Substrate modulation of aldolase B binding in hepatocytes

Abstract

The binding properties of hepatic aldolase (B) were determined in digitonin-permeabilized rat hepatocytes after the cells had been preincubated with either glycolytic or gluconeogenic substrates. In hepatocytes that had been preincubated in medium containing 5 mM glucose as sole carbohydrate substrate, binding of aldolase to the hepatocyte matrix was maximal at low KCl concentrations (20 mM) or bivalent cation concentrations (1 mM Mg2+) and half-maximal dissociation occurred at 50 mM KCl. Preincubation of hepatocytes (for 10-30 min) with glucose or mannose (10-40 mM), fructose, sorbitol, dihydroxyacetone or glycerol (1-10 mM), caused a leftward shift of the salt dissociation curve (maximum binding at 10 mM KCl; half-maximum dissociation at 35 mM KCl) but did not affect the proportion of bound enzyme at low or high KCl concentrations. Galactose and 2-deoxyglucose had no effect on aldolase binding. Inhibitors of glucokinase (mannoheptulose and glucosamine) suppressed the effects of glucose but not the effects of sorbitol, glycerol or dihydroxyacetone. Glucagon suppressed the effects of glucose, fructose and dihydroxyacetone but not glycerol. Poly(ethylene glycol) (PEG) (2-10%), added to the permeabilization medium, increased aldolase binding and caused a rightward shift in the salt dissociation curve. In the presence of PEG (6-8%), the effects of substrates on aldolase dissociation were shifted to higher salt concentrations (50-100 mM versus 35 mM KCl). The effects of substrates (added to the intact cell) on aldolase binding to the permeabilized cell could be mimicked by addition of the phosphorylated derivatives of these substrates to the permeabilized cell. Of the intermediates tested dihydroxyacetone phosphate and fructose 1,6-bisphosphate were the most effective at dissociating aldolase (A50 values of 20 microM and 40 microM respectively). Other effective intermediates in order of decreasing potency were fructose 1-phosphate, glycerol 3-phosphate, glucose 1,6-bisphosphate/fructose 2,6-bisphosphate. These results show that aldolase B binds to the hepatocyte matrix by a salt-dependent mechanism that is influenced by macromolecular crowding and metabolic intermediates. Maximum binding occurs when hepatocytes are incubated in the absence of glycolytic and gluconeogenic substrates and minimum binding occurs in the presence of substrates that are precursors of either fructose 1,6-bisphosphate or triose phosphates. Since the bound form of aldolase represents a kinetically less active state it is proposed that aldolase binding and dissociation may be a mechanism for buffering the concentrations of metabolic intermediates.

Subcellular localization, mobility, and kinetic activity of glucokinase in glucose-responsive insulin-secreting cells.

“…Effects of glucose concentration on cell area. The aforementioned results suggest that cytoplasmic glucokinase shows mobility (measured as diffusion from permeabilized cells) similar to that of phosphoglucoisomerase, which in hepatocytes shows negligible binding (9,27). The lower fractional release of glucokinase during permeabilization of MIN6 with digitonin in the presence of Mg 2+ (Table 1) could be explained by inhibition of depolymerization of the actin filaments by Mg 2+ (9).…”

“…However, as suggested by the finding that the granular staining pattern is preserved after treatment of the cells with a digitonin concentration that releases >85% of phosphoglucoisomerase, we infer that the glucokinase activity released represents mainly the cytoplasmic fraction. Phosphoglucoisomerase was selected as a reference because it is present in very high activity and does not show specific binding in other cells (27). Also, unlike glucokinase and various glycolytic enzymes, it does not bind to the 11.5-kDa zinc-binding protein (32).…”

“…Culture of MIN6 cells. MIN6 cells (passage [16][17][18][19][20][21][22][23][24][25][26][27] were grown in 25-cm 2 flasks in Dulbecco's modified Eagle's medium (DMEM) (25 mmol/l glucose) supplemented with fetal bovine serum (15% vol/vol), ␤-mercaptoethanol (5 µl/l), 75 mg/l penicillin, and 50 mg/l streptomycin. For immunostaining, MIN6 cells were cultured on 13-mm glass coverslips coated with 1% (wt/vol) gelatin, and for determination of glucokinase activity and glucose phosphorylation, they were cultured in 24-well plates.…”

Spatial Clustering of Isozyme-specific Residues Reveals Unlikely Determinants of Isozyme Specificity in Fructose-1,6-bisphosphate Aldolase

“…The interaction between aldolase A and F-actin or GLUT4 can be disrupted by substrates of aldolase, thus implicating structural connectivity of binding residues with residues of the active site (52). Aldolase B has been shown to interact with vacuolar H ϩ -ATPase (51) and the hepatocyte matrix (53). Aldolase C has demonstrated binding to inositol 1,4,5-trisphosphate (54) and S-100 A1 (55) protein, thus implicating aldolase C in calcium signaling.…”

Binding and translocation of glucokinase in hepatocytes

“…However, there is virtually total release of glucokinase in the absence of Mg2+ [ 111. Certain other cytoplasmic enzymes are only partially released at digitonin concentrations that cause almost total release of phosphoglucoisomerase ( [11,16]; L. Agius and M. Peak, unpublished work). These include lactate dehydrogenase, aldolase, glyceraldehyde phosphate dehydrogenase, phosphoglycerate kinase and phosphofructokinase.…”