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Plasma protein(s) yields met-enkephalin-related peptides in near-micromolar concentrations when treated with pepsin
Abstract:
Treatment of animal and human plasmas with pepsin yielded large quantities of immunoreactive methionine5-enkephalin (i-met-ENK). The concentrations measured after pepsin treatment were 0.1-0.5 microM, about 1000 times the normal circulating level of i-met-ENK (0.03-0.3 nM). The reaction was shown to be time and pH dependent and to involve the action of pepsin on a protein(s) of about 65,000 mol wt. Pepsin-generated i-met-ENK from rat plasma gave three major peaks during reverse phase HPLC, one of which (approximately 25% of the total) coeluted with methionine5-enkephalin sulfoxide and also completed in a radioreceptor assay for opiate-related substances. In addition, this material produced met-ENK-like effects on vascular permeability in rat skin and inhibited electrically induced contractions of the isolated guinea pig ileum in a naloxone-sensitive manner. The plasma substrate(s) that yielded i-met-ENK was distinguished from adrenal proenkephalins, since partially purified plasma substrate(s) did not liberate i-met-ENK upon digestion with trypsin and carboxypeptidase B. Although it is possible that these peptides differ from met-ENK in amino acid sequence, the results presented here suggest that met-ENK-related substances might be formed physiologically by the action of a pepsin-related processing enzyme(s) on plasma substrate(s). Such a mechanism would be analogous to that used in the renin-angiotensin system.
Insights
Pepsin treatment of plasma releases large amounts of immunoreactive methionine-5-enkephalin (i-met-ENK), significantly increasing its concentration. This suggests a physiological role for pepsin-like enzymes in generating enkephalins from plasma proteins.
Area of Science:
- Biochemistry
- Neuroendocrinology
- Peptide Processing
Background:
- Methionine-5-enkephalin (met-ENK) is an endogenous opioid peptide with known physiological roles.
- The circulating levels and processing of met-ENK in plasma are not fully understood.
- Previous studies have focused on adrenal proenkephalins, leaving plasma-derived sources less explored.
Purpose of the Study:
- To investigate the potential for generating immunoreactive methionine-5-enkephalin (i-met-ENK) from plasma proteins.
- To characterize the enzyme and substrate involved in this potential plasma-based processing.
- To explore the physiological relevance of pepsin-mediated enkephalin generation.
Main Methods:
- Treatment of animal and human plasma with pepsin.
- Quantification of i-met-ENK using radioimmunoassay and radioreceptor assays.
- Reverse-phase high-performance liquid chromatography (HPLC) for separation and characterization.
- Bioassays for met-ENK-like effects on vascular permeability and smooth muscle contraction.
Main Results:
- Pepsin treatment of plasma yielded significantly high concentrations of i-met-ENK (0.1-0.5 microM), approximately 1000-fold higher than normal circulating levels (0.03-0.3 nM).
- The reaction was time- and pH-dependent, involving pepsin action on a ~65,000 mol wt plasma protein.
- Generated i-met-ENK exhibited biological activity, including effects on vascular permeability and naloxone-sensitive inhibition of guinea pig ileum contractions.
Conclusions:
- Plasma contains a substrate that, upon digestion by pepsin, releases substantial amounts of i-met-ENK.
- The processing mechanism differs from that of adrenal proenkephalins, suggesting a distinct pathway.
- These findings suggest a potential physiological role for pepsin-related enzymes in generating met-ENK-like substances from plasma precursors, analogous to the renin-angiotensin system.