Nucleotidase activities in the soluble fraction of rat liver homogenate

151

1. Cell-surface 5'-nucleotidase was assayed by incubating whole-cell suspensions with 5'[3H]-AMP in iso-osmotic buffer and measuring [3H]adenosine production. The activity of cell-surface 5'-nucleotidase in hepatocytes, adipocytes and lymphocytes isolated from the rat was 15.0, 0.5 and 0.8pmol/min per cell at 37 degrees C respectively. 2. Disruption of the cells by vigorous mechanical homogenization or detergent treatment exposed additional 5'-nucleotidase activity, which represented 52%, 25% and 21% of the total activity in the three cell types respectively. This increase in 5'-nucleotidase activity which occurred when the cells were homogenized was due to a second pool of 5'-nucleotidase within the cell, rather than activation of the cell-surface enzyme. 3. In hepatocytes the intracellular 5'-nucleotidase activity was membrane-bound, indistinguishable from cell-surface 5'-nucleotidase in its inhibition by rabbit anti-(rat liver 5'-nucleotidase) serum and its kinetics with AMP, and was located on the extracytoplasmic face of vesicles within the cell. 4. The cell-surface 5'-nucleotidase of rat hepatocytes was rapidly inhibited when rabbit anti-(rat liver 5'-nucleotidase) serum or concanavalin A was added to the medium at 37 degrees C. Incubation with antiserum for 5 min at 37 degrees C inhibited 83 +/- 3% of the cell-surface enzyme. 5. Incubation of hepatocytes with exogenous antiserum or concanavalin A for 30 min at 37 degrees C resulted in over 50% inhibition of the intracellular enzyme. This inhibition was not prevented by disruption of the cytoskeleton or by ATP depletion. 6. Incubation of hepatocytes with exogenous antiserum or concanavalin A for up to 2h at 0 degrees C caused little or no inhibition of the intracellular enzyme, but over 75% inhibition of the cell-surface enzyme. 7. When surface-inhibited hepatocytes were washed and resuspended in buffer at 37 degrees C, 5'-nucleotidase was observed to redistribute from the intracellular pool to the cell surface.

Section: '-Nucleotidase Activity (% Of Total)mentioning

confidence: 63%

Subcellular distribution and movement of 5'-nucleotidase in rat cells

Stanley¹,

Edwards²,

Luzio³

1980

151

“…Gel-permeation chromatography (Table 2) indicated that about 15% of the crude supernatant-fraction activity towards UMP was due to enzyme(s) different from the alkaline 5'-nucleotidase. Such enzymes could be cytosolic 5'-nucleotidase and the deoxyribonucleoside-activated nucleotidase, with pH optima 6.3 and 6.0 respectively, which may both exert some activity also at pH 8.1 (Fritzson, 1969;Fritzson & Smith, 1971). Contribution of these enzymes to the supernatant-fraction activity could explain a slightly smaller decrease in UMP and IMP dephosphorylation in regenerating liver compared with AMP dephosphorylation, since both enzymes show increased activity in regenerating liver (Fritzson, 1978;Tjernshaugen & Fritzson, 1984) and exhibit very low activity towards AMP at the substrate concentrations used.…”

Section: Discussionmentioning

confidence: 99%

The presence and activity in normal and regenerating rat liver postmicrosomal supernatant fraction of an enzyme with properties similar to those of membrane-bound 5′-nucleotidase

Fritzson¹,

Haugen²,

Tjernshaugen³

1986

An alkaline 5′-nucleotidase with properties similar to those of membrane-bound 5′-nucleotidase was recovered in soluble form in the postmicrosomal supernatant fraction (cytosol) of rat liver. The enzyme seems to constitute a quantitatively distinct fraction, since the activity in postmicrosomal supernatants was increased by a further 10% by additional homogenization of livers. Lysosomal acid phosphatase activity increased similarly, whereas other membrane-bound marker enzymes alkaline phosphatase, phosphodiesterase I and glucose-6-phosphatase showed no increase when homogenization of liver tissue was continued. Gel-permeation chromatography and pH-dependence studies indicated that enzyme activity in the supernatant fraction with 0.3 mM-UMP or -AMP as substrate at pH 8.1 was about 85 or 100% specific respectively. In regenerating liver the enzyme recovered in soluble form showed decreased specific activity, in contrast with alkaline phosphatase measured for comparison. The nucleotidase activity per mg of cytosolic protein was 2.1 nmol/min with AMP as substrate. The total activity measured in the postmicrosomal supernatant was 1.5% of the homogenate activity measured in the presence of detergent.

“…Peak II also contained 2,3-DPG, which was eluted from the column at NaCl concentrations above 150 mM. 5'-Nucleotidase activities of peak I Further analysis of the 5'-nucleotidase activities present in peak I was performed both at pH 7.2, the physiological pH of erythrocytes, and at pH 6.3, the optimum pH of cytosolic 5'-nucleotidase in various tissues [1][2][3] and of the deoxyribonucleotide-dephosphorylating activity of human erythrocytes [12]. Substrate concentration was 2.5 mm.…”

Section: '-Nucleotidase Activities In Dialysed Haemolysatesmentioning

confidence: 99%

“…The kinetic characteristics of the purine 5'-nucleotidase isolated from human erythrocytes are in many respects similar to those of the cytosolic 5'-nucleotidases of other tissues. This includes an absolute dependency on Mg2", a pH optimum around 6.3-6.5 and a higher affinity for inosine and guanosine 5'-nucleotides than for AMP and other nucleoside monophosphates [1][2][3][4][5]26].…”

Section: Properties Of the Erythrocyte Purine 5'-nucleotidasementioning

confidence: 99%

“…However, more recent studies show that, quite logically, this role is most probably assumed by cytosolic enzymes. First described by Itoh et al [1] and by Fritzson [2], these soluble 5'-nucleotidases have been shown to possess complex regulatory properties, including stimulation by nucleoside triphosphates and inhibition by Pi [3][4][5]. Whether distinct cytoplasmic 5'nucleotidases exist for the specific or preferential degradation of the various 5'-nucleotide species remains, however, less clear.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

5′-Nucleotidase activities in human erythrocytes. Identification of a purine 5′-nucleotidase stimulated by ATP and glycerate 2,3-bisphosphate

Bontemps

Berghe

Hers

1988

A purine 5'-nucleotidase has been separated by DEAE-Trisacryl chromatography from other 5'-nucleotidase activities present in human haemolysates and purified approx. 30,000-fold by subsequent chromatography on Blue Sepharose. The enzyme has an Mr of around 250,000, displays hyperbolic substrate-saturation kinetics and hydrolyses preferentially IMP, GMP and their deoxy counterparts. It is much less active with AMP and dAMP. The purine 5'-nucleotidase is inhibited by Pi, and is strongly stimulated by ATP, dATP and GTP, and by glycerate 2,3-bisphosphate. Stimulators decrease Km and increase Vmax. Glycerate 2,3-bisphosphate is the most potent stimulator of the enzyme and, under physiological conditions, over-rides the influence of the other effectors. Glycerate 2,3-bisphosphate also influences the binding of the enzyme to DEAE-Trisacryl, as evidenced by the different elution profile obtained with fresh as compared with outdated blood. It is concluded that the glycerate 2,3-bisphosphate-stimulated purine 5'-nucleotidase is responsible for the dephosphorylation of IMP and GMP, but not of AMP, in human erythrocytes.