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Major localization of aminopeptidase M in rat brain microvessels
Abstract:
The localization of two enkephalin-hydrolysing aminopeptidases i.e. aminopeptidase M (aminopeptidase N, EC 3.4.11.2) relatively insensitive to puromycin (Ki = 78 microM), and a puromycin-sensitive aminopeptidase (Ki = 1 microM) was studied in rat brain. The two aminopeptidases were differentially identified and/or localized using polyclonal anti-aminopeptidase M antibodies displaying anticatalytic activity and the inhibitors puromycin, bestatin and amastatin. Microvessels represent a major localization of cerebral aminopeptidase M as shown by the intense immunostaining of their walls in sections from various regions as well as in a fraction isolated from cerebral cortex homogenates by a sieving procedure. As compared to the starting homogenate, aminopeptidase M activity was enriched about twenty fold in this microvascular fraction. Aminopeptidase M was identified in this fraction by comparing the inhibitory potencies of antibodies and peptidase inhibitors towards the hydrolysis of [tyrosyl-3,5-3H, Met5]enkephalin to those found for the purified enzyme. A rather high aminopeptidase M activity was also localized in choroid plexuses. Following differential and gradient centrifugation analysis of cerebral cortex homogenates, aminopeptidase M activity was also enriched (by five to six fold) in fractions containing synaptic membranes. No significant soluble aminopeptidase M activity could be detected. These data suggest a dual localization of cerebral aminopeptidase M in microvessels and synaptic membranes consistent with its roles in preventing the access of circulating peptides to brain as well as in inactivating neuropeptides released from cerebral neurones. In comparison, puromycin-sensitive aminopeptidase activity, which is about 100 fold higher than aminopeptidase M activity in brain, was relatively low in microvessels and non-detectable in fractions enriched in synaptic membranes, being almost entirely restricted to soluble fractions.
Insights
Cerebral aminopeptidase M is found in brain microvessels and synaptic membranes, regulating peptide access and inactivation. A distinct puromycin-sensitive aminopeptidase is primarily soluble.
Area of Science:
- Neuroscience
- Biochemistry
- Enzymology
Background:
- Enkephalin-hydrolyzing aminopeptidases play crucial roles in the central nervous system.
- Understanding their precise localization is key to elucidating their functions in neuropeptide metabolism and blood-brain barrier integrity.
Purpose of the Study:
- To determine the specific localization of two key aminopeptidases, aminopeptidase M (APM) and a puromycin-sensitive aminopeptidase, within the rat brain.
- To investigate the functional implications of APM localization in relation to peptide regulation.
Main Methods:
- Differential localization using polyclonal anti-APM antibodies with anticatalytic activity.
- Enzyme activity assays and inhibition studies with puromycin, bestatin, and amastatin.
- Isolation and analysis of microvascular and synaptic membrane fractions from rat cerebral cortex.
Main Results:
- Aminopeptidase M (APM) was highly enriched in isolated cerebral microvessels (approx. 20-fold) and also found in choroid plexuses and synaptic membranes (5-6 fold enrichment).
- APM activity was predominantly membrane-bound, with no significant soluble fraction detected.
- Puromycin-sensitive aminopeptidase activity, significantly higher than APM, was mainly localized in soluble fractions and minimally in microvessels, absent in synaptic membranes.
Conclusions:
- Cerebral APM exhibits a dual localization in microvessels and synaptic membranes, suggesting roles in controlling peripheral peptide entry and inactivating neuronal neuropeptides.
- The distinct localization patterns highlight the specialized functions of different aminopeptidases in brain peptide homeostasis.