Pyridoxal phosphate protects against an irreversible temperature-dependent inactivation of hepatic delta-aminolevulinic acid synthase.
The stability of hepatic delta-aminolevulinic acid synthase (ALAS), the first and rate-limiting enzyme of the heme biosynthetic pathway, was investigated. Incubation of the mitochondrial matrix fraction obtained from either control or allylisopropylacetamide-induced rats at 37 degrees C in Tris-Cl, pH 7.4, EDTA, and dithiothreitol resulted in a rapid decrease in ALAS activity such that 50-70% of the activity was lost after 30 min. Similar decreases in ALAS activity were observed when a cytosolic fraction from the induced animals was incubated at 37 degrees C. Addition of 0.1 mM pyridoxal-P, the cofactor of ALAS, to the preincubation medium completely prevented the observed loss of activity; however, dialysis of the inactive matrix fraction against several changes of buffer containing pyridoxal-P did not restore activity, suggesting that the inactivation was irreversible. These decreases in ALAS activity in the absence of pyridoxal-P were temperature dependent, as a 55% loss of ALAS activity was observed after a 60-min incubation at 30 degrees C, while the enzyme was completely stable when preincubated at 22 degrees C for 60 min. This inactivation of ALAS does not appear to involve proteolytic digestion, as addition of a wide spectrum of protease inhibitors to the preincubation medium in the absence of pyridoxal-P did not protect against the inactivation. The suggestion is made that the cofactor, pyridoxal-P, may dissociate from the enzyme during the preincubation and, consequently, the apoenzyme may be irreversibly inactivated at temperatures above 22 degrees C.
D000624:5-Aminolevulinate Synthetase / Q000378:metabolism*; D000818:Animals; D002021:Buffers / Q000494:pharmacology; D003600:Cytosol / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology*; D008297:Male; D008930:Mitochondria, Liver / Q000201:enzymology; D011480:Protease Inhibitors / Q000494:pharmacology; D011732:Pyridoxal Phosphate / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D011736:Pyridoxine / Q000031:analogs & derivatives / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature
D002021:Buffers; D011480:Protease Inhibitors; D011732:Pyridoxal Phosphate; D000624:5-Aminolevulinate Synthetase; D011736:Pyridoxine