l-Phenylalanyl-l-Glutamate-Stimulated, Chloride-Dependent Glutamate Binding Represents Glutamate Sequestration Mediated by an Exchange System

Stimulation of glutamate binding by the dipeptide L-phenylalanyl-L-glutamate (Phe-Glu) was inhibited by the peptidase inhibitor bestatin, suggesting that the stimulation was caused by glutamate liberated from the dipeptide and not by the dipeptide itself. It further suggests that this form of glutamate binding should be reinterpreted as glutamate sequestration and that stimulation of binding both by dipeptides and after preincubation with high concentrations of glutamate is likely to be due to counterflow accumulation. Several other criteria indicate that most of glutamate binding stimulated by chloride represents glutamate sequestration: Binding is reduced when the osmolarity of the incubation medium is increased, when membranes incubated with [3H]glutamate are lysed before filtration, and when membranes are made permeable by transient exposure to saponin. Moreover, dissociation of bound glutamate after a 100-fold dilution of the incubation medium is accelerated about 50 times by the addition of glutamate to the dilution medium. This result would be anomalous if glutamate were bound to a receptor site; it suggests instead that glutamate is transported in and out of membrane vesicles by a transport system that preferentially mediates exchange between internal and external glutamate. Glutamate binding contains a component of glutamate sequestration even when measured in the absence of chloride. Sequestration is adequately abolished only after treating membranes with detergents; even extensive lysis, sonication, and freezing/thawing may be insufficient.     

Binding structure of the leucine aminopeptidase inhibitor microginin FR1

Natural bioactive compounds are of general interest for pharmaceutical research because they may serve as leads in drug development campaigns. Among them, microginins are linear peptides known to inhibit various exopeptidases. The crystal structure of microginin FR1 from Microcystis sp. bound to bovine lens leucine aminopeptidase was established at 1.73 Å resolution. The observed binding structure could be beneficial for the design of potent aminopeptidase inhibitors.     

A practical diastereoselective synthesis of (−)‐bestatin

Diastereoselective addition of nitromethane to Boc‐D‐Phe‐H in the presence of sodium hydride in diethyl ether/hexane containing 15‐crown‐5 and subsequent N,O‐protection with 2,2‐dimethoxypropane gave trans‐oxazolidine in a diastereomeric ratio of >16:1. The oxazolidine was easily separated by column chromatography, which after Nef reaction was coupled to H‐Leu‐OtBu. The 8‐step synthesis afforded (?)‐bestatin in an overall yield of 24.7% after deprotection and ion exchange.     

Separation and partial characterization of two alkaline peptidases from cotyledons of resting kidney beans, Phaseolus vulagaris

Extracts prepared from cotyledons of resting kidney beans ( Phaseolus vulgaris L., cv. Processor) rapidly hydrolyzed two dipeptides, Leu-Tyr and Ala-Gly, with pH optima at 9.2 and 8.5, respectively. On ion exchange chromatography on DEAE-Sephacel the two activities eluted as separate peaks, showing that they were due to two different peptidases. The extracts also hydrolyzed Leu-β-naphthylamide optimally at pH 6.4; this activity eluted as a third peak berween the other peaks. The activity peak acting on Leu-Tyr and Ala-Gly rapidly hydrolized two tripeptides, showing that it was an aminopeptidase, whereas the Ala-Gly hydrolyzing peak acted only on dipeptides. The activities against Leu-Tyr and Ala-Gly were also separated by gel chromatography on Sephacryl S-300 with elution positions corresponding to M, values of about 360 000 and 105 000. The aminopeptidase was inhibited by bestatin, and the dipeptidase was inhibited by p-hydroxymercuribenzoate. Both enzymes were inhibited by o-phenanthroline. In most of their properties the two kidneys bean enzymes resembled the alkaline aminopeptidase and the dipeptidase earlier characterized from barley grains.     

Monocyte activation by an oral immunomodulator (bestatin) in lymphoma patients following autologous bone marrow transplantation

 Bestatin (ubenimex), an inhibitor of aminopeptidase, is an oral immunomodulator that binds to CD13 (aminopeptidase N) on macrophages/monocytes. To examine its immunomodulatory effect after high-dose therapy and autologous bone marrow transplantation (BMT), a dose-finding phase Ib trial was conducted with 30 Hodgkin’s disease and non-Hodgkin’s lymphoma patients who received no drug (control), 10 and 30 mg (low dose), or 90 and 180 mg (high dose) of bestatin daily for 60 days following autologous BMT. Bestatin administration was initiated when the absolute neutrophil count was greater than 250/mm3 on 2 consecutive days. The serum neopterin levels, an indicator of monocyte/macrophage activation, increased in the high-dose group compared to the control group (not significantly) and the low-dose group (significantly). Similarly, the colony-stimulating activity in the sera was significantly increased in the high-dose group compared to the control and low-dose groups. We also examined the expression of cell-surface markers on monocytes in these patients by fluorescent cytometry analysis. There was no significant difference either in the frequency or absolute number of monocytes (CD14⁺) among the three groups at any time. However, a significant increase in the frequency of CD16(FcgRIII)-positive monocytes (a marker of activation) was observed in the high-dose group compared to controls from day 14 to day 60 after the start of bestatin administration. Further, the frequency of HLA-DR⁺ monocytes (another marker of activation) was significantly increased in the high-dose group. These results indicate that bestatin at higher doses (90 and 180 mg daily), but not lower doses, activates macrophages/monocytes, as demonstrated by phenotypic marker (HLA-DR and CD16) up-regulation, and this provides augmentation of neopterin and colony-stimulating activity in the serum of patients following autologous BMT. Received: 24 June 1996 / Accepted 13 September 1996     

Catabolism of neurotensin by neural (neuroblastoma clone N1E115) and extraneural (HT29) cell lines

The mechanisms by which neurotensin (NT) was inactivated by differentiated neuroblastoma and HT29 cells were characterized. In both cell lines, the sites of primary cleavages of NT were Pro⁷-Arg⁸, Arg⁸-Arg⁹ and Pro¹⁰-Tyr¹¹ bonds. The cleavage at the Pro⁷-Arg⁸ bond was totally inhibited by N-benzyloxycarbonyl-Prolyl-Prolinal and therefore resulted from the action of proline endopeptidase. This peptidase also contributed in a major way to the cleavage at the Pro¹⁰-Tyr¹¹ bond. However the latter breakdown was partly due to an NT-degrading neutral metallopeptidase. Finally, we demonstrated the involvement of a recently purified rat brain soluble metalloendopeptidase at the Arg⁸-Arg⁹ site by the use of its specific inhibitor N-[1(R,S)-carboxy-2-Phenylethyl]-alanylalanylphenylalanine-p-aminobenzoate. The secondary processing of NT degradation products revealed differences between HT29 and N1E115 cells. Angiotensin converting enzyme was shown to degrade NT_1–10 and NT_1–7 in N1E115 cells but was not detected in HT29 cells. A post-proline dipeptidyl aminopeptidase activity converted NT_9–13 into NT_11–13 in HT29 cells but not in N1E115 cells. Finally bestatin-sensitive aminopeptidases rapidly broke down NT_11–13 to Tyr in both cell lines. Models for the inactivation of NT in HT29 and N1E115 cells are proposed and compared to that previously described for purified rat brain synaptic membranes.     

Amastatin potentiates the behavioral effects of vasopressin and oxytocin in mice

Vasopressin and oxytocin cause behavioral excitation after intracerebroventricular injection in mice. This effect is short-lasting, suggesting that the peptides are rapidly inactivated in the brain. Co-injection of microgram amounts of amastatin, an aminopeptidase inhibitor, prolonged the effect of both vasopressin and oxytocin. Amastatin did not induce large vasopressin-like behavioral effects by itself, nor did it significantly potentiate the action of 1-deamino[1,6-dicarba, 8-arginine] vasopressin (Asu-AVP), an analog that lacks the N-terminal amino group. The effect of Asu-AVP, but not that of vasopressin, was potentiated by phosphoramidon, an inhibitor of neutral metalloendopeptidase ("enkephalinase A"). These results support previous suggestions that vasopressin and oxytocin are inactivated mainly by aminopeptidase action following intracerebroventricular injection.     

注射Bestatin对幼年大鼠分辨学习的影响

以Ｓｐｒａｑｕｅ－Ｄａｗｌｅｙ大鼠为实验动物,从出生后第１天起,每日皮下分别注射氨基肽酶的抑制剂Ｂｅｓｔａｔｉｎ（１ｍｇ／ｋｇ（ｂ．ｗ））以及钠络酮（２ｍｇ／ｋｇ（ｂ．ｗ）＋Ｂｅｓｔａｉｎｇ（１ｍｇ／ｋｇ（ｂ．ｗ））,对照组注射等量的生理盐水,连续注射１４天,观察：１６日龄幼鼠的吸乳迷津分辨学习（ＡＤＬ）,３０日龄Ｙ迷津明暗分辨学习（ＡＤＬ）,３０日龄Ｙ迷津明暗分辨学习（ＢＤＬ）行为,４５日龄幼鼠     

Physiological role of brain angiotensin III in drinking behavior in the rat

We examined the physiologic role of endogenous brain angiotensin III (AIII), an active degradative product of angiotensin II, in drinking behavior. Adult, male spontaneously hypertensive (SH) and Wistar-Kyoto normotensive (WKY) rats that were instrumented with an intracerebroventricular (i.c.v.) cannula connected to an osmotic minipump for chronic infusion were used. 7-day i.c.v. infusion of the specific AIII antagonist, Ile⁷-AIII (10 or 100 pmol/min), resulted in no significant alteration in daily (24 h), diurnal (8:00 a.m.-8:00 p.m.) or nocturnal (8:00 p.m.-8:00 a.m.) basal water intake in both SH and WKY rats. Similar results were obtained with i.c.v. infusion of the aminopeptidase inhibitor, bestatin (150 or 300 pmol/min), given alone or simultaneously with Ile⁷-AIII (10 pmol/min). Rats that were water-deprived for the first 3 days of 7-day infusion of Ile⁷-AIII consumed significantly less water during the first 2 h after water became available. Furthermore, the accumulated water intake during the first 24 h was appreciably greater in SH than WKY rats. We interpret these results to suggest that the endogenous brain AIII may not be tonically involved in fluid homeostasis. Instead, it must be activated under conditions of dehydration, such as water deprivation, particularly in the SHRs, to initiate drinking behavior.