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An improved assay for hexokinase activity in human tissue homogenates
ANALYTICAL
BIOCHEMISTRY
91, 451-463 (1978)
An Improved Assay for Hexokinase Activity In Human Tissue Homogenates W. DOUGLAS Department
SCHEER,
H. PETER LEHMANN.
AND MYRTON
of Pathology, LSU Medical Cenrer. 1542 Tulane New, Orleans. Louisiana 70112
F. BEELER
Avenue,
Received June 12, 1978 An improved method has been developed for the assay of hexokinase (EC 2.7.1.1) levels in human tissue homogenates. The enzyme is quantitated by the spectrophotometric measurement. at 340 nm, of NADPH formed according to the reaction scheme: Hexokinase glucose + ATP A
glucose 6-phosphate + ADP
glucose 6-phosphate + NADP+&
G6PD 6-phosphogluconolactone
+ NADPH + H+
In tissue homogenates a number of enzymes are present which can interfere with the assay by reacting with substrates or products of the assay reactions. In the described procedure hexokinase is assayed directly in homogenates under conditions in which the effect of possible contaminating enzymes (glucose dehydrogenase. EC 1.1.1.47; glucose 6-phosphatase, EC 3.1.3.9: glucose phosphate isomerase, EC 5.3.1.9; 6-phosphogluconate dehydrogenase EC 1.1.1.44; and NADP-reducing enzymes) are eliminated. Precision studies on the assay gave within-day reproducibility of 4.3% (CV) on a tissue having a mean activity of 1.68 U/g of tissue, and day-to-day variability of 15% (CV) for a tissue averaging 1.83 U/g of tissue.
In a number of studies on normal and neoplastic rat tissue it was found that the level of hexokinase activity paralleled the glycolytic rate (l-5), as well as the growth rate (l-5) and degree of histological differentiation (2), of the neoplastic tissues. Although studies on human tissues are not as extensive as those on rats several studies have shown elevated levels of hexokinase in some tumors as compared to the respective normal tissues (6-8). Thus, the hexokinase activity in tissue may represent a means of quantitatively characterizing neoplastic tissues. Hexokinase’ (HK) (EC 2.7.1 .I.) is one of the rate-limiting enzymes of ’ Abbreviations used: HK, hexokinase; 6GPD. glucose 6-phosphate dehydrogenase; 6PGAD, 6-phosphogluconate dehydrogenase; 6PGA, 6-phosphogluconate; GDH. glucose dehydrogenase: GPI, glucose phosphate isomerase; G6p’tase, glucose 6-phosphatase. 451
0003-2697/78/0912-0451$02.00/O Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
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AND
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glycolysis catalyzing the first reaction of the pathway; the phosphorylation of glucose to glucose 6-phosphate. with ATP as the phosphate donor: glucose + ATP -
Hexokinase
glucose 6-phosphate
h‘fgz+
+ ADP.
[II
There are four enzymes able to catalyze this reaction: hexokinase isoenzymes I, II, and III, which are widely distributed in mammalian tissues. and glucokinase (EC 2.7.1.2.) which is unique to the liver. Katzen and Schimke found, in studies on rats, that not only were the isoenzymes present in constant proportions for a given tissue, but that the properties of each isoenzyme were uniform from tissue to tissue (9). Studies on substrate specificities of the isoenzymes have shown glucose to be the preferred substrate in all cases, having K, values of 10m5, 10m4. 10V6, and lo+ mol/liter for types I, II, III, and glucokinase, respectively (10,ll). We have investigated the assay of the total activity of the three low K, isoenzymes (I, II, and III) of hexokinase in human normal and neoplastic tissues as part of a project to study enzyme levels and isoenzyme distributions as a potential diagnostic aid in judging the degree of histological differentiation of tumors. In reviewing the methods for the assay of hexokinase we found that none of the procedures had the capacity to correct quantitatively for interfering enzymatic activity in the assayed specimens. The most frequently used hexokinase assay, based on the procedure introduced by Slein et al. (12), couples the hexokinase reaction [I] with that of yeast glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49), in which the glucose 6-phosphate is oxidized by NADP+ in the presence of the enzyme producing 6-phosphogluconolactone NADPH and H+: glucose 6-phosphate G6PD + NADP+ -6phosphogluconolactone
+ NADPH
+ H+.
[II]
The rate of NADPH production, which is formed on a mole for mole basis with glucose 6-phosphate, can be measured spectrophotometrically at 340 nm. The 6-phosphogluconolactone formed in reaction [II] is unstable in the alkaline conditions of the assay (pH 7.4) and in the presence of the tissue enzyme lactonase (EC 3.1.1.17) and is spontaneously hydrolyzed to 6-phosphogluconate: 6-phosphogluconoIactone + H,O -
lactonase OH-
6-phosphogluconate.
[III]
The 6-phosphogluconate acts as substrate for the tissue enzyme S-phosphogluconate dehydrogenase (6PGAD) (EC 1.1.1.44), and in the
ASSAYFORHEXOKINASE
presence of NADP+ produces phosphogluconate present:
453
1 mol of NADPH
for each mole of 6-
6-phosphogluconate + NADP+
-
6PGAD
. rlbulose 5-phosphate
+ NADPH
+ H+.
[IV]
This can lead to values up to twice the true hexokinase activity if all the 6-phosphogluconate is oxidized. We have developed a procedure by which tissue hexokinase activity can be accurately quantitated and, at the same time, the effect of interfering enzymatic activity can be measured for each specimen. MATERIALS Homogenizing
AND METHODS
Solution
The homogenizing solution consisted of potassium chloride ( 150 mmoli liter), magnesium chloride (5 mmol/liter), ethylenediaminetetraacetic acid (EDTA; 5 mmol/liter), and 2-mercaptoethanol (5 mmoliliter). To 100 ml of deionized water in a 200-ml volumetric flask add 2.24 g of potassium chloride, 0.20 g of magnesium chloride hexahydrate. 0.34 g of EDTA, and 0.07 ml of 2-mercaptoethanol. Fill to the mark with deionized water. Reagents Tris (hydroxymethyl) aminomethune bufir. The buffer was 0.2 moliliter. pH 7.4 at 30°C. Weigh out 4.8 g of Trizmabase (Sigma Chemical Company. reagent grade) and dissolve in 190 ml of deionized water. Adjust to pH 7.4 with concentrated hydrochloric acid (Mallinckrodt Chemical Works, reagent grade) and make up to 200 ml with deionized water. Magnesium chloride (42 mmollliter). Weigh out 0.17 g of magnesium chloride hexahydrate. transfer to a 200-ml of volumetric flask and fill to the mark with deionized water. Nicotinamide
adenine dinucleotide
phosphate
(NADP’,
6.5 mmollliter).
Weigh out 0.01 I g of NADP+ (Sigma Chemical Company, Sigma grade). transfer to a2-ml volumetric flask, and fill to the mark with deionized water. This is sufficient for two complete assays. The reagent is unstable and must be freshly prepared for each assay run. Adenosine 5’-triphosphate (ATP, 42 mmollliter). Weigh out 0.52 g of ATP (Sigma Chemical Company, Sigma grade), transfer to a 2-ml volumetric flask, and fill to the mark with deionized water. Adjust to pH 7.0 with a few drops of 5 mol/liter sodium hydroxide (Mallinckrodt Chemical Works, reagent grade). This is sufficient for three assays. The reagent is unstable and must be freshly prepared for each assay run. Glucose (4.17 mmoliliter). Weight out 0.075 g of glucose (Sigma Chemical Company, Grade III), transfer to a lOO-ml volumetric flask and fill to
454
SCHEER,
LEHMANN.
AND
BEELER
the mark with deionized water. The solution remained stable for 1 month when stored refrigerated at 4°C. Glucose &phosphate dehydrogenase (25 unitslml). Transfer 250 units of glucose 6-phosphate dehydrogenase (Sigma Chemical Company, Type XV from baker’s yeast) to a lo-ml volumetric flask and fill to the mark with deionized water. The solution was aliquoted into l-ml portions and was stable for up to 3 weeks when stored frozen at -20°C. 6-Phosphogluconate (6PGA, 6.75 mmollliter). Weigh out 0.026 g of 6-phosphogluconate (trisodium salt, Sigma Chemical Company, Sigma grade), transfer to a lo-ml volumetric flask, and fill to the mark with deionized water. The solution remained stable for 1 month when stored refrigerated at 4°C. 2-Mercaptoethanol(120 mmollliter). Add 0.09 ml of 2-mercaptoethanol to 5 ml of deionized water in a IO-ml volumetric flask. Mix and make up to the mark with deionized water. Procedure A 1: 10 (weight:volume) homogenate was made by adding the appropriate volume of homogenizing solution to the minced tissue and homogenizing, until a homogenous solution was obtained, at 4°C with a Thomas Teflon pestle homogenizer powered by an electric motor. The homogenate was centrifuged at 100,OOOg for 1 h at 4°C. Two tubes (blank, A, and sample, B) were made up as shown in Table 1 for each sample assayed. After 5 min of incubation in a water bath at 30°C for temperature equilibration, 0.05 ml of sample was pipetted into each tube at timed intervals. The enzyme activity was measured by following the rate of NADPH production at 340 nm in a l-cm cuvet at 3o”C, for 10 min. The contaminating 6-phosphogluconate dehydrogenase activity was quantitated by adding a third tube (C) to the assay (see Table 1). The contents of tube C are the same as tube B except that no ATP, glucose, or glucose 6-phosphate dehydrogenase is present. Calculations The hexokinase
activity
was calculated
using the expression:
U/g of tissue = (AAbs B - AAbs A) - (AAbs C - AAbs A) x 2 5 x 2. x 1. 6.22 x 1 where: U = enzyme activity in international units (micromoles of NADPH formed per minute), AAbs B = the absorbance change per minute in cuvet B, AAbs A = the absorbance change per minute in cuvet A, AAbs C = the absorbance change per minute in cuvet c, 6.22 = the milli-
455
ASSAY FOR HEXOKINASE TABLE REACTION
Tube A. blank Reactants
Volume (ml)
Tris (pH 7.4) MgCI, NADP ATP Glucose 6PGA Mercaptoethanol G6PD (2.5 units) Sample Water
0.6 0.6 0.3 0.0 0.3 0.0 0.1 0.1 0.05 0.45
I
MIXTURES
Tube B. sample
mmoVliter 47 10 0.8 0.0 0.5 0.0 5.0
Volume (ml)
mmoliliter
0.6 0.6 0.3 0.3 0.3 0.1 0.1 0.1 0.05 0.05
47 10 0.8 5.0 0.5 0.27 5.0
Tube C Volume (ml) 0.6 0.6 0.3 0.0 0.0 0. I 0.1 0.0 0.05 0.75
mmol/liter 47 10 0.8 0.0 0.0 0.27 5.0 /
molar absorptivity of NADPH at 340 nm (1 mmol-l .crn-‘), I = optical path length (cm), 2.5 = correction factor for the final solution volume, 20 = correction factor for the sample volume (0.05 ml), and 10 = correction factor for the dilution of homogenization. The 6-phosphogluconate dehydrogenase activity was calculated using the expression: U/g of tissue =
AAbs C - AAbs A x 2.5 x 20 x 10. 6.22 x 1 RESULTS
Hexokinase
and 6-Phosphogluconate
Dehydrogenase
Activity
In the 84 tumors and 15 normal tissues studied the hexokinase activity ranged from 0.08 to 2.72 U/g of tissue with a mean of 0.89 U/g of tissue. The 6-phosphogluconate dehydrogenase activity ranged from 0.00 to 4.98 U/g of tissue with a mean of 0.94 U/g of tissue. For those specimens that had both hexokinase and 6-phosphogluconate dehydrogenase activity the ratio HK/6PGAD ranged from 0.2 to 4.0. Erythrocyte Hexokinase and 6-Phosphogluconate Dehydrogenase Activity The activities of erythrocyte hexokinase and 6-phosphogluconate dehydrogenase were determined to observe the effect of the presence of blood in the tissue on tissue hexokinase activity. A blood sample was centrifuged and an aliquot of serum was removed. The blood cells were
456
SCHEER,
LEHMANN,
AND
BEELER
then hemolyzed by several freeze-thaw cycles and the hemolyzed blood was centrifuged. The supernatant and the serum were then assayed for hexokinase and 6-phosphogluconate dehydrogenase. The hemoglobin concentration of the hemolyzed specimen, measured spectrophotometrically at 540 nm, was found to be 85 g/liter (4.25 mg/0.05-ml sample volume). This is much higher than the maximum hemoglobin concentration found in the tissue homogenates, which was approximately 5 g/liter (0.25 mg/O.OSml sample volume). The hemolyzed serum sample was then diluted to yield a solution having a hemoglobin concentration of 4.1 g/liter (0.20 mg/0.05-ml sample volume) and 0.05 ml of this specimen was assayed for both erythrocyte hexokinase and 6-phosphogluconate dehydrogenase activities. While no hexokinase activity was detected, a small amount of 6phosphogluconate dehydrogenase activity was observed (3.65 U/g of hemoglobin corresponding to 0.73 mU/0.05-ml sample volume). Glucose
Dehydrogenase
Glucose dehydrogenase activity (EC 1.1.1.47) (GDH) was identified in the commercial preparation of baker’s yeast glucose 6-phosphate dehydrogenase and in mammalian liver. The enzyme uses glucose and NADP+ as substrates and forms NADPH and gluconolactone as products, according to reaction [VI: glucose + NADP+
GDH ->gluconolactone
+ NADPH+
+ H+.
[VI
Increasing the glucose concentration of the assay mixture from 0.05 to 80 mmol/liter resulted in no increase in the absorbance change per minute at 340 nm, indicating to NADPH production from yeast glucose dehydrogenase activity was taking place under these conditions. Therefore, under the reaction conditions and glucose concentration used in this study (0.5 mmol/liter), it was not necessary to correct for glucose dehydrogenase activity. Substrate
Concentration
In order to ensure that the substrate and cofactor concentrations were sufficient to maintain zero order kinetics under the reaction conditions described, the glucose, NADP, ATP, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate were each increased in turn to observe any change in enzyme activity resulting from this. The results in Table 2 show that increasing the concentration of the constituents had no significant effect on the measured enzyme activity. 6Phosphogluconate
Inhibition
An experiment was performed to determine whether 6-phosphogluconate inhibits the hexokinase reaction. A sample, which had been shown to have
457
ASSAY FOR HEXOKINASE TABLE EFFECT
OF INCREASED AND
Reactant
Tube
Glucose
B
NADP ATP G6PD 6PGA
REACTANT
6-PHOSPHOGLUCONATE
B B B C
2
CONCENTRATIONS DEHYDROGENASE
ON HEXOKINASE ACTIVITY
U/g of tissue
Percentage change
0.5 1.0
2.16” 2.00
-7.4%
0.8 1.1
6.30” 6.08h
-3.5Q
5.0 6.7
2.32” 2.16”
-6.9%
1 unit/ml 2 unit/ml
I .36” 1.28”
-5.9%
0.27 0.54
2.40’ 2.40’
Concentration (mmol/liter)
0.0%
a Activity of HK corrected for 6PGAD activity. * Activity of HK + 6PGAD. CActivity of 6PGAD.
no 6-phosphogluconate dehydrogenase activity, was assayed in the presence and absence of 0.27 mmollliter of 6-phosphogluconate. There was no inhibition; the hexokinase activity was found to be 0.48 U/g of tissue in both cases. Linen rity
The linearity of the reaction with the time, when carried out under the described conditions, is illustrated in Fig. 1. A homogenate of a squamous cell carcinoma of the lung, having relatively high enzyme activity, was assayed and readings taken every 2 min for 10 min. The linearity observed for total enzyme activity (3.34 U/g of tissue), 6-phosphogluconate dehydrogenase activity (2.32 U/g of tissue), and hexokinase activity (1.04 U/g of tissue) showed that the enzymes were being analyzed under optimum conditions over this time period. Figure 2 shows the effect on the assay of increasing the amount of enzyme. Different amounts of homogenate of a squamous cell carcinoma of the lung were added to the assay system and absorbance readings taken every 2 min for 10 min as previously described. It was found that the total enzyme activity (tube B) was linear at least up to a AAbslmin of 0.064 (enzyme activity of 5.12 U/g tissue). The 6-phosphogluconate dehydrogenase activity was linear up to a AAbs/min = 0.05 (4.0 U/g tissue), and, by difference, the hexokinase levels were linear up to a AAbsimin = 0.028
458
SCHEER,
LEHMANN,
AND
BEELER
.700 q
Hexoktnose+G-PGAD
0 C-PGAD a Hexokinose
600 .500 I
0
2
4
s
Incubation
FIG. I. Effect dehydrogenase.
of incubation
time on the reaction
8
IO
12
ttme,minutes
rate of hexokinase
(2.24 U/g of tissue). These are relatively measured.
and 6-phosphogluconate
high activities
for the tissues
Precision
The precision of the method was determined for within-day variability by performing 10 assays on a single tumor tissue (renal cell carcinoma) .1(X,100
uo Hexokirase+G-PGAD
i B .B $
080 -
0 6-PGAD A Hexokinase
060-
2 y
.040-
0
FIG.
2. Effect
4 002 Ob2
of enzyme
I 0.04 0.66 0.06 008 0.64 Sample volume, ml concentration
/ 0.10 o.io
on the reaction
1 012
rate.
ASSAY
FOR
TABLE WITHIN-DAY
AND DAY-TO-DAY
459
HEXOKINASE 3
PRECISION Total
OF THE HEXOKINASE
(HK)
ASSAY
enzyme” activity (U/g of tissue)
HK” activity (U/g of tissue)
Within day Number Mean SD cv
10 2.16 0.09 4.2%
IO 1.68 0.07 4.3%
Day-to-day Number Mean SD cv
12 2.76 0.45 16.2%
12 1.83 0.27 15.0%
homogenate. The mean value obtained was 1.60 U/g of tissue (SD = 0.07 U/g of tissue; CV = 4.3%), Table 3. The day-to-day variability was determined on a single frozen tissue stored at -20°C. A fresh homogenate was prepared on each day the tissue was assayed for a total of 12 determinations over a period of 2 months. The mean value obtained was 1.83 U/g of tissue (SD = 0.27 U/g of tissue; CV = IS%), Table 3. The variability shown in this data includes the day-to-day variability of the method as well as tissue site variability. The tissue homogenates were found to be unstable even when frozen at -20°C. The tissues were, therefore, stored frozen and homogenized immediately before analysis. The relatively low day-to-day precision (CV 15%) may be due in part to variability in the cellular composition of the tissue aliquots. DISCUSSION
There are several published modifications of the basic hexokinase assay procedure of Slein et al. (12) which describe a variety of reaction conditons (different buffer and substrate concentrations) in order to optimize conditions for the particular tissue being assayed (13- 17). Most of the procedures employ a blank, containing no ATP, which quantitates the endogenous NADP+ reduction. In addition, a few procedures also eliminate glucose from the blank in order to quantitate endogenous formation of glucose 6-phosphate. Several of the methods include procedures for detecting and quantitating 6-phosphogluconate dehydrogenase activity, although not for individual specimens. The assay used in this study was based on the procedure described by
460
SCHEER,
LEHMANN,
AND
BEELER
Salas (13), modified to take into consideration a number of potential interfering enzymes which may be found in tissue homogenates. In establishing optimum substrate concentrations the properties of several possible contaminating enzymes were considered. GPhosphogluconate
Dehydrogenase
This enzyme catalyzes reaction [IV]. The substrate for this enzyme (6-phosphogluconate) is the final product of the assay reactions for hexokinase (I, II, and III). Therefore, the presence of the enzyme in tissue can give falsely elevated hexokinase activities by reacting with 6phosphogluconate and thereby increasing the rate of NADPH production. The wide range of 6-phosphogluconate dehydrogenase activity and the lack of correlation with the hexokinase activity indicates that there is contaminating 6-phosphogluconate dehydrogenase present and that the degree of contamination is not constant and, therefore, must be corrected for each sample assayed. Several correction procedures for 6-phosphogluconate dehydrogenase contamination of hexokinase assays have been reported (13,16,17). Yeast 6-phosphogluconate dehydrogenase has been added to the sample assay mixture after the hexokinase reaction has proceeded for a certain period of time (17). If there is sufficient endogenous 6-phosphogluconate dehydrogenase present to react with all of the 6-phosphogluconate formed in the hexokinase assay reactions (I, II, III) there should be no increase in the rate of NADPH formation. In this case a factor of 0.5 has been applied to the final measured enzyme activity to correct for the contaminating 6-phosphogluconate dehydrogenase activity, since for each mole of glucose 6-phosphate formed 2 mol of NADPH are generated. The activity of the sample hexokinase and the length of time the enzyme is allowed to react before the addition of exogenous 6-phosphogluconate dehydrogenase are important factors since they will determine the amount of 6phosphogluconate present in the assay mixture. If there is no 6-phosphogluconate dehydrogenase in the sample, the lack of an increased formation of NADPH on the addition of the yeast 6-phosphogluconate dehydrogenase will be misinterpreted. Another problem arises when there is an increase in the rate of NADPH formation on the addition of yeast 6-phosphogluconate dehydrogenase. This indicates that the amount of the endogenous 6-phosphogluconate dehydrogenase is insufficient to react with all of the 6-phosphogluconate formed. It does not, however, quantitate the amount of 6-phosphogluconate which has reacted and it is, therefore, not possible to make a correction in these cases. Another technique which has been used for the detection of contaminating 6-phosphogluconate dehydrogenase activity is the substitution of
ASSAY
FOR
HEXOKINASE
461
glucose 6-phosphate for glucose as the substrate (16). Due to the excess glucose 6-phosphate dehydrogenase present in such a system most of this substrate is immediately converted to 6-phosphogluconate (reactions II and III) with the production of NADPH. When assaying hexokinase by the described procedure the rate of formation of 6-phosphogluconate is limited by the activity of the hexokinase producing the glucose 6-phosphate. Addition of glucose 6-phosphate as substrate will yield a higher activity of 6-phosphogluconate dehydrogenase than in the actual hexokinase assay, and no true measure of the 6-phosphogluconate dehydrogenase contamination is obtained. Glucose dehydrogenase. The presence of this enzyme could cause substrate (glucose) depletion in reaction [I] and an increased rate of NADPH production through reaction [V] resulting in falsely elevated measured hexokinase activities. Glucose dehydrogenase is primarily a mammalian liver enzyme but was found to be present in the commercial preparation of yeast glucose 6-phosphate dehydrogenase used in reaction [II]. It does not interfere with the assay of the low K, hexokinase being measured in this study since the concentration of glucose used (0.5 mmol/liter) is too low to elicit significant glucose dehydrogenase activity (K, for glucose 100 mmol/liter). Glucose phosphate isomerase (GPZ), EC 5.3.1.9). This enzyme catalyzes the reaction: D-glucose 6-phosphate
z
D-fructose 6-phosphate.
WI
GPI is the second enzyme in the glycolytic pathway and is present in tissue at approximately 100 times the levels of hexokinase. The effect on the assay could be through the removal of glucose 6-phosphate from reaction [II], resulting in a decrease in the overall rate of NADPH formation. The equilibrium for the reaction lies to the left (K = 0.30) and since glucose 6-phosphate dehydrogenase is added in excess the assay reaction [II] predominates over reaction [VI]. Glucose bphosphatase (G6p’tase. EC 3.1.3.9). G6P’Tase catalyzes the reaction: glucose 6-phosphate
+ H,O
G6p’tase
l
glucose + orthophosphate.
[VIII
Glucose 6-phosphatase is a second enzyme present in tissue which can act on glucose 6-phosphate, thereby decreasing the measured hexokinase activity. It is a microsomal enzyme which is, therefore, removed from the homogenate by the centrifugation prior to the hexokinase assay (18). Also as in the case of glucose dehydrogenase the excess glucose 6-phosphate dehydrogenase added to the assay system minimizes the effect of this enzyme.
462
SCHEER,
LEHMANN,
AND
BEELER
NADP+ cofactor enzymes. Enzymes which used NADP+ catalyze hydrogen atom transfer and react according to:
as a cofactor
substrate (red) + NADP+
enzymes -substrate
(oxid) + NADPH
+ H+.
[VIII]
The effect of endogenous reducing enzymes can be measured by utilizing a blank assay system containing no ATP, so that the only activity is due to the NADP+ reducing enzymes, tube A, Table 1. These reactions also occur in tubes B and C and are therefore automatically corrected for in the calculation of the hexokinase activity. The concentration of magnesium chloride was increased to 10 mmol/liter to give a magnesium chloride, ATP ratio of 2:l in order to prevent the inhibition of hexokinase that occurs when the molar ratio of ATP to magnesium is greater than 1 (I I). The lack of any increase in hexokinase activity when the amounts of glucose 6-phosphate dehydrogenase, and ATP, are increased (Table 2) show that with these changes the reaction is still being carried out under optimum conditions, zero-order kinetics, with respect to these compounds. The procedure presented in this study has been shown to be linear (Figs. 1 and 2) and reproducible, and accounts for the contaminating 6-phosphogluconate dehydrogenase activity in a quantitative manner. The amount of 6-phosphogluconate added is high enough to give zero order kinetics and at the same time is not inhibitory for the hexokinase reaction. The lack of correlation between the hexokinase and 6-phosphogluconate dehydrogenase levels also indicates that a common factor cannot be used, but individual correction must be made for interfering enzymes in each sample assayed. ACKNOWLEDGMENTS This work was supported in part by USPHS Grant AH ooOO2-02 and by a grant from the Cancer Association of New Orleans and was performed in partial fulfillment of the dissertation requirements for the Ph.D. degree by W. Douglas Scheer.
REFERENCES I. Knox, W. E. (1967) Cancer Res. 10, 117-161. 2. Knox, W. E., Jamdar, S. C., and Davis, P. A. (1970) Cancer Res. 30, 2240-2244. 3. Sharma, R. M., Sharma, C., Donnelly, A. J., Morris, H. P., and Weinhouse, S. (1965) Cancer Res. 24, 193-198. 4. Weber, G. (1974) in The Molecular Biology of Cancer (Busch, H., ed.) p. 487, Academic Press. New York. 5. Weinhouse. S. (1966) Gann Monograph 1, 99- 115. 6. Bar, U., Schmidt, E., and Schmidt, F. W. (1963) K/in. Wochschr. 41, 977-988. 7. Pedersen. S. N. (1975) Cancer 35, 469-474. 8. Kikuchi, U.. Sato, S., and Sugimura. T. (1972) Cancer 30, 444-447.
ASSAY FOR HEXOKINASE
463
9. Katzen, H. M. and Schimke, R. T. (1965) Proc. Nuf. Acad. Sci. USA 54, 1218-1225. 10. Katzen, H. M., Soderman, D. D., and Nitowsky, H. M. (1965)Biochem. Biophys. Res. Commun. 19, 311-382. 11. Grossbard, L., and Schmike, R. T. (1966) J. Biol. Chem. 241, 3546-3560. 12. Slein. M. W., Cori, G. T., and Cori, C. F. (1950) J. Biol. Chem. 186, 763-780. 13. Salas, M.. Vinuela. E., and Sols, A (1963) J. Biol. Chem. 238, 3535-3538. 14. Joshi, M. D., and Jagannathan, V. (1966)in Methods of Enzymology (Wood, W. A., ed.) p. 371, Academic Press, New York. 15. Shonk, C. E., and Boxer, G. E. (1964) Cancer Rrs. 24, 709-721. 16. Sharma, C., Manjeshwar, R., and Weinhouse, S. (1963)5. Bio1. Chem. 238, 3840-3845. 17. Jamdar, S. C., and Greengard, 0. (1970) J. Biol. Chem. 245, 2779-2783. 18. Walker, D. G. (1966) in Essays in Biochemistry (Campbell. P., IV, and Greville, G. D.. eds.), Vol. 2, pp. 33-62, Academic Press, New York.
BIOCHEMISTRY
91, 451-463 (1978)
An Improved Assay for Hexokinase Activity In Human Tissue Homogenates W. DOUGLAS Department
SCHEER,
H. PETER LEHMANN.
AND MYRTON
of Pathology, LSU Medical Cenrer. 1542 Tulane New, Orleans. Louisiana 70112
F. BEELER
Avenue,
Received June 12, 1978 An improved method has been developed for the assay of hexokinase (EC 2.7.1.1) levels in human tissue homogenates. The enzyme is quantitated by the spectrophotometric measurement. at 340 nm, of NADPH formed according to the reaction scheme: Hexokinase glucose + ATP A
glucose 6-phosphate + ADP
glucose 6-phosphate + NADP+&
G6PD 6-phosphogluconolactone
+ NADPH + H+
In tissue homogenates a number of enzymes are present which can interfere with the assay by reacting with substrates or products of the assay reactions. In the described procedure hexokinase is assayed directly in homogenates under conditions in which the effect of possible contaminating enzymes (glucose dehydrogenase. EC 1.1.1.47; glucose 6-phosphatase, EC 3.1.3.9: glucose phosphate isomerase, EC 5.3.1.9; 6-phosphogluconate dehydrogenase EC 1.1.1.44; and NADP-reducing enzymes) are eliminated. Precision studies on the assay gave within-day reproducibility of 4.3% (CV) on a tissue having a mean activity of 1.68 U/g of tissue, and day-to-day variability of 15% (CV) for a tissue averaging 1.83 U/g of tissue.
In a number of studies on normal and neoplastic rat tissue it was found that the level of hexokinase activity paralleled the glycolytic rate (l-5), as well as the growth rate (l-5) and degree of histological differentiation (2), of the neoplastic tissues. Although studies on human tissues are not as extensive as those on rats several studies have shown elevated levels of hexokinase in some tumors as compared to the respective normal tissues (6-8). Thus, the hexokinase activity in tissue may represent a means of quantitatively characterizing neoplastic tissues. Hexokinase’ (HK) (EC 2.7.1 .I.) is one of the rate-limiting enzymes of ’ Abbreviations used: HK, hexokinase; 6GPD. glucose 6-phosphate dehydrogenase; 6PGAD, 6-phosphogluconate dehydrogenase; 6PGA, 6-phosphogluconate; GDH. glucose dehydrogenase: GPI, glucose phosphate isomerase; G6p’tase, glucose 6-phosphatase. 451
0003-2697/78/0912-0451$02.00/O Copyright Q 1978 by Academic Press, Inc. All rights of reproduction in any form reserved.
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glycolysis catalyzing the first reaction of the pathway; the phosphorylation of glucose to glucose 6-phosphate. with ATP as the phosphate donor: glucose + ATP -
Hexokinase
glucose 6-phosphate
h‘fgz+
+ ADP.
[II
There are four enzymes able to catalyze this reaction: hexokinase isoenzymes I, II, and III, which are widely distributed in mammalian tissues. and glucokinase (EC 2.7.1.2.) which is unique to the liver. Katzen and Schimke found, in studies on rats, that not only were the isoenzymes present in constant proportions for a given tissue, but that the properties of each isoenzyme were uniform from tissue to tissue (9). Studies on substrate specificities of the isoenzymes have shown glucose to be the preferred substrate in all cases, having K, values of 10m5, 10m4. 10V6, and lo+ mol/liter for types I, II, III, and glucokinase, respectively (10,ll). We have investigated the assay of the total activity of the three low K, isoenzymes (I, II, and III) of hexokinase in human normal and neoplastic tissues as part of a project to study enzyme levels and isoenzyme distributions as a potential diagnostic aid in judging the degree of histological differentiation of tumors. In reviewing the methods for the assay of hexokinase we found that none of the procedures had the capacity to correct quantitatively for interfering enzymatic activity in the assayed specimens. The most frequently used hexokinase assay, based on the procedure introduced by Slein et al. (12), couples the hexokinase reaction [I] with that of yeast glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.49), in which the glucose 6-phosphate is oxidized by NADP+ in the presence of the enzyme producing 6-phosphogluconolactone NADPH and H+: glucose 6-phosphate G6PD + NADP+ -6phosphogluconolactone
+ NADPH
+ H+.
[II]
The rate of NADPH production, which is formed on a mole for mole basis with glucose 6-phosphate, can be measured spectrophotometrically at 340 nm. The 6-phosphogluconolactone formed in reaction [II] is unstable in the alkaline conditions of the assay (pH 7.4) and in the presence of the tissue enzyme lactonase (EC 3.1.1.17) and is spontaneously hydrolyzed to 6-phosphogluconate: 6-phosphogluconoIactone + H,O -
lactonase OH-
6-phosphogluconate.
[III]
The 6-phosphogluconate acts as substrate for the tissue enzyme S-phosphogluconate dehydrogenase (6PGAD) (EC 1.1.1.44), and in the
ASSAYFORHEXOKINASE
presence of NADP+ produces phosphogluconate present:
453
1 mol of NADPH
for each mole of 6-
6-phosphogluconate + NADP+
-
6PGAD
. rlbulose 5-phosphate
+ NADPH
+ H+.
[IV]
This can lead to values up to twice the true hexokinase activity if all the 6-phosphogluconate is oxidized. We have developed a procedure by which tissue hexokinase activity can be accurately quantitated and, at the same time, the effect of interfering enzymatic activity can be measured for each specimen. MATERIALS Homogenizing
AND METHODS
Solution
The homogenizing solution consisted of potassium chloride ( 150 mmoli liter), magnesium chloride (5 mmol/liter), ethylenediaminetetraacetic acid (EDTA; 5 mmol/liter), and 2-mercaptoethanol (5 mmoliliter). To 100 ml of deionized water in a 200-ml volumetric flask add 2.24 g of potassium chloride, 0.20 g of magnesium chloride hexahydrate. 0.34 g of EDTA, and 0.07 ml of 2-mercaptoethanol. Fill to the mark with deionized water. Reagents Tris (hydroxymethyl) aminomethune bufir. The buffer was 0.2 moliliter. pH 7.4 at 30°C. Weigh out 4.8 g of Trizmabase (Sigma Chemical Company. reagent grade) and dissolve in 190 ml of deionized water. Adjust to pH 7.4 with concentrated hydrochloric acid (Mallinckrodt Chemical Works, reagent grade) and make up to 200 ml with deionized water. Magnesium chloride (42 mmollliter). Weigh out 0.17 g of magnesium chloride hexahydrate. transfer to a 200-ml of volumetric flask and fill to the mark with deionized water. Nicotinamide
adenine dinucleotide
phosphate
(NADP’,
6.5 mmollliter).
Weigh out 0.01 I g of NADP+ (Sigma Chemical Company, Sigma grade). transfer to a2-ml volumetric flask, and fill to the mark with deionized water. This is sufficient for two complete assays. The reagent is unstable and must be freshly prepared for each assay run. Adenosine 5’-triphosphate (ATP, 42 mmollliter). Weigh out 0.52 g of ATP (Sigma Chemical Company, Sigma grade), transfer to a 2-ml volumetric flask, and fill to the mark with deionized water. Adjust to pH 7.0 with a few drops of 5 mol/liter sodium hydroxide (Mallinckrodt Chemical Works, reagent grade). This is sufficient for three assays. The reagent is unstable and must be freshly prepared for each assay run. Glucose (4.17 mmoliliter). Weight out 0.075 g of glucose (Sigma Chemical Company, Grade III), transfer to a lOO-ml volumetric flask and fill to
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the mark with deionized water. The solution remained stable for 1 month when stored refrigerated at 4°C. Glucose &phosphate dehydrogenase (25 unitslml). Transfer 250 units of glucose 6-phosphate dehydrogenase (Sigma Chemical Company, Type XV from baker’s yeast) to a lo-ml volumetric flask and fill to the mark with deionized water. The solution was aliquoted into l-ml portions and was stable for up to 3 weeks when stored frozen at -20°C. 6-Phosphogluconate (6PGA, 6.75 mmollliter). Weigh out 0.026 g of 6-phosphogluconate (trisodium salt, Sigma Chemical Company, Sigma grade), transfer to a lo-ml volumetric flask, and fill to the mark with deionized water. The solution remained stable for 1 month when stored refrigerated at 4°C. 2-Mercaptoethanol(120 mmollliter). Add 0.09 ml of 2-mercaptoethanol to 5 ml of deionized water in a IO-ml volumetric flask. Mix and make up to the mark with deionized water. Procedure A 1: 10 (weight:volume) homogenate was made by adding the appropriate volume of homogenizing solution to the minced tissue and homogenizing, until a homogenous solution was obtained, at 4°C with a Thomas Teflon pestle homogenizer powered by an electric motor. The homogenate was centrifuged at 100,OOOg for 1 h at 4°C. Two tubes (blank, A, and sample, B) were made up as shown in Table 1 for each sample assayed. After 5 min of incubation in a water bath at 30°C for temperature equilibration, 0.05 ml of sample was pipetted into each tube at timed intervals. The enzyme activity was measured by following the rate of NADPH production at 340 nm in a l-cm cuvet at 3o”C, for 10 min. The contaminating 6-phosphogluconate dehydrogenase activity was quantitated by adding a third tube (C) to the assay (see Table 1). The contents of tube C are the same as tube B except that no ATP, glucose, or glucose 6-phosphate dehydrogenase is present. Calculations The hexokinase
activity
was calculated
using the expression:
U/g of tissue = (AAbs B - AAbs A) - (AAbs C - AAbs A) x 2 5 x 2. x 1. 6.22 x 1 where: U = enzyme activity in international units (micromoles of NADPH formed per minute), AAbs B = the absorbance change per minute in cuvet B, AAbs A = the absorbance change per minute in cuvet A, AAbs C = the absorbance change per minute in cuvet c, 6.22 = the milli-
455
ASSAY FOR HEXOKINASE TABLE REACTION
Tube A. blank Reactants
Volume (ml)
Tris (pH 7.4) MgCI, NADP ATP Glucose 6PGA Mercaptoethanol G6PD (2.5 units) Sample Water
0.6 0.6 0.3 0.0 0.3 0.0 0.1 0.1 0.05 0.45
I
MIXTURES
Tube B. sample
mmoVliter 47 10 0.8 0.0 0.5 0.0 5.0
Volume (ml)
mmoliliter
0.6 0.6 0.3 0.3 0.3 0.1 0.1 0.1 0.05 0.05
47 10 0.8 5.0 0.5 0.27 5.0
Tube C Volume (ml) 0.6 0.6 0.3 0.0 0.0 0. I 0.1 0.0 0.05 0.75
mmol/liter 47 10 0.8 0.0 0.0 0.27 5.0 /
molar absorptivity of NADPH at 340 nm (1 mmol-l .crn-‘), I = optical path length (cm), 2.5 = correction factor for the final solution volume, 20 = correction factor for the sample volume (0.05 ml), and 10 = correction factor for the dilution of homogenization. The 6-phosphogluconate dehydrogenase activity was calculated using the expression: U/g of tissue =
AAbs C - AAbs A x 2.5 x 20 x 10. 6.22 x 1 RESULTS
Hexokinase
and 6-Phosphogluconate
Dehydrogenase
Activity
In the 84 tumors and 15 normal tissues studied the hexokinase activity ranged from 0.08 to 2.72 U/g of tissue with a mean of 0.89 U/g of tissue. The 6-phosphogluconate dehydrogenase activity ranged from 0.00 to 4.98 U/g of tissue with a mean of 0.94 U/g of tissue. For those specimens that had both hexokinase and 6-phosphogluconate dehydrogenase activity the ratio HK/6PGAD ranged from 0.2 to 4.0. Erythrocyte Hexokinase and 6-Phosphogluconate Dehydrogenase Activity The activities of erythrocyte hexokinase and 6-phosphogluconate dehydrogenase were determined to observe the effect of the presence of blood in the tissue on tissue hexokinase activity. A blood sample was centrifuged and an aliquot of serum was removed. The blood cells were
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then hemolyzed by several freeze-thaw cycles and the hemolyzed blood was centrifuged. The supernatant and the serum were then assayed for hexokinase and 6-phosphogluconate dehydrogenase. The hemoglobin concentration of the hemolyzed specimen, measured spectrophotometrically at 540 nm, was found to be 85 g/liter (4.25 mg/0.05-ml sample volume). This is much higher than the maximum hemoglobin concentration found in the tissue homogenates, which was approximately 5 g/liter (0.25 mg/O.OSml sample volume). The hemolyzed serum sample was then diluted to yield a solution having a hemoglobin concentration of 4.1 g/liter (0.20 mg/0.05-ml sample volume) and 0.05 ml of this specimen was assayed for both erythrocyte hexokinase and 6-phosphogluconate dehydrogenase activities. While no hexokinase activity was detected, a small amount of 6phosphogluconate dehydrogenase activity was observed (3.65 U/g of hemoglobin corresponding to 0.73 mU/0.05-ml sample volume). Glucose
Dehydrogenase
Glucose dehydrogenase activity (EC 1.1.1.47) (GDH) was identified in the commercial preparation of baker’s yeast glucose 6-phosphate dehydrogenase and in mammalian liver. The enzyme uses glucose and NADP+ as substrates and forms NADPH and gluconolactone as products, according to reaction [VI: glucose + NADP+
GDH ->gluconolactone
+ NADPH+
+ H+.
[VI
Increasing the glucose concentration of the assay mixture from 0.05 to 80 mmol/liter resulted in no increase in the absorbance change per minute at 340 nm, indicating to NADPH production from yeast glucose dehydrogenase activity was taking place under these conditions. Therefore, under the reaction conditions and glucose concentration used in this study (0.5 mmol/liter), it was not necessary to correct for glucose dehydrogenase activity. Substrate
Concentration
In order to ensure that the substrate and cofactor concentrations were sufficient to maintain zero order kinetics under the reaction conditions described, the glucose, NADP, ATP, glucose 6-phosphate dehydrogenase, and 6-phosphogluconate were each increased in turn to observe any change in enzyme activity resulting from this. The results in Table 2 show that increasing the concentration of the constituents had no significant effect on the measured enzyme activity. 6Phosphogluconate
Inhibition
An experiment was performed to determine whether 6-phosphogluconate inhibits the hexokinase reaction. A sample, which had been shown to have
457
ASSAY FOR HEXOKINASE TABLE EFFECT
OF INCREASED AND
Reactant
Tube
Glucose
B
NADP ATP G6PD 6PGA
REACTANT
6-PHOSPHOGLUCONATE
B B B C
2
CONCENTRATIONS DEHYDROGENASE
ON HEXOKINASE ACTIVITY
U/g of tissue
Percentage change
0.5 1.0
2.16” 2.00
-7.4%
0.8 1.1
6.30” 6.08h
-3.5Q
5.0 6.7
2.32” 2.16”
-6.9%
1 unit/ml 2 unit/ml
I .36” 1.28”
-5.9%
0.27 0.54
2.40’ 2.40’
Concentration (mmol/liter)
0.0%
a Activity of HK corrected for 6PGAD activity. * Activity of HK + 6PGAD. CActivity of 6PGAD.
no 6-phosphogluconate dehydrogenase activity, was assayed in the presence and absence of 0.27 mmollliter of 6-phosphogluconate. There was no inhibition; the hexokinase activity was found to be 0.48 U/g of tissue in both cases. Linen rity
The linearity of the reaction with the time, when carried out under the described conditions, is illustrated in Fig. 1. A homogenate of a squamous cell carcinoma of the lung, having relatively high enzyme activity, was assayed and readings taken every 2 min for 10 min. The linearity observed for total enzyme activity (3.34 U/g of tissue), 6-phosphogluconate dehydrogenase activity (2.32 U/g of tissue), and hexokinase activity (1.04 U/g of tissue) showed that the enzymes were being analyzed under optimum conditions over this time period. Figure 2 shows the effect on the assay of increasing the amount of enzyme. Different amounts of homogenate of a squamous cell carcinoma of the lung were added to the assay system and absorbance readings taken every 2 min for 10 min as previously described. It was found that the total enzyme activity (tube B) was linear at least up to a AAbslmin of 0.064 (enzyme activity of 5.12 U/g tissue). The 6-phosphogluconate dehydrogenase activity was linear up to a AAbs/min = 0.05 (4.0 U/g tissue), and, by difference, the hexokinase levels were linear up to a AAbsimin = 0.028
458
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.700 q
Hexoktnose+G-PGAD
0 C-PGAD a Hexokinose
600 .500 I
0
2
4
s
Incubation
FIG. I. Effect dehydrogenase.
of incubation
time on the reaction
8
IO
12
ttme,minutes
rate of hexokinase
(2.24 U/g of tissue). These are relatively measured.
and 6-phosphogluconate
high activities
for the tissues
Precision
The precision of the method was determined for within-day variability by performing 10 assays on a single tumor tissue (renal cell carcinoma) .1(X,100
uo Hexokirase+G-PGAD
i B .B $
080 -
0 6-PGAD A Hexokinase
060-
2 y
.040-
0
FIG.
2. Effect
4 002 Ob2
of enzyme
I 0.04 0.66 0.06 008 0.64 Sample volume, ml concentration
/ 0.10 o.io
on the reaction
1 012
rate.
ASSAY
FOR
TABLE WITHIN-DAY
AND DAY-TO-DAY
459
HEXOKINASE 3
PRECISION Total
OF THE HEXOKINASE
(HK)
ASSAY
enzyme” activity (U/g of tissue)
HK” activity (U/g of tissue)
Within day Number Mean SD cv
10 2.16 0.09 4.2%
IO 1.68 0.07 4.3%
Day-to-day Number Mean SD cv
12 2.76 0.45 16.2%
12 1.83 0.27 15.0%
homogenate. The mean value obtained was 1.60 U/g of tissue (SD = 0.07 U/g of tissue; CV = 4.3%), Table 3. The day-to-day variability was determined on a single frozen tissue stored at -20°C. A fresh homogenate was prepared on each day the tissue was assayed for a total of 12 determinations over a period of 2 months. The mean value obtained was 1.83 U/g of tissue (SD = 0.27 U/g of tissue; CV = IS%), Table 3. The variability shown in this data includes the day-to-day variability of the method as well as tissue site variability. The tissue homogenates were found to be unstable even when frozen at -20°C. The tissues were, therefore, stored frozen and homogenized immediately before analysis. The relatively low day-to-day precision (CV 15%) may be due in part to variability in the cellular composition of the tissue aliquots. DISCUSSION
There are several published modifications of the basic hexokinase assay procedure of Slein et al. (12) which describe a variety of reaction conditons (different buffer and substrate concentrations) in order to optimize conditions for the particular tissue being assayed (13- 17). Most of the procedures employ a blank, containing no ATP, which quantitates the endogenous NADP+ reduction. In addition, a few procedures also eliminate glucose from the blank in order to quantitate endogenous formation of glucose 6-phosphate. Several of the methods include procedures for detecting and quantitating 6-phosphogluconate dehydrogenase activity, although not for individual specimens. The assay used in this study was based on the procedure described by
460
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Salas (13), modified to take into consideration a number of potential interfering enzymes which may be found in tissue homogenates. In establishing optimum substrate concentrations the properties of several possible contaminating enzymes were considered. GPhosphogluconate
Dehydrogenase
This enzyme catalyzes reaction [IV]. The substrate for this enzyme (6-phosphogluconate) is the final product of the assay reactions for hexokinase (I, II, and III). Therefore, the presence of the enzyme in tissue can give falsely elevated hexokinase activities by reacting with 6phosphogluconate and thereby increasing the rate of NADPH production. The wide range of 6-phosphogluconate dehydrogenase activity and the lack of correlation with the hexokinase activity indicates that there is contaminating 6-phosphogluconate dehydrogenase present and that the degree of contamination is not constant and, therefore, must be corrected for each sample assayed. Several correction procedures for 6-phosphogluconate dehydrogenase contamination of hexokinase assays have been reported (13,16,17). Yeast 6-phosphogluconate dehydrogenase has been added to the sample assay mixture after the hexokinase reaction has proceeded for a certain period of time (17). If there is sufficient endogenous 6-phosphogluconate dehydrogenase present to react with all of the 6-phosphogluconate formed in the hexokinase assay reactions (I, II, III) there should be no increase in the rate of NADPH formation. In this case a factor of 0.5 has been applied to the final measured enzyme activity to correct for the contaminating 6-phosphogluconate dehydrogenase activity, since for each mole of glucose 6-phosphate formed 2 mol of NADPH are generated. The activity of the sample hexokinase and the length of time the enzyme is allowed to react before the addition of exogenous 6-phosphogluconate dehydrogenase are important factors since they will determine the amount of 6phosphogluconate present in the assay mixture. If there is no 6-phosphogluconate dehydrogenase in the sample, the lack of an increased formation of NADPH on the addition of the yeast 6-phosphogluconate dehydrogenase will be misinterpreted. Another problem arises when there is an increase in the rate of NADPH formation on the addition of yeast 6-phosphogluconate dehydrogenase. This indicates that the amount of the endogenous 6-phosphogluconate dehydrogenase is insufficient to react with all of the 6-phosphogluconate formed. It does not, however, quantitate the amount of 6-phosphogluconate which has reacted and it is, therefore, not possible to make a correction in these cases. Another technique which has been used for the detection of contaminating 6-phosphogluconate dehydrogenase activity is the substitution of
ASSAY
FOR
HEXOKINASE
461
glucose 6-phosphate for glucose as the substrate (16). Due to the excess glucose 6-phosphate dehydrogenase present in such a system most of this substrate is immediately converted to 6-phosphogluconate (reactions II and III) with the production of NADPH. When assaying hexokinase by the described procedure the rate of formation of 6-phosphogluconate is limited by the activity of the hexokinase producing the glucose 6-phosphate. Addition of glucose 6-phosphate as substrate will yield a higher activity of 6-phosphogluconate dehydrogenase than in the actual hexokinase assay, and no true measure of the 6-phosphogluconate dehydrogenase contamination is obtained. Glucose dehydrogenase. The presence of this enzyme could cause substrate (glucose) depletion in reaction [I] and an increased rate of NADPH production through reaction [V] resulting in falsely elevated measured hexokinase activities. Glucose dehydrogenase is primarily a mammalian liver enzyme but was found to be present in the commercial preparation of yeast glucose 6-phosphate dehydrogenase used in reaction [II]. It does not interfere with the assay of the low K, hexokinase being measured in this study since the concentration of glucose used (0.5 mmol/liter) is too low to elicit significant glucose dehydrogenase activity (K, for glucose 100 mmol/liter). Glucose phosphate isomerase (GPZ), EC 5.3.1.9). This enzyme catalyzes the reaction: D-glucose 6-phosphate
z
D-fructose 6-phosphate.
WI
GPI is the second enzyme in the glycolytic pathway and is present in tissue at approximately 100 times the levels of hexokinase. The effect on the assay could be through the removal of glucose 6-phosphate from reaction [II], resulting in a decrease in the overall rate of NADPH formation. The equilibrium for the reaction lies to the left (K = 0.30) and since glucose 6-phosphate dehydrogenase is added in excess the assay reaction [II] predominates over reaction [VI]. Glucose bphosphatase (G6p’tase. EC 3.1.3.9). G6P’Tase catalyzes the reaction: glucose 6-phosphate
+ H,O
G6p’tase
l
glucose + orthophosphate.
[VIII
Glucose 6-phosphatase is a second enzyme present in tissue which can act on glucose 6-phosphate, thereby decreasing the measured hexokinase activity. It is a microsomal enzyme which is, therefore, removed from the homogenate by the centrifugation prior to the hexokinase assay (18). Also as in the case of glucose dehydrogenase the excess glucose 6-phosphate dehydrogenase added to the assay system minimizes the effect of this enzyme.
462
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NADP+ cofactor enzymes. Enzymes which used NADP+ catalyze hydrogen atom transfer and react according to:
as a cofactor
substrate (red) + NADP+
enzymes -substrate
(oxid) + NADPH
+ H+.
[VIII]
The effect of endogenous reducing enzymes can be measured by utilizing a blank assay system containing no ATP, so that the only activity is due to the NADP+ reducing enzymes, tube A, Table 1. These reactions also occur in tubes B and C and are therefore automatically corrected for in the calculation of the hexokinase activity. The concentration of magnesium chloride was increased to 10 mmol/liter to give a magnesium chloride, ATP ratio of 2:l in order to prevent the inhibition of hexokinase that occurs when the molar ratio of ATP to magnesium is greater than 1 (I I). The lack of any increase in hexokinase activity when the amounts of glucose 6-phosphate dehydrogenase, and ATP, are increased (Table 2) show that with these changes the reaction is still being carried out under optimum conditions, zero-order kinetics, with respect to these compounds. The procedure presented in this study has been shown to be linear (Figs. 1 and 2) and reproducible, and accounts for the contaminating 6-phosphogluconate dehydrogenase activity in a quantitative manner. The amount of 6-phosphogluconate added is high enough to give zero order kinetics and at the same time is not inhibitory for the hexokinase reaction. The lack of correlation between the hexokinase and 6-phosphogluconate dehydrogenase levels also indicates that a common factor cannot be used, but individual correction must be made for interfering enzymes in each sample assayed. ACKNOWLEDGMENTS This work was supported in part by USPHS Grant AH ooOO2-02 and by a grant from the Cancer Association of New Orleans and was performed in partial fulfillment of the dissertation requirements for the Ph.D. degree by W. Douglas Scheer.
REFERENCES I. Knox, W. E. (1967) Cancer Res. 10, 117-161. 2. Knox, W. E., Jamdar, S. C., and Davis, P. A. (1970) Cancer Res. 30, 2240-2244. 3. Sharma, R. M., Sharma, C., Donnelly, A. J., Morris, H. P., and Weinhouse, S. (1965) Cancer Res. 24, 193-198. 4. Weber, G. (1974) in The Molecular Biology of Cancer (Busch, H., ed.) p. 487, Academic Press. New York. 5. Weinhouse. S. (1966) Gann Monograph 1, 99- 115. 6. Bar, U., Schmidt, E., and Schmidt, F. W. (1963) K/in. Wochschr. 41, 977-988. 7. Pedersen. S. N. (1975) Cancer 35, 469-474. 8. Kikuchi, U.. Sato, S., and Sugimura. T. (1972) Cancer 30, 444-447.
ASSAY FOR HEXOKINASE
463
9. Katzen, H. M. and Schimke, R. T. (1965) Proc. Nuf. Acad. Sci. USA 54, 1218-1225. 10. Katzen, H. M., Soderman, D. D., and Nitowsky, H. M. (1965)Biochem. Biophys. Res. Commun. 19, 311-382. 11. Grossbard, L., and Schmike, R. T. (1966) J. Biol. Chem. 241, 3546-3560. 12. Slein. M. W., Cori, G. T., and Cori, C. F. (1950) J. Biol. Chem. 186, 763-780. 13. Salas, M.. Vinuela. E., and Sols, A (1963) J. Biol. Chem. 238, 3535-3538. 14. Joshi, M. D., and Jagannathan, V. (1966)in Methods of Enzymology (Wood, W. A., ed.) p. 371, Academic Press, New York. 15. Shonk, C. E., and Boxer, G. E. (1964) Cancer Rrs. 24, 709-721. 16. Sharma, C., Manjeshwar, R., and Weinhouse, S. (1963)5. Bio1. Chem. 238, 3840-3845. 17. Jamdar, S. C., and Greengard, 0. (1970) J. Biol. Chem. 245, 2779-2783. 18. Walker, D. G. (1966) in Essays in Biochemistry (Campbell. P., IV, and Greville, G. D.. eds.), Vol. 2, pp. 33-62, Academic Press, New York.