Myron Benuck

Enzymatic inactivation of substance p by a partially purified enzyme from rat brain

Summary Substance P, a unidecapeptide with potent CNS action, is rapidly inactivated by rat brain homogenate with release of all amino acids including Met.NH 2 . The inactivating enzyme present in the 100,000 g supernatant was purified on DEAE-cellulose and was eluted in parallel with a neutral endopeptidase degrading hemoglobin and histone. Breakdown was followed by measurement of amino acids released using a method capable of resolving Met.NH 2 and Leu-Met. NH 2 . The preferential release of Phe and Leu indicated cleavage at two or more internal sites (-Gln 6 -Phe 7 - or -Phe 7 -Phe 8 - and -Gly 9 -Leu 10 -) with release of intermediate peptidyl products. The presence of free Met following inactivation resulted from the deamidation of Met.NH 2 after its liberation from Substance P. A slow release of Arg 1 and Pro 2 indicated that the Arg 1 -Pro 2 bond is only slowly cleaved by brain aminopeptidases.

Co-identity of brain angiotensin converting enzyme with a membrane bound dipeptidyl carboxypeptidase inactivating Met-enkephalin

Abstract The distribution in rat brain of angiotensin converting enzyme (EC3.4.15.1) using hippuryl-His-Leu as substrate was identical to a dipeptidyl carboxypeptidase present in membranes assayed with Met-enkephalin as substrate. Highest activity occurred in pituitary, followed by cerebellum, corpus striatum, midbrain, pons-medulla, hypothalamus, cerebral cortex and spinal cord. The ratio of products His-Leu/Tyr-Gly-Gly was identical for all regions but differed from His-Leu/Tyr. Angiotensin converting enzyme purified by immunoaffinity chromatography gave a K m for hippuryl-His-Leu of 0.5mM and for Met-enkephalin of 0.1 mM. In the presence of the specific inhibitor of angiotensin converting enzyme, SQ 14,225, the Ki value was 10 −7 M. Present data point to the co-identity of brain angiotensin converting enzyme with the dipeptidyl carboxypeptidase inactivating enkephalin.

Differences in the degradation of hypothalamic releasing factors by rat and human serum

Abstract Rat serum is 2–5 fold more active than human serum in cleaving the three hypothalamic releasing hormones, luteinizing hormone releasing hormone (LH-RH), thyrotropin releasing hormone (TRH), and somatostatin. LH-RH was degraded by two distinct enzymatic mechanisms; 1) endopeptidase cleavage, 2) C-terminal cleavage. The C-terminal cleaving enzyme was active in rat serum but present only in trace levels in human. These mechanisms were substantiated by the use of suitably substituted analogs; D-Ala at position 6 of LH-RH prevented cleavage at the -Tyr 5 -D-Ala 6 -Leu 7 -site and the presence of ethylamide (C 2 H 5 NH 2 ) at position 10 inhibited significantly the action of the second enzyme. These analogs have an enhanced biological activity in vivo which correlates well with their decreased rate of degradation. Somatostatin was degraded by endopeptidase cleavage at one or more sites. D-Trip in position 8 blocked cleavage of the -Trp 8 -Lys 9 -bond, reducing significantly the rate of degradation. This also correlates well with the enhanced biological potency of the (D-Trp 8 )-somatostatin analog. TRH was degraded by cleavage of the pyroGlu-His and His-Pro.NH 2 bonds with the release of free His and Pro. The analog (3-Me-His 2 )-TRH was degraded by a similar mechanism with the release of 3-Me-His.

Receptor systems participating in nicotine-specific effects

It is generally accepted that self-administration of drugs is prompted primarily by a reward system driven by an increase in extracellular dopamine in the nucleus accumbens. Recent findings that dopamine increase in the accumbens can be caused by many other factors, among them stress, suggest a more complex mechanism, and possibly differences in the reward system for different compounds. In the present paper we compare the effects of receptor-specific antagonists on the increase of dopamine induced by nicotine with that induced by cocaine in the nucleus accumbens in conscious rats. The compounds alone or together were injected intravenously, and dopamine level changes were measured via microdialysis. When administered together the effect of nicotine and cocaine on the level of dopamine in the accumbens was additive. Apparently there is some interaction between the two compounds, since nicotine had no effect after combined nicotine and cocaine administration. Perhaps the available dopamine pool was exhausted by the prior administration. The nicotinic antagonist mecamylamine, the muscarinic antagonist atropine, and the NMDA glutamate receptor antagonist MK-801 each blocked nicotine-induced dopamine release in the accumbens, indicating the participation of more than a single receptor system in the nicotine-induced effect. These three antagonists did not inhibit cocaine-induced dopamine increase in the accumbens, indicating the lack of a role of these receptors in the cocaine effect under our experimental conditions. SCH-23390, a dopamine D1 receptor antagonist, blocked both nicotine- and cocaine-induced effects, indicating the possible role of this receptor in these reward effects. The results indicate that there are differences in some of the receptors mediating the central effects of the two compounds examined, nicotine and cocaine, although each influences dopamine levels, and that the two compounds interact.

Met-enkephalin-Arg6-Phe7 metabolism: conversion to Met-enkephalin by brain and kidney dipeptidyl carboxypeptidases.

Abstract Enzymes degrading Met-enkephalin-Arg6-Phe7, an endogenous brain peptide with enhanced opiate activity in vivo, were isolated from membrane preparations of rabbit kidney and brain, and their specificity compared. A preparation from kidney or brain containing the angiotensin converting enzyme (EC 3.4.15.1) released with time Arg-Phe, Met-enkephalin, Phe-Met and Tyr-Gly-Gly. Kinetic analysis revealed a product precursor relationship with conversion of hepta- to pentapeptide (Met-enkephalin) followed by release of Tyr-Gly-Gly and Phe-Met indicating sequential cleavage at the Met5-Arg6 and Gly3-Phe4 bonds. A second preparation devoid of angiotensin converting enzyme activity released the same products and in addition a tetrapeptide Phe-Met-Arg-Phe. Release of products with time indicated cleavage at Gly3-Phe4 by an endopeptidase and at the Met5-Arg6 and Gly3-Phe4 bonds by a dipeptidyl carboxypeptidase. The dipeptidyl carboxypeptidases thus provide a mechanism for the formation of Met-enkephalin from a potential precursor.

Changes in Brain Protease Activity in Aging

Abstract: We measured changes in protease activity with aging, conducting assays of cathepsin D and calpain II activities and the rate of degradation of cytoskeletal proteins, preparing the enzymes and substrates from young and aged brains. Calpain preparations added to the young and to the aged substrates were standardized with casein as substrate so that age‐related changes in calpain specificity and substrate susceptibility were measured. Several age‐related differences were observed in substrate susceptibility and in enzyme activity. With respect to substrate, the neurofilament protein from young animals was somewhat more susceptible to calpain action than that from older animals. With respect to enzyme activity, calpain from aged brain cleaved neurofilament protein at a faster rate than did calpain from young. With neurofilaments, the most rapid breakdown usually occurred when enzyme from aged tissue was incubated with substrate from young. Kidney enzyme of aged rats incubated with neurofilament substrate of aged rats resulted in a more rapid breakdown than enzyme of young kidney incubated with substrate of young. The age dependence of tubulin breakdown was somewhat different from that of neurofilament breakdown. The most rapid breakdown usually occurred when using enzyme from young with tubulin from young. Incubation of neurofilament protein or tubulin with cathepsin D did not reveal any differences with aging. These studies suggest that an increase in enzyme activity observed previously during aging may also include changes in the properties of the enzyme (substrate specificity) and/or in the properties of their endogenous substrates (susceptibility to breakdown).

Effect of food deprivation on glutathione and amino acid levels in brain and liver of young and aged rats

The effect of short-term food deprivation on glutathione (GSH) and amino acid levels in brain regions of young and aged rats was compared with changes observed in liver. Animals aged 3 months and 24 months were deprived of food for 48 h. GSH and amino acid levels from cerebral cortex, cerebellum, pons medulla, and liver were assayed and compared with levels in animals of the same age fed normal diets. In liver in both young and old rats, GSH levels fell 30%, from 13 mumol/g tissue to 8.7 mumol/g tissue. Significant changes were observed in other amino acids, including an increase of 30-50% in methionine, glycine, and glutamine, and a decrease of 30-50% in alanine in liver of both young and aged rats, and a 4-fold increase in taurine in young. In brain, little change was observed upon food deprivation. No decrease was observed in GSH, and only small changes were observed in other amino acids. In the aged animal aspartate, glutamate, and alanine levels were slightly lower; tyrosine in cerebellum was reduced by 30%, and both glycine and tyrosine in the pons medulla were reduced by 20-30%. In the brain areas examined, levels of GSH ranged from 1-2 mumol/g in young and 0.8-1.4 mumol/g in old; with levels in pons medulla being lower than those in cerebral cortex. In brain, in contrast to liver, levels were scarcely affected by short-term food deprivation.(ABSTRACT TRUNCATED AT 250 WORDS)

Dopamine releasing effect of phenylbiguanide in rat striatal slices

SummaryThe present study explored the mechanisms underlying the dopamine releasing effect of phenylbiguanide, a compound commonly used as a 5-HT3 receptor agonist. Phenylbiguanide, and also serotonin and 2-methyl-serotonin, enhanced the outflow of radioactivity from superfused rat striatal slices preloaded with [3H]dopamine. The presence of the dopamine uptake blocker nomifensin prevented the increase in outflow. The effect of phenylbiguanide was not antagonized by 5-HT3 receptor antagonists, did not require the presence of Ca2+ in the superfusion buffer, and also occurred in reserpinized preparations with depleted dopamine stores. Phenylbiguanide caused a greater shift in the distribution of superfusate radioactivity from DOPAC to dopamine than did nomifensin. All these results are in agreement with an exchange mechanism by which phenylbiguanide promotes the efflux of dopamine by operation of the uptake carrier in the reversed direction. In consonance, phenylbiguanide, and also serotonin and 2-methyl-serotonin, inhibited the binding of [3H]CFT to dopamine uptake sites, although the rank order for promoting outflow, serotonin > phenylbiguanide > 2-methyl-serotonin, differed from that for inhibiting [3H]CFT binding to dopamine uptake sites, 2-methylserotonin ∼ serotonin > phenylbiguanide. The present results raised the possibility that phenylbiguanide has an additional activity in releasing vesicular dopamine into the cytoplasmic pool.

Evidence for the involvement of Na+/Ca2+ exchange in the stimulation of inositol phospholipid hydrolysis by sodium channel activation and depolarization

Amiloride, an inhibitor of the Na+/Ca2+ exchanger, blocked the hydrolysis of inositol phospholipids in mouse cerebrocortical slices induced by the sodium channel activator veratridine, by KCl, or by the sodium ionophore monensin; there was no inhibition by A 23187, a Ca2+ ionophore, or by serotonin. It is concluded that agents that increase intracellular Na2+ stimulate inositide hydrolysis by an indirect effect via Na+/Ca2+ exchange.

Characterization of a distinct membrane bound dipeptidyl carboxypeptidase inactivating enkephalin in brain

Abstract A dipeptidyl carboxypeptidase distinct from the angiotensin converting enzyme (EC 3.4.15.1) was isolated from membrane preparations of rabbit brain. The enzyme cleaved enkephalin at the Gly-Phe bond, releasing either Phe-Leu from Leu-enkephalin or Phe-Met from Met-enkephalin, and also acted on bradykinin, releasing the terminal dipeptide Phe-Arg. In contrast to the converting enzyme, however, this dipeptidyl carboxypeptidase did not act on angiotensin-1, and it did not degrade hippuryl-His-Leu. Chloride ions did not affect its activity, but the enzyme was inhibited by metal chelating agents. The enzyme was not inhibited by captopril (SQ 14225) or by SQ 20881. Kinetic studies indicated a Km for this enzyme of 0.14 mM with Leu-enkephalin and 0.12 mM with bradykinin as substrates. Present data indicate that more than one enzyme is present in brain membrane fractions acting as dipeptidyl carboxypeptidases inactivating enkephalin; these data suggest multiple roles for such enzymes in the regulation of peptide metabolism.