Increased activity of cyclic AMP phosphodiesterase from frozen-thawed rat liver. A role of lysosomal protease in enzyme activation.
The activity of cyclic AMP phosphodiesterase (3':5'-cyclic-nucleotide 5'-nucleotidohydrolase, EC 3.1.4.17) in 105 000 X g supernatant fraction from frozen-thawed rat liver was 2.5 times higher than the corresponding preparation from fresh liver. This increased activity of frozen liver enzyme was accompanied by a decreased sensitivity of the enzyme to known activators such as alpha-tocopheryl phosphate and trypsin. Neither membrane-bound cyclic AMP phosphodiesterase, nor supernatant cyclic GMP phosphodiesterase increased in frozen liver preparation. It is unlikely that the activator protein of phosphodiesterase participated in the observed change of enzyme activity. Among rat tissues so far tested, the increased level of cyclic AMP phosphodiesterase was noted only in tissues rich in lysosome content. In the recombination experiment where phosphodiesterase from fresh liver was incubated with lysosomal fraction, stimulation of the enzyme activity was observed with a concomitant loss of sensitivity to above-mentioned activators. Since the stimulation by lysosomal fraction was effectively inhibited by cathepsin B1 inhibitors, leupeptin and antipain, it was deduced cathepsin-B1 (EC 3.4.12.3) type protease(s) was the main causative of activating the cyclic AMP phosphodiesterase. The freezing-thawing process of rat liver made the lysosomal membrane more permeable, and hence lysosomal proteases were released into soluble fraction during phosphodiesterase preparation. These results provide a warning not to use frozen liver for phosphodiesterase preparation, otherwise altered properties of the enzymes will be seen.
D015105:3',5'-Cyclic-AMP Phosphodiesterases / Q000378:metabolism*; D015106:3',5'-Cyclic-GMP Phosphodiesterases / Q000378:metabolism; D000818:Animals; D004789:Enzyme Activation; D005615:Freezing; D007976:Leupeptins / Q000494:pharmacology; D008099:Liver / Q000201:enzymology*; D008247:Lysosomes / Q000201:enzymology*; D008297:Male; D010447:Peptide Hydrolases / Q000378:metabolism*; D051381:Rats; D014357:Trypsin / Q000494:pharmacology; D014810:Vitamin E / Q000494:pharmacology
D007976:Leupeptins; D014810:Vitamin E; D015105:3',5'-Cyclic-AMP Phosphodiesterases; D015106:3',5'-Cyclic-GMP Phosphodiesterases; D010447:Peptide Hydrolases; D014357:Trypsin