inhibited, and less significantly, the dipeptide absorption by isolated cells. At 10-4M bestatin inhibited half the dipeptide absorption, but only minimally inhibited the absorption of constituent amino acids. In the presence of bestatin at substantial concentrations the isolated cells took up a significant amount of the intact dipeptide, which otherwise appeared entirely in the form of free amino acids. These results are interpreted to substantiate a notion that a dual mechanism is operative for the absorption of readily-hydrolysable peptides: the peptide hydrolysis followed by uptake of thereby released amino acids, and the peptide transport followed by cytosolic hydrolysis.

Degradation of abnormal proteins in intact mouse reticulocytes: accumulation of intermediates in the presence of bestatin.

Incubation of intact mouse reticulocytes with bestatin (a competitive inhibitor of aminopeptidases) produced the accumulation of low molecular weight intermediates in the degradation of puromycinyl-peptides or analog-containing proteins that had been pulse labeled with L-[1-14C]leucine. A large fraction of the radioactive protein was degraded to trichloroacetic acid-soluble products within 10 min. In the presence of bestatin (0.5 mg/ml), one-fourth of these products appeared to be dipeptides, tripeptides, or both: they were resistant to ninhydrin at acid pH (a treatment that decarboxylates only free amino acids) except after intensive acid hydrolysis, and they eluted from a Sephadex G-10 column with an apparent average size of 300 daltons. These radioactive products did not appear if incorporation of the tracer was prevented by prior treatment with cycloheximide, demonstrating that they originated from polypeptide precursors. Thus, a peptidase inhibitor has been successfully used in the production of low molecular weight intermediates in the in vivo degradation of cellular proteins.