Bestatin-sensitive Aminopeptidase Research Articles

Neurokinin B is hydrolysed by synaptic membranes and by endopeptidase-24.11 (‘enkephalinase’) but not by angiotensin converting enzyme

Endopeptidase-24.11 (‘enkephalinase’, EC 3.4.24.11) from pig kidney hydrolysed neurokinin B at 4 sites: ▪ The major site of hydrolysis was the Gly 8-Leu 9 bond. Angiotensin converting enzyme (peptidyl dipeptidase A, EC 3.4.15.1) from pig kidney hydrolysed substance P releasing the C-tenninal tripeptide Gly-Leu-MetNH 2 but failed to hydrolyse neurokinin B. Pig brain striatal synaptic membranes hydrolysed neurokinin B producing a similar pattern of products as did endopeptidase-24.11. Substantial inhibition of this activity was achieved with the selective inhibitor phosphoramidon. A combination of phosphoramidon and bestatin abolished the hydrolysis of neurokinin B by synaptic membranes. Thus, a bestatin-sensitive aminopeptidase may play a role in the synaptic metabolism of neurokinin B in addition to endopeptidase-24.11. This aminopeptidase appears to be distinct from aminopeptidase N (EC 3.4.11.2).

Hydrolysis of the C-terminal octapeptide of cholecystokinin by rat kidney membranes: Characterization of the cleavage by solubilized endopeptidase - 24.11

Rat kidney membranes were solubilized by Triton X-100 and the CCK-8 degrading peptidases were resolved by chromatography on DEAE-cellulose. Four proteases were detected: two phosphoramidon-sensitive endopeptidases (EC 3.4.24.11), a bestatin-sensitive aminopeptidase and an unidentified enzyme. The pattern of cleavage of CCK-8 and shorter C-terminal fragments by endopeptidase 24.11 was investigated and indicated that the Gly 29-Trp 30-Met 31 and Asp 32-Phe 33 were scissile bonds. However, the cleavage pattern differed markedly from one CCK peptide to another: in the penta- and hexapeptide of CCK the bonds hydrolyzed were either Asp-Phe and Trp-Met or, Asp-Phe and Gly-Trp, respectively. The presence of the sulfate group on the tyrosine residue of CCK-8 influence markedly the nature of the major cleavage fragments produced by the endopeptidase. The major bonds cleaved were Asp-Phe, Trp-Met and Gly-Trp for unsulfated CCK-8, whilst for the sulfated octapeptide, the Trp-Met bond became a minor cleavage site.

Bestatin, a microbial aminopeptidase inhibitor, inhibits DNA synthesis induced by insulin or epidermal growth factor in primary cultured rat hepatocytes.

Bestatin, a microbial aminopeptidase inhibitor, inhibited insulin- or epidermal growth factor-induced DNA synthesis and cell proliferation in primary cultured hepatocytes of rats. The aminopeptidase inhibitor also affected the growth of FM3A or LOBN cells of mice, when it is included in the culture media at the concentration of 0.5 mg/ml. These results suggest the important role of the bestatin-sensitive aminopeptidase(s) in the process of DNA synthesis and proliferation of animal cells.

Hydrolytic deactivation of kyotorphin by the rodent brain homogenates and sera.

A simple analytical assay method for kyotorphin was developed by the use of amino acid analyzer and the deactivation of kyotorphin in biological media was studied with this method. Kyotorphin was hydrolyzed rapidly by enzymes in serum and brain of rat and mouse, but L-tyrosyl-D-arginine was not. The hydrolysis activity was much higher in brain than in serum. The apparent Michaelis' constants, Km, were of the order of 10(-5) for brain and 10(-4) M for serum in both species. The hydrolysis of kyotorphin was strongly inhibited by bestatin (Ki = 0.1 microM). Thus the hydrolysis might be attributed to aminopeptidase(s). A dose of 50 micrograms of bestatin administered intracisternally potentiated the analgesic activity of kyotorphin by 4.8 times or so. Thus kyotorphin seems to be deactivated by bestatin-sensitive aminopeptidase(s) in brain.

The Role of Bestatin-Sensitive Aminopeptidase, Angiotensin Converting Enzyme and Thiorphan-Sensitive "Enkephalinase" in the Potency of Enkephalins in the Guinea-Pig Ileum

The role of each enkephalin-hydrolyzing peptidase in the inhibitory potency of exogenously added enkephalins in the myenteric plexus-longitudinal muscle preparation of guinea-pig ileum was studied by using the relatively specific inhibitor of each enzyme. Results showed that three distinct enzymes, bestatin-sensitive aminopeptidase(s), angiotensin converting enzyme, and thiorphan-sensitive "enkephalinase", played a critical role in the inactivation of enkephalins. Additionally, these enzymes are likely to be located close to opioid receptors, since they produce a significant concentration difference of enkephalin between the surrounding organ bath and the vicinity of opioid receptors. In contrast to these three enzymes, both L-tyrosyl-L-tyrosine-sensitive dipeptidyl aminopeptidase and D-phenylalanine-sensitive carboxypeptidase are indicated not to be involved significantly in the degradation of exogenously added enkephalins in the guinea-pig ileum.

Heterogeneous distribution of enkephalin-degrading peptidases between neuronal and glial cells

Cultured neurones, astroblasts and astrocytes from murine brain have been screened with specific tests for the presence of peptidases capable of degrading enkephalin. Bestatin-sensitive aminopeptidases represent the major enkephalin-degrading activity in all cases. The dipeptidylaminopeptidasic activity is much higher in the neuronal than the glial cultures, whereas the opposite is true for the metallopeptidase called "enkephalinase". Only trace amounts of the dipeptidylcarboxypeptidase "angiotensin-converting enzyme" have been found. We conclude that bestatin-sensitive aminopeptidases on nerve cells are probable candidates for enkephalin-inactivating enzymes, whereas the "enkephalinase" on glial cells more likely serves a scavenger function.

Peptidases involved in the inactivation of exogenous and endogenous enkephalins.

Among the various cerebral enzyme activities able to hydrolyse the enkephalins into inactive fragments only two seem responsible for the metabolism of the endogenous opioid peptides: a dipeptidylcarboxypeptidase ("enkephalinase"), and a bestatin-sensitive aminopeptidase. Their inhibition by thiorphan and bestatin results in an antinociceptive effect observed in tests in which the nociceptive stimulation is probably accompanied by a concomittent release of enkephalins.

Inhibition of enkepha,in metabolism by, and antinociceptive activity of, bestatin, an aminopeptidase inhibitor

In the presence of thiorphan an ‘enkephalinase’ inhibitor, bestatin an aminopeptidase inhibitor of bactreial origin potently inhibited the hydrolysis of [ 3H][Leu 5]enkephalin by slices from rat striatum with an IC 50 value of about 0.2 μM whereas puromycin was approximately 1000 times less potent on this preparation. In vivo bestatin or thiorphan (but not puromycin) significantly protected [ 3H][Met 5]enkephalin administered intracerebroventricularly to mice from hydrolysis and co-administration of these two peptides inhibitors resulted in strong reduction in the appearance of hydrolysis products in brain. In a parallel fashion the antinociceptive activity of [Met 5]enkephalin in the mouse hot-plate test was additively potentiated by bestatin and thiorphan but not by puromycin. Finally both bestatin and thiorphan themselves displayed antinociceptive properties on either the hot-plate jump test or the phenyl-benzoquinone writhing test. It is concluded that a bestatin-sensitive aminopeptidase activity together with the ‘enkephalinase’ activity plays a critical role in the inactivation of both exogenous and endogenous enkephalins.

Enkephalin metabolism in brain and its inhibition

Various cerebral peptidases are able to hydrolyse the enkephalins into inactive fragments. Among these enzymes enkephalin-dipeptidylcarboxypeptidase (“enkephalinase”) inhibited by thiorphan and a bestatin-sensitive aminopeptidase activity seem to play a keyrole as “inactivating neuropeptidases”. Their inhibition, in vitro as well as in vivo, leads to a protection of endogenous enkephalins and to antinociceptive effects.