Limited proteolysis of liver and muscle aldolases: Effects of subtilisin, cathepsin B, and Staphylococcus aureus protease

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The molecular architecture of the rabbit skeletal muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) tetramer has been determined to 2.7-A resolution. Solution of the three-dimensional structure of rabbit muscle aldolase utilized phase information from a single isomorphous Pt(CN)4-derivative, which was combined with iterative-phase refinement based upon the noncrystallographic 222-fold symmetry exhibited by the tetramer subunits. The electron-density map calculated from the refrned phases (mf = 0.72) was interpreted on the basis of the known amino acid sequence (363 amino acids per subunit). The molecular architecture of the aldolase subunit corresponds to a singly wound fl-barrel of the parallel a/fl class structures as has been observed in triose phosphate isomerase, pyruvate kinase, phosphogluconate aldolase, as well as others. Close contacts between tetramer subunits are virtually all between regions of hydrophobic residues. Contrary to other ,8-barrel structures, the known active-site residues are located in the center of the ,8-barrel and are accessible to substrate from the COOH side of the fl-barrel. Biochemical and crystallographic data suggest that the COOH-terminal region of aldolase covers the active-site pocket from the COOH side of the fl-barrel and mediates access to the active site. On the basis of sequence studies, active-site residues as well as residues lining the active-site pocket have been totally conserved throughout evolution. By comparison, homology in the COOH-terminal region is minimal. It is suggested that the amino acid sequence of the COOH-terminal region may be, in part, the basis for the variable specific activities aldolases exhibit toward their substrates.Aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) is an ubiquitous and abundant glycolytic enzyme that plays a central and pivotal role in glycolysis and fructose metabolism. Aldolases from all species catalyze the reversible aldol cleavage of fructose 1,6-bisphosphate (Fru-1,6-P2) into the triose phosphates, Dglyceraldehyde 3-phosphate and dihydroxyacetone phosphate. Catalysis proceeds by two distinct chemical pathways in aldolases. In class I aldolases, found in plants and higher animals, catalysis depends upon Schiff-base formation with the substrate (1), whereas in class II aldolases, found mostly in molds and bacteria, catalysis requires a metal cofactor such as Zn2+ (2). Demonstrable activity by aldolases also exists toward substrates such as fructose 1-phosphate (Fru-1-P), and the differential activity by aldolases toward Fru-1,6-P2 and Fru-1-P has been used as a basis to discriminate between the various isozymes in vertebrates (3). In rabbit tissues, aldolase A has been isolated from muscle, aldolase B from liver, and aldolase C from brain. The three forms have been purified to homogeneity and extensively characterized (3-5). The enzymes have a relative molecular mass (Mr) of approximately 158,000 and a tertiary structure composed of...

mentioning

confidence: 99%

Molecular architecture of rabbit skeletal muscle aldolase at 2.7-A resolution.

Sygusch

Beaudry²,

Allaire³

1987

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“…recently, several of these endogenous proteinases have been identified, including cathepsin B from rat (13) or human (10) liver, carboxypeptidase A from rat liver (13), .and cathepsin M from rabbit liver (3). A similar accumulation of inactive crossreacting aldolase has been reported by Gershon and Gershon (14) in livers of aged mice, but the molecular basis for this decreased activity has not yet been determined.…”

Section: Discussionmentioning

confidence: 68%

“…The COOH terminus of rabbit liver aldolase has been shown to contain the following sequence: Ser-Thr-Glu-Ser-Leu-Phe-Thr-Ala-Ser-Tyr-ThrTyrOH (10). Digestion with subtilisin releases peptides containing both of the tyrosine residues (10), thus providing a simple and convenient method for evaluating prior modification of this region of the molecule.…”

Section: Effect Of Homogenization Conditions and Intraportalmentioning

confidence: 99%

Characterization of the inactive form of fructose-1,6-bisphosphate aldolase isolated from livers of fasted rabbits.

Pontremoli¹,

Melloni²,

Michetti³

et al. 1982

The accumulation of an inactive, immunologically crossreactive form offructose-1,6-bisphosphate aldolase (EC 3.1.3.11) in livers of fasted rabbits has now been related to limited proteolysis at the COOH terminus. The extent of modification of this region of the molecule, determined by analysis of tyrosine residues in the peptides released by digestion with subtilisin, agrees with the observed decrease in the specific activity of the enzyme purified from livers of fasted rabbits. The following evidence supports the conclusion that the modified form is produced in vivo and not during the isolation of the enzyme from the liver homogenates: (i) liver homogenates prepared in isotonic sucrose contained negligible amounts of soluble lysosomal proteinases; (ii) the decreased aldolase activity after fasting was observed in the homogenates and no change in aldolase activity occurred when the homogenates were -incubated for 2 hr at 37C; (iii) the modified enzyme was also isolated from the livers of fasted rabbits when leupeptin was injected intraportally before the animals were sacrificed or when the inhibitor was added to the homogenization solution. On the other hand, homogenization oflivers in hypotonic medium resulted in release of lysosomal proteinases and also in decreases in catalytic activity and COOH-terminal modification of liver aldolase, similar to those observed in livers from fasted rabbits. We attribute the changes in activity and structure of aldolase isolated from livers of fasted rabbits to the action in vivo of cathepsin M.We have previously reported the accumulation of an inactive, immunologically crossreactive form of fructose-1,6-bisphosphate aldolase (EC 3.1.3.11) in livers offasted rabbits (1, 2). We now show that the loss of activity is due to proteolytic modification of the COOH terminus and provide evidence that this modification is not the result of proteolysis during the isolation ofthe enzyme from the homogenates. The loss ofpeptides from the COOH terminus can be attributed to the action ofa recently identified lysosomal proteinase, designated cathepsin M (3), which was shown to catalyze a similar modification ofrabbit liver aldolase in vitro (4). The present results, which establish that this modification occurs during fasting in vivo, also support the concept that in the living cell some lysosomal proteinases may be accessible to the cytosol, accounting for at -least partial proteolysis of cytosolic enzymes without their entry into the lysosomes. EXPERIMENTAL PROCEDURESMaterials. Except as indicated below, these were isolated as described or from sources previously identified (3).Methods. Aldolase was isolated from the liver homogenates (5) and assayed (6) as described. Digestion of the purified enzyme with subtilisin and analysis of the acid-soluble peptides for tyrosine was carried out with dialyzed solutions of the purified aldolase (4). For the evaluation of release of lysosomal proteinases, the rate of hydrolysis of benzoyl-L-arginyl-2-naphthylamide (BANA) was determined. Other...

“…The solutions were lyophilized and analyzed for free amino acids. For qualitative identification ofthe peptides released, aliquots were analyzed by reverse-phase HPLC as described (13).…”

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confidence: 99%

“…Subtilisin was also used to evaluate the modification of the COOH terminus of rabbit liver aldolase after incubation with cathepsin M. Subtilisin has been shown to release two peptides specifically from this region of the molecule, including the COOH-terminal tetrapeptide Ser-Tyr-Thr-Tyr (13). Aliquots (0.5 mg of aldolase) were removed from the reaction mixtures, treated with leupeptin (final concentration, 10 uM) to stop the digestion, and dialyzed for 6 hr against 10 mM diethanolamine/10 mM triethanolamine, pH 7.5.…”

mentioning

confidence: 99%

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

Pontremoli¹,

Melloni²,

Michetti³

et al. 1982

Cathepsin M, which catalyzes inactivation of both rabbit liver fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) and rabbit liver fructose 1,6-bisphosphatase (Fru-P2ase; EC 3.1.3.11), has been characterized as a peptidyl peptidase. Modification of the COOH terminus of aldolase by cathepsin M or by Fru-P2ase converting enzyme 2 abolishes its ability to bind to phosphocellulose P11 and to form the complex with Fru-P2ase. On the other hand, modification ofthe COOH terminus of Fru-P2ase does not affect its interaction with aldolase. This property is lost, however, when Fru-P2ase is modified in the NH2-terminal region by the converting enzyme or by subtilisin. The results suggest that interaction of aldolase and Fru-P2ase may involve the exposed COOH-terminal region of the former and an exposed proteinasesensitive region located between residues 57 and 67 of the latter.During fasting, the activity of fructose-1,6-bisphosphate aldolase (EC 4.1.2.13) in rabbit liver decreases (1, 2) and an immunologically crossreacting material accumulates (2, 3). We have identified several lysosomal proteinases that catalyze inactivation of purified rabbit liver aldolase; limited proteolysis by one or more of these proteinases may be responsible for the changes observed in vivo. A new proteinase, designated cathepsin M, is ofparticular interest because (i) it is partially associated with lysosomal membranes, (ii) fasting increases the activity of the membrane-bound form, and (iii) it is expressed by intact lysosomes at neutral pH (4). Cathepsin M also catalyzes inactivation of rabbit liver fructose 1,6-bisphosphatase (Fru-P2ase) (4).In this paper, we report that, for both aldolase and Fru-P2ase, inactivation by cathepsin M is associated with loss of a segment of the COOH terminus. Complex formation between the two enzymes (5, 6) is affected by this modification of aldolase but not by similar modification ofthe COOH terminus ofFru-P2ase. On the other hand, modification of Fru-P2ase by either subtilisin or Fru-P2ase converting enzyme 2 (CE 2) does abolish its ability to form the complex with aldolase. Fru-P2ase modified by these enzymes shows substantially increased catalytic activity at pH 9.2, and this change in catalytic properties appears to be associated with nicking of one or more peptide bonds in a susceptible region located near the NH2 terminus, between residues 57 and 67 (7,8).MATERIALS AND METHODS Materials. Aldolase (5) and Fru-P2ase (9) were purified from livers of fed rabbits as described. The enzymes were stored at 2°C as suspensions in 80% saturated (NH4)2SO4. Before use, aliquots were centrifuged and the precipitates were dissolved in 10 mM sodium acetate (pH 6.0) and dialyzed overnight against the same buffer. Cathepsin M (4) and CE 2 (10) were purified from lysosome-rich rabbit liver heavy particle fraction as described. Ultrogel AcA34 was purchased from LKB.Enzyme Assays. Aldolase and Fru-P2ase activities were assayed as described by Gracy et al. (11) and Traniello et al. (12), respectively, the latter in the...