Subcellular distribution and properties of aldehyde dehydrogenase in the rat liver

The subcellular distribution and certain properties of rat liver aldehyde dehydrogenase are investigated. The enzyme is shown to be localized in fractions of mitochondria and microsomes. Optimal conditions are chosen for detecting the aldehyde dehydrogenase activity in the mentioned fractions. The enzyme of mitochondrial fraction shows the activity at low (0,03-0.05 mM; isoenzyme I) and high (5 mM; isoenzyme II) concentrations of the substrate. The seeming Km and V of aldehyde dehydrogenase from fractions of mitochondria and microsomes of rat liver are calculated, the acetaldehyde and NAD+ reaction being used as a substrate.     

5'-Nucleotidase activity and adenosine production in rat liver mitochondria.

The controversial subject of mitochondrial 5'-nucleotidase in the liver was studied employing density gradient fractionation combined with a method for analyzing the distribution profiles of marker enzymes based on multiple regression analysis. Triton WR-1339 was used to improve the separation of mitochondria from lysosomes by the gradient centrifugation technique. Adenosine production was examined further using acetate to increase intramitochondrial AMP, and thus adenosine production, in incubations with gradient centrifugation-purified mitochondria. Distribution analysis of the crude homogenate showed that 5'-nucleotidase activity exists in the mitochondrial fraction. To increase the resolution of this approach with respect to mitochondria, a crude mitochondrial fraction was also studied. In this case the relative mitochondrial activity decreased but 5'-nucleotidase activity was still clearly detectable. The mitochondrial 5'-nucleotidase exhibited a Km of 94 microM and a Vmax of 31 nmol/min per mg protein for AMP. The kinetic data for the Mg2+, ATP, ADP and AOPCP sensitivity of the enzyme showed that it differs from the plasma membrane, lysosome and cytosol 5'-nucleotidases. AOPCP was only a moderate inhibitor, and ATP was a more potent inhibitor than ADP at a 1 mM concentration. The enzyme also showed a requirement of Mg2+. Acetate caused the conversion of intramitochondrial adenylates to AMP and the formation of adenosine. Adenosine concentration increased in the extramitochondrial space in a time-dependent manner, but only trace amounts of nucleotides were detected. The data show that 5'-nucleotidase activity producing adenosine exists in rat liver mitochondria and a concentration-dependent adenosine output from mitochondria by diffusion or facilitated diffusion is also suggested.     

Monamine oxidase in outer membrane of skeletal muscle mitochondria.

(1) Monoamine oxidase (EC 1.4.3.4) is present in rat skeletal muscle mitochondria. (2) A radioassay procedure for the assay of monoamine oxidase in muscle mitochondria is described. It is based on teh procedure using side-chain [2-14C]-tryptamine as substate described by Wurtman, R.J. and Axelrod, J. (1963) Biochem. Pharmacol. 12, 1439--1441 and employs a pH of 8.0 and a substrate concentration of 0.25 mM. (3) The Km of the muscle mitochondrial enzyme at pH 8.0 is 1.34 - 10(-5) M and that of the liver enzyme under the same conditions is 2.5 - 10(-5) M. Muscle mitochondria contain only one quarter of the activity of enzyme present in liver mitochondria. (4) Monoamine oxidase is shown to be in the outer membrane of skeletal muscle mitochondria and thus to be a suitable marker enzyme for use in the fractionation of these mitochondria.     

Characterization of the mitochondrial Na+-H+ exchange. The effect of amiloride analogues

The kinetic properties and inhibitor sensitivity of the Na+-H+ exchange activity present in the inner membrane of rat heart and liver mitochondria were studied. (1) Na+-induced H+ efflux from mitochondria followed Michaelis-Menten kinetics. In heart mitochondria, the Km for Na+ was 24 +/- 4 mM and the Vmax was 4.5 +/- 1.4 nmol H+/mg protein per s (n = 6). Basically similar values were obtained in liver mitochondria (Km = 31 +/- 2 mM, Vmax = 5.3 +/- 0.2 nmol H+/mg protein per s, n = 4). (2) Li+ proved to be a substrate (Km = 5.9 mM, Vmax = 2.3 nmol H+/mg protein per s) and a potent competitive inhibitor with respect to Na+ (Ki approximately 0.7 mM). (3) External H+ inhibited the mitochondrial Na+-H+ exchange competitively. (4) Two benzamil derivatives of amiloride, 5-(N-4-chlorobenzyl)-N-(2',4'-dimethyl)benzamil and 3',5'-bis(trifluoromethyl)benzamil were effective inhibitors of the mitochondrial Na+-H+ exchange (50% inhibition was attained by approx. 60 microM in the presence of 15 mM Na+). (5) Three 5-amino analogues of amiloride, which are very strong Na+-H+ exchange blockers on the plasma membrane, exerted only weak inhibitory activity on the mitochondrial Na+-H+ exchange. (6) The results indicate that the mitochondrial and the plasma membrane antiporters represent distinct molecular entities.     

Influence of adenosine and Nagarse on palmitoyl-CoA synthetase in rat heart and liver mitochondria

1. A Q₁₀ of about 3 for palmitoyl-CoA synthetase (EC 6.2.1.3) in rat heart and liver mitochondria is found.

2. In heart mitochondria Nagarse (EC 3.4.4.16) destroys the ability to activate palmitate. When, however, heart mitochondria are oxidizing palmitate, they are protected from the inactivating action of Nagarse.

3. Although treatment of liver mitochondria with Nagarse causes the loss of about 95 % of the palmitoyl-CoA synthetase activity, no influence is observed on palmitate oxidation.

4. Adenosine inhibits palmitoyl-CoA synthetase in liver and heart mitochondria. Adenosine is a competitive inhibitor with respect to ATP with an apparent K_i of 0.1 mM. The residual palmitoyl-CoA synthetase in Nagarse-treated liver mitochondria is much less sensitive to adenosine.

5. 2 mM adenosine or 2 mM adenosinesulfate inhibit palmitate oxidation (in the presence of 2.5 mM ATP) in heart mitochondria 60–90 %.

6. The data obtained are consistent with the concept of a palmitoyl-CoA synthetase localized on the outside of the outer membrane of rat heart and liver mitochondria, with an additional locus of (ATP-dependent) palmitoyl-CoA synthesis in the inner membrane matrix compartment of liver mitochondria.     

Properties of a DNA-dependent ATPase from rat mitochondria.

A DNA-dependent ATPase has been highly purified from rat liver mitochondria and characterized. The enzyme catalyzes the hydrolysis of ATP or dATP in the presence of single-stranded DNA cofactor and a divalent cation. The Km values for ATP and dATP are 0.15 mM and 0.35 mM, respectively. The enzyme activity is highly sensitive to N-ethylmaleimide. The sedimentation coefficient of the enzyme is 8.3 S in a glycerol gradient. From this and data on Sephadex G-200 gel filtration, the molecular weight of the native enzyme was calculated to be about 190,000. All the natural single-stranded DNAs tested were equally effective for the ATPase activity, but synthetic deoxyhomopolymer poly(dC) was found to be more effective than natural single-stranded DNAs. Synthetic and natural RNAs had no effect on the activity.     

Relationship between 5'-nucleotidase, adenosine deaminase, AMP deaminase, ATP-(Mg2+)-ase activities and dTMP kinase activity in rat liver mitochondria.

The activities of dTMP kinase (ATP-deoxythymidine monophosphate phosphotransferase, EC 2.7.4.9), 5'-nucleotidase (5'-ribonucleoside phosphohydrolase, EC 3.1.3.5), adenosine deaminase (adenosine aminohydrolase, EC 3.5.4.4), AMP deaminase (AMP aminohydrolase, EC 3.5.3.6) and ATP-(Mg2+)-ase (ATP phosphohydrolase, EC 3.6.1.3) were assayed in mitochondria of normal and regenerating rat liver. In regenerating mitochondria, the dTMP kinase activity increased 20 times, 5'-nucleotidase (5'Nase) activity for dTMP diminished by 65% and its activity for other nucleoside monophosphates did not change; adenosine deaminase activity for adenosine (AR) increased by 40%, but for deoxyadenosine (AdR) decreased by 70%. AMP deaminase and ATP-(Mg2+)-ase activities behaved similarly in mitochondria from regenerating liver, decreasing by 70 and 64% respectively. The changes of the amount of dTMP in mitochondria depend on enzyme activities which regulate the AdR concentration.     

Changes in AMP deaminase activities in the hearts of diabetic rats

AMP deaminase from normal and diabetic rat hearts was separated on cellulose phosphate and quantitated by HPLC. From soluble fractions three different AMP deaminase activities, according to KCl elution from cellulose phosphate and percent of total activity were: 170 mM (85%), 250 mM (8%) and 330 mM (7%) KCl. The AMP deaminase activity which eluted with 170 mM KCl was resolved to two distinct peaks by HPLC anionic exchange. After 4 weeks of diabetes the heart enzyme profile change to: 170 mM (10%), 250 mM (75%) and 330 mM (15%). Once purified the four activities were kinetically distinct: 170 mM KCl cytosolic, AMP Km = 1.78, stimulated by ATP, GTP, NADP and strongly inhibited by NAD; 170 mM KCl mitochondria AMP Km = 17.9, stimulated by ATP, ADP; 250 mM KCl isozyme, AMP Km = 0.66, stimulated by ADP; and 330 mM KCl isozyme, AMP Km = 0.97, inhibited by ATP, NAD(P).     

Liver phosphorylase b kinase. Cyclic-AMP-mediated activation and properties of the partially purified rat-liver enzyme.

Phosphorylase b kinase was extensively purified from rat liver. It was located in a form which could be activated 20--30-fold by a preincubation with adenosine 3':5'-monophosphate (cyclic AMP) and ATP-Mg. This activation was time-dependent, and was paralleled by a simultaneous incorporation of 32P from [gamma-32P]ATP into two polypeptides which comigrated in sodium dodecyl sulfate gel electrophoresis with the alpha and beta subunits of rabbit skeletal muscle phosphorylase b kinase. The liver enzyme was eluted from Sepharose 4B and Bio-Gel A-50m columns at the same place as muscle phosphorylase b kinase, which is indicative of a molecular weight of 1.3 x 10(6). After activation, the most purified liver preparation had a specific activity about 10-fold less than the homogeneous muscle enzyme at pH 8.2. The inactive enzyme form had a pronounced pH optimum around pH 6.0, whereas the activated form was mostly active above neutral pH. The activation of the enzyme reduced the Km for its substrate phosphorylase b severalfold. Liver phosphorylase b kinase was shown to be partially dependent on Ca2+ ions for its activity: addition of 0.5 mM [ethylenebis-(oxoethylenenitrilo)]tetraacetic acid (EGTA) to the phosphorylase b kinase assay increased the Km for phosphorylase b about twofold for both the inactive and the activated form of liver phosphorylase b kinase, but affected the V of the inactive species only.     

Different forms of rat liver aldehyde dehydrogenase and their subcellular distribution.

1. The properties and distribution of the NAD-linked unspecific aldehyde dehydrogenase activity (aldehyde: NAD+ oxidoreductase EC 1.2.1.3) has been studied in isolated cytoplasmic, mitochondrial and microsomal fractions of rat liver. The various types of aldehyde dehydrogenase were separated by ion exchange chromatography and isoelectric focusing. 2. The cytoplasmic fraction contained 10-15, the mitochondrial fraction 45-50 and the microsomal fraction 35-40% of the total aldehyde dehydrogenase activity, when assayed with 6.0 mM propionaldehyde as substrate. 3. The cytoplasmic fraction contained two separable unspecific aldehyde dehydrogenases, one with high Km for aldehydes (in the millimolar range) and the other with low Km for aldehydes (in the micromolar range). The latter can, however, be due to leakage from mitochondria. The high-Km enzyme fraction contained also all D-glucuronolactone dehydrogenase activity of the cytoplasmic fraction. The specific formaldehyde and betaine aldehyde dehydrogenases present in the cytoplasmic fraction could be separated from the unspecific activities. 4. In the mitochondrial fraction there was one enzyme with a low Km for aldehydes and another with high Km for aldehydes, which was different from the cytoplasmic enzyme. 5. The microsomal aldehyde dehydrogenase had a high Km for aldehydes and had similar properties as the mitochondrial high-Km enzyme. Both enzymes have very little activity with formaldehyde and glycolaldehyde in contrast to the other aldehyde dehydrogenases. They are apparently membranebound.     

Hepatic pyruvate kinase. Regulation by glucagon, cyclic adenosine 3'-5'-monophosphate, and insulin in the perfused rat liver.

A reversible interconversion of two kinetically distinct forms of hepatic pyruvate kinase regulated by glucagon and insulin is demonstrated in the perfused rat liver. The regulation does not involve the total enzyme content of the liver, but rather results in a modulation of the substrate dependence. The forms of pyruvate kinase in liver homogenates are distinguished by measurements of the ratio of the enzyme activity at a subsaturating concentration of P-enolpyruvate (1.3 mM) to the activity at a saturating concentration of this substrate (6.6 mM). A low ratio form of pyruvate kinase (ratio between 0.1 and 0.2) is obtained from livers perfused with 10(-7) M glucagon or 0.1 mM adenosine 3':5'-monophosphate (cyclic AMP). A high ratio form of the enzyme is obtained from livers perfused with no hormone (ratio = 0.35 to 0.45). The regulation of pyruvate kinase by glucagon and cyclic AMP occurs within 2 min following the hormone addition to the liver. Insulin (22 milliunits/ml) counteracts the inhibition of pyruvate kinase caused by 5 X 10(-11) M glucagon, but has only a slight influence on the enzyme properties in the absence of the hyperglycemic hormone. The low ratio form of pyruvate kinase obtained from livers perfused with glucagon or cyclic AMP is unstable in liver extracts and will revert to a high ratio form within 10 min at 37 degrees or within a few hours at 0 degrees. Pyruvate kinase is quantitatively precipitated from liver supernatants with 2.5 M ammonium sulfate. This precipitation stabilizes the enzyme and preserves the kinetically distinguishable forms. The kinetic properties of the two forms of rat hepatic pyruvate kinase are examined using ammonium sulfate precipitates from the perfused rat liver. At pH 7.5 the high ratio form of the enzyme has [S]0.5 = 1.6 +/- 0.2 mM P-enolpyruvate (n = 8). The low ratio form of enzyme from livers perfused with glucagon or cyclic AMP has [S]0.5 = 2.5 +/- 0.4 mM P-enolpyruvate (n = 8). The modification of pyruvate kinase induced by glucagon does not alter the dependence of the enzyme activity on ADP (Km is approximately 0.5 mM ADP for both forms of the enzyme). Both forms are allosterically modulated by fructose 1,6-bisphosphate, L-alanine, and ATP. The changes in the kinetic properties of hepatic pyruvate kinase which follow treating the perfused rat liver with glucagon or cyclic AMP are consistent with the changes observed in the enzyme properties upon phosphorylation in vitro by a clyclic AMP-stimulated protein kinase (Ljungstr?m, O., Hjelmquist, G. and Engstr?m, L. (1974) Biochim. Biophys. Acta 358, 289--298). However, other factors also influence the enzyme activity in a similar manner and it remains to be demonstrated that the regulation of hepatic pyruvate kinase by glucagon and cyclic AMP in vivo involes a phosphorylation.     

Purification and characterization of rat liver mitochondrial phenylalanine pyruvate aminotransferase.

Phenylalanine pyruvate aminotransferase in rat liver was found in both the mitochondrial and supernatant fractions. Phenylalanine pyruvate aminotransferase was purified from rat liver mitochondria. The purified enzyme was specific for pyruvate, exhibiting no activity with 2-oxoglutarate as aminoacceptor, and utilized a wide range of amino acids as amino donors. Amino acids were effective in the following order of activity: L-phenylalanine > L-tyrosine > L-histidine > 3,4-dihydroxy-DL-phenylalanine. Very little activity was observed with L-tryptophan and 5-hydroxy-L-tryptophan. The apparent Km values for L-phenylalanine and L-histidine were 2.6 mM and 2.7 mM, respectively. The Km values for pyruvate were 5.0 mM and 1.5 mM with phenylalanine and histidine as amino donors, respectively. The pH optimum was near 9.0. Sucrose density gradient centrifugation gave a molecular weight of approximately 68,000. On the basis of subcellular distributions, substrate specificities, substrate inhibition, pH optima, polyacrylamide gel electrophoresis and some other properties, it was suggested that mitochondrial phenylalanine pyruvate aminotransferase was identical with mitochondrial histidine pyruvate aminotransferase.     

Glutathione peroxidase activities from rat liver

There are two enzymes in rat liver with glutathione peroxidase activity when cumene hydroperoxide is used as substrate. One is the selenium-requiring glutathione peroxidase (glutathione:hydrogen-peroxide oxidoreductase, EC 1.11.1.9) and the other appears to be independent of dietary selenium. Activities of the two enzymes vary greatly among tissues and among animals. The molecular weight of the enzyme with selenium-independent glutathione peroxidase activity was estimated by gel filtration to be 35 000, and the subunit molecular weight was estimated by dodecyl sulfate-polyacrylamide gel electrophoresis to be 17 000. Double reciprocal plots of enzyme activity as a function of substrate concentration produced intersecting lines which are suggestive of a sequential reaction mechanism. The Km for glutathione was 0.20 mM and the Km for cumene hydroperoxide was 0.57 mM. The enzyme was inhibited by N-ethylmaleimide, but not by iodoacetic acid. Inhibition by cyanide was competitive with respect to glutathione and the Ki for cyanide was 0.95 mM. This selenium-independent glutathione peroxidase also catalyzes the conjugation of glutathione to 1-chloro-2,4-dinitrobenzene. Along with other similarities to glutathione S-transferase, this suggests that the selenium-independent glutathione peroxidase and glutathione S-transferase activities in rat liver are of the same enzyme.     

Properties and subcellular localization of adenosine diphosphatase in rat heart

Some properties and subcellular localization of adenosine diphosphatase (ADPase) activity from rat heart have been investigated. The pH optimum was 7.4, maximal activity was found with 5 mM MgCl2, and the apparent Km was 20 microM. ADPase activity was strongly inhibited by NaF and AppNHp, and to a lesser extent by AMP and GppNHp. The enzyme was not inhibited by p-nitrophenylphosphate, beta-glycerophosphate, or pyridoxal phosphate. The distribution of ADPase activity in subcellular fractions obtained by differential centrifugation parallel ouabain-sensitive (Na+-K+)ATPase and 5'-nucleotidase activities, suggesting a plasma membrane-bound localization. The functional significance of ADPase in adenosine production and hemostasis is discussed.     

Characterization of sheep liver and lung microsomal ethylmorphine N-demethylase

Ethylmorphine N-demethylase activity of the sheep liver and lung microsomes was reconstituted in the presence of solubilized microsomal cytochrome P-450, NADPH-cytochrome c reductase and synthetic lipid, phosphatidylcholine dilauroyl. The Km of the lung microsomal ethylmorphine N-demethylase was calculated to be 4.84 mM ethylmorphine from its Lineweaver-Burk graph and lung enzyme was inhibited by its substrate, ethylmorphine, when its concn was 25 mM and above, reaching to 67% inhibition at 50 mM concn. The Lineweaver-Burk and Eadie-Hofstee plots of the liver enzyme were found to be curvilinear. From these graphs, two different Km values were calculated for the liver enzyme as 4.17 mM and 0.40 mM ethylmorphine. Ethylmorphine N-demethylase activities of both liver and lung microsomes were inhibited by NiCl2, CdCl2 and ZnSO4. Ethylalcohol inhibited N-demethylation of ethylmorphine in lung and liver microsomes. Acetone (5%) slightly enhanced the N-demethylase activity of the liver enzyme, whereas 5% acetone completely inhibited the lung enzyme. Phenylmethylsulfonyl fluoride at 0.10 mM and 0.25 mM concn had no effect on liver enzyme activity, while at these concns, it inhibited the activity of the lung enzyme by about 35%.     

A simple procedure for isolating adenosine triphosphatase from mitochondria

A simple method for isolation of adenosine triphosphatase (EC 3.6.1.3) from mitochondria is described. The enzyme is released from mitochondrial Lubrol particles by drastic sonication and purified by gel filtration on Sepharose 6-B. The described procedure is effective in isolating adenosine triphosphatase from rat liver as it is from beef heart mitochondria. The enzyme isolated from beef heart has a specific activity of 120 μmol P/min per mg protein and enzyme isolated from rat liver has a specific activity of 70 μmol P/min per mg protein when measured as a release of inorganic phosphate.     

Adenosine aminohydrolase activity in the regenerating rat liver

The specific activity of adenosine aminohydrolase in the regenerating rat liver is significantly increased 12 h after partial hepatectomy. There is a twofold increase in enzyme activity at 48 h, after which the activity begins to decline. However, increased values still persist 7 days postsurgery. The enzyme is located mainly in the soluble supernatant (90-95%) of the cell. The purified enzyme from 48-h regenerating liver and control liver has similar kinetic properties (Km 54-58 microM for adenosine), similar molecular weights (30,000-35,000), and are equally inhibited by an irreversible transition-state analog and a reversible competitive inhibitor. It is concluded that adenosine aminohydrolase in regenerating liver is an integral component of a salvage pathway designed for the reutilization of nucleotides, and thus helps maintain a "growth state" for the regenerating liver.     

Inhibitory effects of histidine and their reversal. The roles of pyruvate carboxylase and N10-formyltetrahydrofolate dehydrogenase.

1. N10-Formyltetrahydrofolate dehydrogenase was purified to homogeneity from rat liver with a specific activity of 0.7--0.8 unit/mg at 25 degrees C. The enzyme is a tetramer (Mw = 413,000) composed of four similar, if not identical, substrate addition and give the Km values as 4.5 micron [(-)-N10-formyltetrahydrofolate] and 0.92 micron (NADP+) at pH 7.0. Tetrahydrofolate acts as a potent product inhibitor [Ki = 7 micron for the (-)-isomer] which is competitive with respect to N10-formyltetrahydrofolate and non-competitive with respect to NADP+. 3. Product inhibition by NADPH could not be demonstrated. This coenzyme activates N10-formyltetrahydrofolate dehydrogenase when added at concentrations, and in a ratio with NADP+, consistent with those present in rat liver in vivo. No effect of methionine, ethionine or their S-adenosyl derivatives could be demonstrated on the activity of the enzyme. 4. Hydrolysis of N10-formyltetrahydrofolate is catalysed by rat liver N10-formyltetrahydrofolate dehydrogenase at 21% of the rate of CO2 formation based on comparison of apparent Vmax. values. The Km for (-)-N10-folate is a non-competitive inhibitor of this reaction with respect to N10-formyltetrahydrofolate, with a mean Ki of 21.5 micron for the (-)-isomer. NAD+ increases the maximal rate of N10-formyltetrahydrofolate hydrolysis without affecting the Km for this substrate and decreases inhibition by tetrahydrofolate. The activator constant for NAD+ is obtained as 0.35 mM. 5. Formiminoglutamate, a product of liver histidine metabolism which accumulates in conditions of excess histidine load, is a potent inhibitor of rat liver pyruvate carboxylase, with 50% inhibition being observed at a concentration of 2.8 mM, but has no detectable effect on the activity of rat liver cytosol phosphoenolpyruvate carboxykinase measured in the direction of oxaloacetate synthesis. We propose that the observed inhibition of pyruvate carboxylase by formiminoglutamate may account in part for the toxic effect of excess histidine.     

Mitochondrial and cytosolic hexokinase from rat brain: one and the same enzyme?

Rat brain mitochondrial hexokinase (ATP: D-hexose 6-phosphotransferase, EC 2.7.1.1) was solubilized by treatment of the mitochondria with glucose 6-phosphate and partly purified. The solubilized enzyme was compared with the cytosolic enzyme fraction. The solubilized and cytosolic enzymes were also compared with the enzyme bound to the mitochondrial membrane. The following observations were made. 1. There is no difference in electrophoretic mobility on cellulose-acetate between the cytosolic and the solubilized enzyme. Both fractions are hexokinase isoenzyme I. 2. There is no difference in kinetic parameters between the cytosolic or solubilized enzymes (P less than 0.001). For the cytosolic enzyme Km for glucose was 0.067 mM (S.E. = 0.024, n = 7); Km for MgATP2- was 0.42 mM (S.E. = 0.13, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.084 mM (S.E. = 0.011, n = 5). For the solubilized enzyme Km for glucose was 0.071 mM (S.E. = 0.021, n = 6); Km for MgATP2- was 0.38 mM (S.E. = 0.11, n = 6) and Ki,app for glucose 1,6-diphosphate was 0.074 mM (S.E. = 0.010, n = 5). However when bound to the mitochondrial membrane, the enzyme has higher affinities for its substrates and a lower affinity for the inhibitor glucose 1,6-diphosphate. For the mitochondrial fraction Km for glucose was 0.045 mM (S.E. = 0.013, n = 7); Km for MgATP2- was 0.13 mM (S.E. = 0.02, n = 7) and Ki,app for glucose 1,6-diphosphate was 0.33 mM (S.E. = 0.03, n = 5). 3. The cytosolic and solubilized enzyme could be (re)-bound to depleted mitochondria to the same extent and with the same affinity. Limited proteolysis fully destroyed the enzyme's ability to bind to depleted mitochondria. 4. Our data support the hypothesis that soluble- and solubilizable enzyme from rat brain are one and the same enzyme, and that there is a simple equilibrium between the enzyme in these two pools.