Abstract
Morphine is an analgesic alkaloid used to relieve severe pain, and irreversible binding of morphine to specific unknown proteins has been previously observed. In the brain, changes in the expression of energy metabolism enzymes contribute to behavioral abnormalities during chronic morphine treatment. Creatine kinase B (CK-B) is a key enzyme involved in brain energy metabolism. CK-B also corresponds to the imidazoline-binding protein I2 which binds dopamine (a precursor of morphine biosynthesis) irreversibly. Using biochemical approaches, we show that recombinant mouse CK-B possesses a μM affinity for morphine and binds to morphine in vitro. The complex formed by CK-B and morphine is resistant to detergents, reducing agents, heat treatment and SDS-polyacrylamide gel electrophoresis (SDS-PAGE). CK-B-derived peptides CK-B1–75 and CK-B184–258 were identified as two specific morphine binding-peptides. In vitro, morphine (1–100 μM) significantly reduces recombinant CK-B enzymatic activity. Accordingly, in vivo morphine administration (7.5 mg/kg, i.p.) to mice significantly decreased brain extract CK-B activity compared to saline-treated animals. Together, these results show that morphine strongly binds CK-B and inhibits its activity in vitro and in vivo.
Highlights
Morphine, an alkaloid from Papaver somniferum, is used to relieve pain in multiple clinical settings
The binding of mouse Recombinant mouse CK-B (rCK-B), produced in E. coli to increasing concentrations (1.56 μM to 400 μM) of morphine, codeine, M3G or M6G was tested by Enzyme-Linked ImmunoSorbent Assay (ELISA)
The results show that mouse rCK-B binds morphine (Figure 1A) with a calculated Kd of 10.8 μM
Summary
An alkaloid from Papaver somniferum, is used to relieve pain in multiple clinical settings. In addition to its analgesic properties, morphine decreases intestinal motility, suppresses cough and has vasodilatory effects (Andersen et al, 2003). It influences many other physiological processes and notably decreases ATP availability in specific brain structures (Nasello et al, 1973). Endogenous morphine has been characterized in numerous mammalian cells and tissues, and its structure is identical to that of morphine isolated from the poppy (for review Laux-Biehlmann et al, 2013). Our laboratory has Abbreviations: ASB9, ankyrin repeat and SOCS box protein 9; CK-B, brain creatine kinase; CK-M, muscular creatine kinase; CNS, central nervous system; I2B, I2-binding; M3G, morphine-3-glucuronide; M6G, morphine-6-glucuronide; PEBP, phosphatidylethanolamine-binding protein; SEM, standard error of the mean
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