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Inactivity of enkephaline on human serum esterase
Life Sciences Vol. 19, pp . 479-482, 1976 . Printed in the U.S .A .
Pergamon Prese
INACTIVITY OF ENKEPHALINE ON HUMAN SERUM ESTERASE Alexander Gero Department of Pharmacology, Hannemann Medical College, Philadelphia, Pa . 19102, USA (Received in final form April 15, 1976) While opioid agonists accelerate the hydrolytic action of human serum esterase, and opioid antagonists competitively antagonize this acceleration, the endogenous morphine-like factor methionine-enkephaline neither accelerates the enzyme nor competes with an opioid accelerator. It is proposed that the pentapeptide enkephaline is only the prosthetic group of the true endogenous morphine-like factor and too short a peptide chain to have a stable enough secondary structure for opioid action in the presence of dissolved protein . For a number of years this writer and his associates have studied the action of opiates on human serum esterase and have established that this enzyme contains a morphine receptor site for Which opioid drugs have affinity and attached to which opioid agonista accélerate enzymatic action, while others attach to the receptor site silently and thus function as competitive antagonists (1) . It has also been possible to interpret the agonistic properties of opiates at the esterase receptor as a function of the distortion of the receptor by a rigid drug molecule : the observed enzyme acceleration would then be an allosteric effect of the receptor distortion (2) . Drugs which attach silently to the receptor either are not rigid and can adjust their conformation to that of the receptor, or if they are rigid, their structure is such that they force no change in the conformation of the receptor . In view of the recent discovery of enkephaline, an endogenous substance which, like morphine and other opioid drugs, depresses electrically induced contractions of the guinea-pig ileum (3), it appeared interesting to find out whether enkepha line behaves as an opiate on serum esterase as well . This experiment was performed, and the present paper reports the results obtained . For experimental details the reader is referred to our earlier publications listed in reference (1) . Me~ionine-enke~haline over a concentration range of
3 x 10- M to 3 x 10 - M was allowed to interact with an esterase
solution under standard conditions (1) and its ability to accelerate the activity of the enzyme was examined . As Table 1 shows, the results were entirely negative : at no concentration studied did enkephaline exert a perceptible acceleration on the enzyme, much in contrast to our standard acceleratory opiate, 479
Enicephaline and H~mman Serum Esterase
480
Vol . 19, No . 4
dihydromorphinone ,. whic$ accelerates the enzyme very clearly at a concentration of 3x10 - b M. TABLE 1 Teat of Acceleratory Effect of Enkephaline on Human Serum Esterase -5 -5 3x10 Enkephaline, 10 molarity 3x10 -6 Effect, v/vo*
10-4
3x10 -~
1 .06+ .01 .97± .02 .98± .04 .96± .07 1 .02± .02
e e ec e e ne as v v , w ere vo is é vé~ocity o~ the enzyme-catalyzed hydrolysis o$ procaine under standard conditions, and v the same velocity in the presence of enkephaline . The possibility still remained, however, that enkephaline may attach to the morphine receptor site, not as an agonist, but silently . In this case it should function as a competitive antagonist of an opioid agoniat and, present together with dihydromorphinone, diminish the a~celeratory effect of the latter . Accordingly, 10 -5M and 10 - M dihydromorphinone . was added to the enzyme and allowed to act on the substrate, procaine, both alone and in the presence of 3x10 -5 enkephaline . As Table 2 shows, the results were again negative : the effect of both dihydromorphinone-enkephaline combinations was within experimental error the same as that of dihydromorphinone alone . TABLE 2 Teat of Antagonism of Dihydromorphinone by Enkephaline
Dihydromorphinone, molarity
10 -5
10-4
Effect, alone
1 .25± .11
1 .81+ .05
Effect in the presence of 3x10 -5M enkephaline
1 .30_+ .04
1 .77± .01
This complete lack of agonistic activity, and even of affinity for the receptor, in enkephaline is sharply at variance with its potency in inhibiting the electrically driven guinea-pig ileum (3) but it is in harmony with the absence of any reports of an analgesic action of enkephaline other than weak analgesia on direct intraventricular administration (4) . The situation we face is, therefore, that enkephaline is fifteen times as potent as morphine in inhibiting electrically stimulated contractions of the guinea-pig ileum, one-tenth as potent an analgesic as morphine on intraventricular administration, and capable neither of inducing analgesia when administered by other routes nor of interacting with the opiate receptor site in human serum esterase . In attempting to interpret these observations, the possibility
Vol . 19, No . 4
Ealcephaline and Human Serum Esterase
481
might be considers$ that enkephaline is not the real endogenous morphine-like factor but perhaps only its prosthetic group . As long as enkephaline is part of a large alpha helix, it will share the stability of the latter and thus will be rigid enough to force a distortion of the receptor and have an effect . But as an isolated pentapeptide, enkephaline is too short to have a stable secondary structure (5) . In an in-vitro experiment on the guinea-pig ileum it will retain itsco~rmation and act like the true morphine-like factor, but in the presence of a protein solution, as in blood or in the presence of the esterase, the dissolved protein will break open whatever internal hydrogen bonding the pentapeptide has and form new hydrogen bonds with it in a conformation too different from its native conformation for enkephaline to retain its former ability to act on the receptor . Only in the presence of a bloodless isolated organ such as the guinea-pig ileum, or when brought close to its cerebral site of action in an intraventricular injection, without passing through the blood stream, is enough of the original conformation of enkephaline preserved so that it can still act as a weak analgesic agent . If this reasoning is valid, then the heptapeptide recently synthesized by Goldstein (6) also should be inactive both as an analgesic and as an accelerator of serum esterase . But as the terminal part of a large peptide, made up of perhaps 15-20 amino acid residues, it should have potency . Acknowledgment . The author is grateful to Dr . H . McGregor of ~iye a ors oriel, Radnor, Pa ., for a sample of methionineenkephaline . References 1.
A . GERO and R . J. CAPETOLA, Life Sçi . 16, 1821-1822 (1975), References to earlier publics ons are-Iisted there .
2.
A . GERD, Arch . Int . . Pharmacodyn . 206, 41-46 (1973) .
3.
J . HUGHES, T . W. SMITH, H . W . KOSTERLITZ L . A . FOTHERGILL, B . A . MDRGAN and H . R . MORRIS, Nature 2~, 577-579 (1975) " J . D . BELLUZZI, N. GRANT, V . GARSKY, D . SARANTAKIS, C . D . WISE and L . STEIN, Nature in press .
5.
J . A . SC~"T .MAN ~ Com tes rendus Lab . Carlsberg , Ser. chim . 230-259 (1955~ -
6.
A . GOLDSTEIN, J . S . GOLDSTEIN, B. M. COX, Life Sçi . ~, 1643-1654 (1975) "
Pergamon Prese
INACTIVITY OF ENKEPHALINE ON HUMAN SERUM ESTERASE Alexander Gero Department of Pharmacology, Hannemann Medical College, Philadelphia, Pa . 19102, USA (Received in final form April 15, 1976) While opioid agonists accelerate the hydrolytic action of human serum esterase, and opioid antagonists competitively antagonize this acceleration, the endogenous morphine-like factor methionine-enkephaline neither accelerates the enzyme nor competes with an opioid accelerator. It is proposed that the pentapeptide enkephaline is only the prosthetic group of the true endogenous morphine-like factor and too short a peptide chain to have a stable enough secondary structure for opioid action in the presence of dissolved protein . For a number of years this writer and his associates have studied the action of opiates on human serum esterase and have established that this enzyme contains a morphine receptor site for Which opioid drugs have affinity and attached to which opioid agonista accélerate enzymatic action, while others attach to the receptor site silently and thus function as competitive antagonists (1) . It has also been possible to interpret the agonistic properties of opiates at the esterase receptor as a function of the distortion of the receptor by a rigid drug molecule : the observed enzyme acceleration would then be an allosteric effect of the receptor distortion (2) . Drugs which attach silently to the receptor either are not rigid and can adjust their conformation to that of the receptor, or if they are rigid, their structure is such that they force no change in the conformation of the receptor . In view of the recent discovery of enkephaline, an endogenous substance which, like morphine and other opioid drugs, depresses electrically induced contractions of the guinea-pig ileum (3), it appeared interesting to find out whether enkepha line behaves as an opiate on serum esterase as well . This experiment was performed, and the present paper reports the results obtained . For experimental details the reader is referred to our earlier publications listed in reference (1) . Me~ionine-enke~haline over a concentration range of
3 x 10- M to 3 x 10 - M was allowed to interact with an esterase
solution under standard conditions (1) and its ability to accelerate the activity of the enzyme was examined . As Table 1 shows, the results were entirely negative : at no concentration studied did enkephaline exert a perceptible acceleration on the enzyme, much in contrast to our standard acceleratory opiate, 479
Enicephaline and H~mman Serum Esterase
480
Vol . 19, No . 4
dihydromorphinone ,. whic$ accelerates the enzyme very clearly at a concentration of 3x10 - b M. TABLE 1 Teat of Acceleratory Effect of Enkephaline on Human Serum Esterase -5 -5 3x10 Enkephaline, 10 molarity 3x10 -6 Effect, v/vo*
10-4
3x10 -~
1 .06+ .01 .97± .02 .98± .04 .96± .07 1 .02± .02
e e ec e e ne as v v , w ere vo is é vé~ocity o~ the enzyme-catalyzed hydrolysis o$ procaine under standard conditions, and v the same velocity in the presence of enkephaline . The possibility still remained, however, that enkephaline may attach to the morphine receptor site, not as an agonist, but silently . In this case it should function as a competitive antagonist of an opioid agoniat and, present together with dihydromorphinone, diminish the a~celeratory effect of the latter . Accordingly, 10 -5M and 10 - M dihydromorphinone . was added to the enzyme and allowed to act on the substrate, procaine, both alone and in the presence of 3x10 -5 enkephaline . As Table 2 shows, the results were again negative : the effect of both dihydromorphinone-enkephaline combinations was within experimental error the same as that of dihydromorphinone alone . TABLE 2 Teat of Antagonism of Dihydromorphinone by Enkephaline
Dihydromorphinone, molarity
10 -5
10-4
Effect, alone
1 .25± .11
1 .81+ .05
Effect in the presence of 3x10 -5M enkephaline
1 .30_+ .04
1 .77± .01
This complete lack of agonistic activity, and even of affinity for the receptor, in enkephaline is sharply at variance with its potency in inhibiting the electrically driven guinea-pig ileum (3) but it is in harmony with the absence of any reports of an analgesic action of enkephaline other than weak analgesia on direct intraventricular administration (4) . The situation we face is, therefore, that enkephaline is fifteen times as potent as morphine in inhibiting electrically stimulated contractions of the guinea-pig ileum, one-tenth as potent an analgesic as morphine on intraventricular administration, and capable neither of inducing analgesia when administered by other routes nor of interacting with the opiate receptor site in human serum esterase . In attempting to interpret these observations, the possibility
Vol . 19, No . 4
Ealcephaline and Human Serum Esterase
481
might be considers$ that enkephaline is not the real endogenous morphine-like factor but perhaps only its prosthetic group . As long as enkephaline is part of a large alpha helix, it will share the stability of the latter and thus will be rigid enough to force a distortion of the receptor and have an effect . But as an isolated pentapeptide, enkephaline is too short to have a stable secondary structure (5) . In an in-vitro experiment on the guinea-pig ileum it will retain itsco~rmation and act like the true morphine-like factor, but in the presence of a protein solution, as in blood or in the presence of the esterase, the dissolved protein will break open whatever internal hydrogen bonding the pentapeptide has and form new hydrogen bonds with it in a conformation too different from its native conformation for enkephaline to retain its former ability to act on the receptor . Only in the presence of a bloodless isolated organ such as the guinea-pig ileum, or when brought close to its cerebral site of action in an intraventricular injection, without passing through the blood stream, is enough of the original conformation of enkephaline preserved so that it can still act as a weak analgesic agent . If this reasoning is valid, then the heptapeptide recently synthesized by Goldstein (6) also should be inactive both as an analgesic and as an accelerator of serum esterase . But as the terminal part of a large peptide, made up of perhaps 15-20 amino acid residues, it should have potency . Acknowledgment . The author is grateful to Dr . H . McGregor of ~iye a ors oriel, Radnor, Pa ., for a sample of methionineenkephaline . References 1.
A . GERO and R . J. CAPETOLA, Life Sçi . 16, 1821-1822 (1975), References to earlier publics ons are-Iisted there .
2.
A . GERD, Arch . Int . . Pharmacodyn . 206, 41-46 (1973) .
3.
J . HUGHES, T . W. SMITH, H . W . KOSTERLITZ L . A . FOTHERGILL, B . A . MDRGAN and H . R . MORRIS, Nature 2~, 577-579 (1975) " J . D . BELLUZZI, N. GRANT, V . GARSKY, D . SARANTAKIS, C . D . WISE and L . STEIN, Nature in press .
5.
J . A . SC~"T .MAN ~ Com tes rendus Lab . Carlsberg , Ser. chim . 230-259 (1955~ -
6.
A . GOLDSTEIN, J . S . GOLDSTEIN, B. M. COX, Life Sçi . ~, 1643-1654 (1975) "