Fructose 1,6-Bisphosphatase: The Role of Lysosomal Enzymes in the Modification of Catalytic and Structural Properties

Abstract

Seasonal variations in the properties of rabbit-liver fructose 1 ,6-bisphosphatase have now been linked to corresponding changes in the levels of proteolytic activity in the liver extracts. Incubation of native fructose 1,6-bisphosphatase with purified liver lysosomes causes a 3-fold increase in catalytic activity at pH 9.2, with a smaller, and variable, decrease in activity tested at pH 7. (7), and the mitochondrial fraction was carefully layered on top of a linear 45-60% sucrose gradient containing 1 mM EDTA. The purified lysosome and mitochondria-enriched fractions, respectively, were pooled, diluted to a final sucrose concentration of 0.35 M, and recovered by centrifugation at 10,000 X g. Rabbit liver lysosomes were used in the experiments reported here, but identical results were obtained with lysosomal preparations from mouse liver.Proteolytic activities of crude liver extracts were assayed with bovine-serum albumin as the substrate, by measurement of the amount of ninhydrin-positive material in the supernatant solution after precipitation with trichloroacetic acid. RESULTSEffect of the lysosome fraction on "neutral" FbPase When purified intact lysosomes were incubated at pH 6.5 with native FbPase the enzyme showed a rapid increase in of purified "neutral" FbPase, and 4 mg of lysosomal fraction, in a total volume of 1.0 ml. Aliquots (0.2 ml) were removed as indicated and analyzed for catalytic activity at pH 7.5 and 9.2 (1), and for mobility in Na dodecyl sulfate-polyacrylamide gel electrophoresis. The patterns obtained at various times are illustrated in the lower part of the figure. 303 pH 9.2

Changes in Activity and Molecular Properties of Fructose 1,6-Bisphosphatase During Fasting and Refeeding

“…We have previously shown (11) that the single tryptophan residue is located in the NH2-terminal region of the subunit; the present results suggest that it may lie very close to the NH2-terminus. The second is the appearance of traces of a lighter subunit, which suggests that a modification similar to that observed when the enzyme is exposed to lysosomes in vitro (12) has occurred in vivo. However, in vitro, the accumulation of lighter subunits is far more extensive, reaching at least 50% of the total, and is accompanied by a shift in the pH optimum of the enzyme from neutral to alkaline (12).…”

“…However, while there were no gross changes in molecular weight, we did find evidence for a more subtle proteolytic modification of the NHrterminus, since the enzyme isolated from the livers of fasted animals had lost the single tryptophan residue from this region of the molecule. In several respects, the enzyme isolated from the fasted animals was found to resemble the form previously isolated from the livers of farmbred rabbits in the winter (12 PFK activity was assayed spectrophotometrically at pH 8.2 following the oxidation of NADH in the presence of excess aldolase and glycerol 1-phosphate dehydrogenase-triose phosphate isomerase, as described by Mansour (14). PEPcarboxykinase activity was assayed at 30°by the method of Nordlie and Lardy (15), as modified by Foster et al (16).…”

“…Each subunit contains a single tryptophan residue (7,9,10), located in the NHrterminal region of the molecule (11). Exposure of the purified native enzyme to proteolytic enzymes, or to lysosomal fractions, has been shown to cause marked changes in the catalytic properties of the enzyme (9,12,13), notably a shift from a neutral to an alkaline pH optimum. The changes in catalytic properties appear to be due to the conversion of the native subunits to a lighter form, having a molecular weight of approximately 29,000.…”

Limited proteolysis of liver aldolase and fructose 1,6-bisphosphatase by lysosomal proteinases: effect on complex formation.

“…Values in parentheses are those predicted for the 21-amino acid segment at the COOH terminus of Fru-P2ase (15). HPLC analysis indicated the presence of a number of free amino acids, including lysine, and peptides that emerged in overlapping fractions.…”

“…How (8,10,15). The change in activity at pH 9.2 resulting from the action of GE 2 has been attributed to a conformational change associated with hydrolysis of the Asn-64/Val-65 bond.…”