Study of the bovine erythrocyte

ARCHIVES

OF BIOCHEMISTRY

AND

165, g-20

BIOPHYSICS

(1974)

Study of the Bovine Enzymatic

Activities

Erythrocyte

of the Cell and Its Membrane’,

GEORGE W. DRESDNER,3 SILVIA INES SIEGMUND-MONTEFUSCO Institute

de Bioflsica,

Uniuersidad Received

Austral

October

de Chile,

HEIN,

Vuldiuia,

’

AND

Chile

29, 1973

In bovine red cells, haemolysed and extensively washed, ten different enzyme activities were found to be present. The cells easily release glucose 6-phosphate dehydrogenase, glucose phosphate isomerase, fructose bisphosphate aldolase, and aspartate aminotransferase into the haemolysis medium. An important part of the last two enzymes and all the isocitrate dehydrogenase (NADP linked) are retained in the membrane. The levels of these enzymes in the membrane are strongly dependent on the age of the preparation. The optimal assay conditions have been defined for some of these enzymes. These findings are discussed in relation to red cell and membrane structure.

The red blood cell complex composition, particularly its enzyme composition and metabolic capabilities, are well-established (l-4). This complexity has also been shown to be present in the cell ghost (5-B). Due to this fact, the red cell membrane has been subjected to many studies in recent years in order to define its composition and structure in terms of its components, particularly protein components. Of these, enzymes have been shown to be present in important amounts. The strength of binding of proteins and enzymes to the membrane has been found to be variable. For many enzymes, their activity has been found to be in close association with membrane lipids (6, 9). For others, this has not been found to be the case. Hoogeveen et al. (10) found that about 50% of the total protein of the human red cell membrane can be solubilized using aqueous solutions, by relatively mild procedures, in the absence of calcium ions. The contribution

that these strongly bound and loosely bound proteins make to the membrane structure has not been clearly evaluated, possibly because a precise concept of the physical boundaries of the cell membrane is lacking. Many studies of the red cell proteins and enzymes have been carried out on human erythrocytes. In the present study we have centered our attention on the bovine red cell and its membrane. Studies on the bovine red cell enzymes have been carried out by Burger et al. (6) and Heller and Hanahan (11). We have tried a procedure for preparing red cell membrane using one-step haemolysis followed by washings in buffered solutions of sodium chloride of decreasing osmolarity. We have intended to characterize the membrane preparation thus obtained in terms of its morphological appearance, the electrophoretic behavior of the solubilized membrane components, and in terms of certain enzyme activities present in them. We have studied in some detail three glycolitic enzymes (glucose 6-phosphate dehydrogenase, glucose phosphate isomerase, fructose bisphosphate aldolase), isocitric dehydrogenase, and aspartic amino-

‘A preliminary report of these results was presented at the Meeting of the Sociedad de Biologla de Chile, Cartagena, Chile (1971). * This is paper I of a series. 3 Present address: Institute of Biochemistry, Uppsala University, Uppsala, Sweden. 9 Copyright All rights

0 1974 hy Academic Press, Inc. of reproduction in any form reserved.

10

DRESDNER,

HEIN

AND

transferase. We have studied the optimal assay conditions for these enzymes, their levels in the cells, the ease of their release from them after lysis and extensive washing, their levels in the membranes, and their gross distribution between cell and membrane. MATERIALS

AND

METHODS

Chemicals. All chemicals used, obtained from E. Merck A.G., Darmstadt, Germany, were reagent grade. Chemicals for enzyme assays, purchased from Sigma Chemical Company, St. Louis, Missouri, were Sigma grade. pH measurements. pH measurements were made using a T’lTlc Radiometer apparatus. Blood treatment and preparation of membranes. Blood was collected from *Holstein-Friesian cows, using potassium oxalate as anticoagulant; in some cases, heparin was used as anticoagulant. The collected blood was cooled at 2°C and treated according to the following procedure. It was centrifuged at 3500g for 20 min in a RCZ-B Sorvall refrigerated centrifuge. The cells were suspended and centrifuged twice in 0.15 M NaCl (solution No. 1). In these steps, the buffy coat was removed and discarded. The cells were then successively suspended and centrifuged in NaCl solutions with the following concentrations: 0.05, 0.04, 0.03,0.015, and 0.005 M (solutions Nos. 2-6). All NaCl solutions contained enough Tris [tris(hydroxymethyl)aminomethane] to yield a 0.005 M solution (pH 7.45). After each centrifucation step, the cells were suspended and stirred for 1 hr. All separations were carried out at 2°C. At the end of the preparative procedure, the membranes were pale and had no pink color. The benzidine test was negative. Membrane preparations were kept in solution No. 6, at 2”C, for several weeks. For each ml of packed red cells, 8.2 + 1.2 mg of wet membranes were obtained ( 14 determinations). ’ Electron microscopy. Membrane specimens both for negative staining and sectioning were used. Samples for negative staining suspended in solution No. 6 were deposited on a grid covered with a parlodion film and stained with a drop of 2% phosphotungtate, previously adjusted to pH 7.4 with NaOH, and immediately observed in the electron microscope. Samples for sectioning were fixed for 60 min in Kamovsky solution, postfixed overnight in osmium tetroxide, dehydrated in ethanol, and embedded in Epon 812. Thin sections were stained with lead citrate and uranyl acetate mixture at pH 6.8, rinsed with ‘Uncertainty values in this standard deviation from the where d is the deviation of each the mean and n is the number

work are indicated as mean c d2/(n - l), individual value from of determinations.

SIEGMUND-MONTEFUSCO water, and viewed in an EM 300 Philips electron microscope. Polyacrylamide gel disc electrophoresis. Disc electrophoresis on polyacrylamide gels, according to the procedure of Davis and Ornstein (12, 13) as modified by Schneiderman (5), was carried out in 6 M urea. Membranes were solubilized in 6 M urea at pH 7.4. During the run, a current of 5 PA per tube was used. Samples were stained with Amido Schwarz and decolorized with 7% acetic acid aqueous solution. Water content determination of membranes. Water content of membranes was determined as follows. Wet membrane aliquots were weighed in small flasks by means of an analytical Metler balance, kept at 2O”C, and frozen at -40°C in a de Virtis refrigerated bath, containing ethylene glycol monomethyl ether. They were next Iiophilized using a Virtis freeze mobile unit until complete dryness was achieved, and weighed. From these measurements, the water content of membranes was calculated. Rotein determination. Proteins were determined on membrane suspensions using the procedure of Lowry et al. (14). In order to get uniform results, membranes were subjected prior to the protein determination to ultrasound treatment using a Biosonik probe. Irradiation of samples was performed during 5 min at 20,000 Hz and 105 W. Enzyme assays. Enzyme assays were carried out on duplicate aliquots of membranes, haemolyzate, and wash NaCl-Tris solutions. For enzyme assays, membranes were collected by centrifugation at 35OOg for 30 min, the supernatant discarded, and aliquots for the assay weighed. Spectrophotometric measurements were made in l-cm-path silica cuvettes in a SP.500 Unicam spectrophotometer provided with a thermostated cell housing. Test kits used for some enzyme assays (glucose phosphate isomerase, isocitrate dehydrogenase, and aspartate aminotransferase) were purchased from Sigma Chemical Co. For all enzymes studied, tests were made to check the linearity of activity against enzyme concentration, their activity at different pH values, and the time-dependent decrease of activity in the stored membranes. Glucose B-phosphate dehydrogenase (EC 1 J.1.49). The assay was performed according to the procedure described by Liihr and Waller (15). Duplicates of the reaction mixture contained haemolyzate or amounts of membranes that varied from 10 to 70 mg. The absorbance of the mixture at 340 nm was measured at 25°C. Blanks were made replacing the NADP solution by buffer. The enzyme activity unit used was defined as the amount of enzyme that under these assay conditions oxidizes 1 nmole of glucose 6-phosphate per minute at 25°C. Glucose phosphate isomerase (phosphohenose isomerase, EC, 5.3.1.9) assay. To 2.0 ml of a prewarmed 2.5 mM solution of glucose g-phosphate (pH 7.41, 0.1 ml of sample was added and incubated at

BOVINE

RED

CELL

MEMBRANE

37°C for 30 min. In samples containing membranes, lo-40 mg of this material was used. Next, 2.5 ml of a 5% trichloroacetic acid solution were added and the sample centrifuged. To 2.0 ml of supematant fluid were added 2.0 ml of a 0.1% (w/v) resorcinol solution and 6.0 ml of 10 N HCl. The mixture was then warmed at 80°C for 15 min, cooled and the absorbance measured at 490 nm. To the blank, the enzyme was added immediately after the trichloroacetic acid. An enzyme unit was defined as the amount of enzyme that under these assay conditions forms 1 nmole of fructose 6-phosphate per hour at 37°C. Fructose bisphosphate aldolase (aldolase, EC 4.1.2.13) assay. The calorimetric procedure of Bruns and Bergmeyer (16) or a modification of that of Sibley and Lehninger (17) was used. In the latter procedure, to prewarmed samples, containing 0.70 ml of 0.05 M Tris (pH 8.6), 0.10 ml of 0.56 M hydrazine sulfate (pH 8.6), and 0.10 ml of sample, were added 0.1 ml of 0.05 M fructose 1,6-b&phosphate disodium salt. The sample was incubated at 37 “C for 30 min. Then 1.0 ml of a 10% trichloroacetic acid solution was added and centrifugation carried out. To 0.5 ml of supernatant fluid, 0.5 ml of 0.75 N NaOH was added; the mixture was allowed to stand 10 min at room temperature and 0.5 ml of a 0.1% (w/v) 2.4.dinitrophenyl-hydrazine acid solution added. After incubating at 37°C for 60 min, color was developed with 7.0 ml of a 0.75 N NaOH solution. Readings were made at 540 nm. Samples with membranes contained lo-40 mg of this material. In the blank tubes, substrate was added immediately after trichloroacetic acid. One enzyme unit was defined as the amount of enzyme that under these assay conditions forms 1 pmole of triosephosphate per hour at 37°C. Isocitmte dehydrogenase (EC 1 .l .1.41, 1 .l .1.42) assay. Prewarmed samples, containing 2.0 ml of a 3 mru isocitrate solution (pH 7.5), 0.3 mg of NAD or NADP (disodium salt), and 0.1 ml of a 0.01 M MnCl, solution (prepared in 0.15 M NaCI), were incubated in a spectrophotometer cuvette at 25°C and its absorbance measured at 340 nm for about 30 min. When used, membranes in amounts of lo-50 mg were added. Blanks were made with membranes preheated at 100°C for 5 min. An enzyme unit was defined as the amount of enzyme that under these assay conditions forms 1 nmole of reduced NAD or NADP per hour at 25°C. Aspartate aminotransferase (glutamic-oxaloacetic transaminase, EC 2.6.1 .I) assay. The absorbance at 340 nm of a mixture containing 0.5 ml of a 0.2 M L-aspartate solution (pH 7.5), 200 units of malate dehydrogenase, and 0.2 mg of reduced NAD was read and then 0.2 ml of a 0.1 M a-ketoglutarate solution (pH 7.5) added and the absorbance measured for about 30 min at 25°C. The test cuvette contained lo-30 mg of membranes or haemolyzate solution. A blank without enzyme was used as reference. One

11

ENZYMES

enzyme unit was defined as the amount of enzyme that under these assay conditions converts 1 pmole of aspartate per minute at 25°C. Other enzyme assays. Other enzyme activities besides those already described were detected in the membranes. Acetylcholinesterase (acetylcholine hydrolase, EC 3.1.1.7) was determined using the procedure of Rappaport et al. (18). Malate dehydrogenase (L-malate: NAD oxidoreductase, EC 1.1.1.37) was determined using the procedure of Hohorst (19). Alkaline and acid phosphatases (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1, 3.1.3.2) were determined using the procedure of Bessey et al. (20). Creatin kinase (ATP: creatine phosphotransferase, EC 2.7.3.2) was determined using the procedure of Bemt and Bergmeyer (21). RESULTS

Electron Microscopy An electron micrograph of a thin section from a pellet of bovine red cell membranes is shown in Fig. 1. Membrane fragments constitute vesicles of about 100-400 nm diam. The unit membrane is apparent in many of these microvesicles. In Fig. 2 is shown a magnification of the same preparation, where the unit membrane is clearly seen. The thickness of the membrane is loo-120 A. A negatively stained membrane vesicle can be seen in Fig. 3. A well-defined envelope loo-150 A thick, corresponding to the membrane, surrounds the vesicle. Its interior has a microalveolar structure. Disc Electrophoresis of Solubilized Membranes Polyacrylamide gel disc electrophoresis patterns of solubilized red cell membranes showed the presence of at least 16 bands (Fig. 4). These results agree with those found by Schneiderman (5) in human erythrocytes and Lenard (22) in erythrocytes of several animal species. The complexity of electrophoresis patterns should be related to the complex enzyme composition of these membranes (see below). Dry Weight and Protein Content of Membranes The water content of wet membranes was found to be 94 * 0.3% in 13 determinations. 4 Proteins represented 74.3% of the dry weight.

12

DRESDNER,

FIG. 1. Electron embedded in Epon

micrograph and stained

HEIN

AND

SIEGMUND-MONTEFUSCO

from a thin section of a pellet of bovine erythrocyte with lead citrate and uranyl acetate. Magnification:

membranes, 108,500.

FIG. 2. Electron micrograph from a thin section of a pellet of bovine erythrocyte membranes, embedded in Epon and stained with lead citrate and uranyl acetate. The unit membrane is clearly apparent. Magnification: 126,900.

EEnzyme Activities

of the Membranes

T ‘he following enzyme activities were acetylcholindetc acted in the membranes: este rase, acid phosphatase, alkaline phos-

isophatase, aspartate aminotransferase, citrate dehydrogenase, malate dehydrc lgenase, creatine kinase, glucose 6-phosp Ihate dehydrogenase, phosphohexoseisoma and aldolase. Of these, some were sub-

BOVINE

jetted below. study.

RED

to the more detailed study Other enzymes are now Assay

CELL

MEMBRANE

shown under

Conditions

In determining the assay conditions for some enzymes, freshly prepared membrane preparations were always used. Enzyme activities have been reported with respect to wet weight of membranes. The maximum time period that elapsed between the preparation of the membranes and the test was usually 14 hr. Glucose &phosphate dehydrogenase. In Fig. 5 is shown the relationship between activity and the amount of membranes used in the assay. It can be seen that this is a linear one. This was also found to be the case when the test was made with haemolyzate. Homogenization of the membranes in 0.05 M triethanolamine buffer (pH 7.5), with a Potter-Elvehjem tissue grinder, reduced to less than half the enzyme activity. This effect was also detected in solutions of the enzyme in the same buffer. This shows that the stability of the enzyme to shearing forces is low and is the same in particulate and soluble form. The effect of pH was

13

ENZYMES

studied on the stroma between pH 7.7 and 10.2 and showed a maximum of activity near pH 8.0 (Fig. 6). Glucosephosphate isomerase. The time course of the reaction was studied with samples containing 30 mg of membranes and was found to be linear within a 2-hr period. The reaction was also linear with respect to the amount of membranes present in the assay tube (Fig. 5). The effect of pH was studied between pH 6.0 and 8.6. Maximum activity was found near pH 8.0 (Fig. 6). Fructose bisphosphate aldolase. A linear relationship was found between the activity of this enzyme and the amount of membranes present in the assay mixture (Fig. 5). Activity curves, pH-dependent, were different whether collidine buffer or Tris-Cl was used (Fig. 7). With collidine buffer, maximum activity was near pH 7.0; with Tris-Cl buffer, there was a maximum of activity near pH 9.0 and a minimum near pH 7.0. Isocitrate dehydrogenase. This enzyme was found to be very unstable. It was found that both NADand NADP-dependent dehydrogenase activities were present in

FIG. 3. Electron micrograph from bovine erythrocyte phosphotungstic acid. A microvesicle similar to those envelope is 100-150 A thick. Magnification: 153,900.

membranes seen in Figs.

negatively 1 and Fig.

stained with 2 is shown. Its

14

DRESDNER,

the cell membrane. carried out on the zyme, as the major associated with it.

HEIN

AND

SIEGMUND-MONTEFUSCO

Most of the work was NADP-dependent enpart of the activity was Activity of the NAD-

FIG. 4. Polyacrylamide electrophoresis gel of bovine red cell membrane components stained with Amido Schwarz. For experimental details, see text.

AMCWT

CF

dependent dehydrogenase was found to be about 15% of the total activity at pH 7.0 and at pH 6.2. No activity was found in the haemolyzate or in washing solutions in any of the experimental conditions described below. The specific activity of membranes prepared from blood samples obtained either with potassium oxalate or heparin was the same. What follows refers to the NADP-linked enzyme. The presence of activity in the membranes strongly depended on the duration of the washing procedure. “Crude membranes”, i.e., those obtained directly from the haemolyzate, showed enzyme activity in all cases examined. Membranes prepared according to the procedure described in Materials and Methods and stirred in solution No. 6 for 12 hr at 2°C had enzyme activity in about half of the cases. A total of 24 separate blood samples, obtained from 12 animals, were analyzed. When stirring was carried out for 24 hr at 2”C, the membranes only showed enzyme activity in samples obtained from three of the animals. Samples collected from the same animal did not always show enzyme activity after being stirred in solution No. 6. The average values for “crude”, 12-hr-, and 24-hr-washed membranes were, in units per mg of wet membranes, 2.00,0.27,

MEMBRANE

IN ASSAV

(mg)

5. Relationship between enzyme activity and bovine red cell membrane concentration in assay mixture. A separate ordinate axis is given for each enzyme; (@) glucose 6-phosphate dehydrogenase (G6PD); (0) glucosephosphate isomerase (GPI); (W) fructose bisphosphate aldolase (FDPA); (0) isocitrate dehydrogenase (ICD); (A) aspartate aminotransferase (AAT). For assay conditions and units used. see text. FIG.

BOVINE

RED

CELL

MEMBRANE

15

ENZYMES

P”

FIG. 6. Relationship

between activity of bovine red cell membrane enzymes and pH. A separate ordinate axis is shown for each enzyme; (0) glucose 6-phosphate dehydrogenase (GGPD); (0) glucosephosphate isomerase (GPI); (A) aspartate aminotransferase (AAT).

and 0.17, respectively. A linear relationship was found between the enzyme activity and the amount of membranes present in the assay medium (Fig. 5). Preincubation of the stroma in water for 10 min at 25 “C! decreased its enzyme activity to onethird, whereas preincubation in buffer did not modify it. Assays made at pH values between 7.0 and 8.0 showed a small variation of the enzyme activity, being maximum at pH 7.5 (Fig. 7). Assays at higher pH values could not be performed due to insolubility of manganase ions and precipitate formation. Aspartate aminotransferase. A linear relationship was obtained between enzyme activity and the amount of stroma present in the assay (Fig. 5). No change in the enzyme activity was found after homogenization of the sample. The activity, studied between pH 6.2 and 9.5, showed a maximum value near pH 6.8 and a minimum near pH 8.2 (Fig. 6). Enzyme Activities

FDPA

ICD

~ 0.25.6.~ 2

.,, ’

50

7.0

5.0

9.2

100

P”

FIG. 7. Relationship between enzyme activity of bovine red cell membrane enzymes and pH. A separate ordinate axis is shown for each enzyme; (U) fructose bisphosphate aldolase (FDPA) in collidine buffer; (0) fructose bisphosphate aldolase in Tris-Cl buffer; (0) isocitrate dehydrogenase (ICD).

in Stored Membranes

Most of the activities of these enzymes decreased in the membranes stored in solution No. 6 at 2°C. Observations were made over a period of 18 days (Fig. 8). The rates of decrease of their activities were, however, variable. Isocitrate dehydrogenase was the most labile enzyme and aspar-

tate aminotransferase the most stable one. It should also be observed that the rates of inactivation of the glycolytic enzymes are very similar and differ clearly from those shown by the other two enzymes. The half-life for glycolytic enzymes fluctuated between 6 and 12 days, whereas that of

16

DRESDNER.

HEIN

AND

SIEGMUND-MONTEFUSCO

isocitrate dehydrogenase was less than 1 day and that of asparate aminotransferase was found to be around 23 days. That the decay of enzyme activity of the membranes was not due to enzyme solubilization during storage in solution No. 6 is shown in Table I. It can be seen there that the amounts of solubilized glucose phosphate isomerase and aldolase are negligible during the observation period. No isocitrate dehydrogenase activity was ever detected in solution No. 6. Aspartate aminotransferase controls were not done since 100% of this enzyme activity was present in the stroma during longer observation periods . Controls were made in order to see

whether the decrease of enzyme activities observed was due to bacterial growth during storage of the membranes in solution No. 6. Bacterial growth was detected in stored membranes from 1 day on after the preparation. A simultaneous check was done with membranes stored in solution No. 6 added with thymol. No bacterial growth was detected after 18 days of observation under these conditions. The effect of thymol on enzyme activities was also checked. It was found that thymol did not effect enzyme activities studied nor increased the solubilization of the enzyme from the stroma. Therefore the decrease of enzyme activities observed in the days following the preparation of membranes was not due to bacterial growth. Release of Enzymes During Haemolysis and Washing All the glycolytic enzymes studied and aspartate aminotransferase were almost completely released from the red cells during haemolysis. Their release was virtually complete after the first step of the haemolysis and washing procedure here used (Table II). No isocitrate dehydrogenase activity was detected in the haemolyzate and wash solutions.

FIG. 8. Variation of the activities of enzymes in bovine red cell membranes stored at 2°C in solution No. 6. Activities are represented as percent values of those of the freshly prepared membranes; (0) glucose 6-phosphate dehydrogenase; (0) glucose phosphate isomerase; (W) fructose bisphosphate aldolase; (0) isocitrate dehydrogenase; (A) aspartate aminotransferase. For further explanation, see text.

Enzyme Contents of the Red Cell and Its Membrane A comparison of the total enzyme content of the red cell and its membrane is shown in Table II. It is clear that in all

TABLE ENZYME

ACTIVITIES

PRESENT

IN BOVINE

RED

CELL

I

MEMBRANES

AFTER

STORAGE

IN SOLUTION

No. 6 AT 2°C

Enzyme activity (percent with respect to freshly prepared membranes) Enzyme

Age of preparation (days)

Glucose g-phosphate dehydrogenase Glucose phosphate isomerase Fructose bisphosphate aldolase a Separated

from

solution

No. 6 by centrifugation

Membranes”

Solution

No. 6

10

28

0

11

18

3.5

11

23

2.7

at 35OOg for 30 min.

BOVINE

RED

CELL

MEMBRANE

TABLE AMOUNTS

OF ENZYMES

PRESENT IN WHOLE

Activity

Haemolyzate Solution No. 3 Solution No. 4 Solution No. 5 Solution No. 6 Total in haemolyzate and wash solutions Total in membranes Total in cell Percent of total in membranes

(enzyme

17

ENZYMES

II RED CELL AND THE CELL MEMBRANP

units/ml

packed

cells)

Glucose 6phosphate dehydrogenase

Glucose phosphate isomerase

Fructose bisphosphate aldolase

1,115 10 1 0 0 1,126

152,000 837 876 761 84 154,558

112.4 0.4 0.4 0.2 0.2 113.6

0 0 0 0 0 0

352.00 1.40 0.11 0.05 0.06 353.62

90

1.7

15

3.50

115.3 1.5

15 100

357.12 0.96

0.5 1,126.5 0.04

154,648 0.06

Isocitrate dehydrogenase

” Tris-Cl (pH 7.45) contributes in 0.005 to the total molarity of the haemolyzate solutions, and NaCl the rest. For experimental details see the text. TABLE COMPARISON

Enzyme Glucose 6-phosphate dehydmgenase Glucose 6-phosphate isomerase Fructose bisphosphate aldolase Isocitrate dehydrogenase Aspartate aminotransferase

For

a The difference the calculation

OF ENZYME

Aspartate aminotransferase

and the numbered

wash

III

LEVELS PRESENT IN THE BOVINE RED CELL AND ITS MEMBRANE”

Enzyme level of cell interior (units/ml) 1,126

Enzyme level of cell membrane (units/ml)

Enzyme level ratio (cell interior/ membrane)

58

154,558

10,600

114

211

0

1,820

354

424

between the total cell volume and the membrane volume is designed of its volume, a density of 1.0 was used for the membranes.

cases, except for isocitrate dehydrogenase, the amount of enzyme remaining in the membrane is not greater than 2% of the total. But unless the reduced volume of the membrane (compared with the total cell volume) is considered (less than l%), this data does not provide an accurate picture of the enzyme levels in both the membrane and the cell interior. In Table III enzyme

17.9 14.6 0.54 0 0.84

as cell interior

volume.

concentrations are separately given for the cell interior and the membrane. There are also shown the ratios of the levels found in both cell interior and membrane. It is clear that glucose 6-phosphate dehydrogenase and glucose phosphate isomerase are more concentrated in the cell interior-whatever remains of the cell after excluding the cell membrane-, whereas fructose bisphos-

18

DRESDNER,

HEIN

AND

phate aldolase and aspartate aminotransferase are equally distributed in the cell interior and the membrane. Isocitrate dehydrogenase seems to be exclusively located in the cell membrane. The use of a density of 1.0 for the packed membranes in these calculations is justified since as mentioned above the determined water content of the wet membranes was found to be 94.4 f 0.3%. DISCUSSION

The bovine red cell membrane preparation obtained in this work shows a large complexity, as also found in other cases (22). Evidence for this is provided by the gel electrophoresis patterns obtained from the solubilized membranes in 6 M urea that show at least 16 bands. A large number of enzyme activities can also be measured directly from them. Due to this variability in the number of components and to the proved existence of genetic variants for many of these enzymes, we have limited ourselves to work with a particular breed of animal. Another variable that we had to consider when using this membrane preparation was the fact that the detected enzyme activities decreased with time under storage. The rate of decrease was found to be different for each of the studied enzymes. In general, it would seem necessary that this fact be taken into account for any quantitative estimate of enzyme activities of these membranes and for any purpose of comparing results. However, the importance of this fact seems not to have been adequately stressed in similar studies made with other membrane preparations. Results of similar nature on the decrease of enzyme activities, but in stored whole human erythrocytes, have been described by Lijhr and Waller (1). The rate of enzyme inactivation they found for glucose 6-phosphate dehydrogenase was higher than that found in this study for the corresponding isozyme. The rate of inactivation of aldolase was comparable to that found here. The shape of the decay curves found by them showed a downward concavity rather than an upward one, as found in our case, and a very sharp decrease of activity be-

SIEGMUND-MONTEFUSCO

tween the 7th and 10th day after the collecting of the blood samples. It is difficult, however, to compare both cases since enzymes were stored under very different conditions and also isozymes from different species are involved. A comparison with goose erythrocyte enzymes reveals that, grossly considered, storage decreases aldolase, glucose B-phosphate dehydrogenase, and aspartate aminotransferase activities in a similar way as we found in bovine red cells, but isocitrate dehydrogenase in goose erythrocytes, when compared with its bovine counterpart, is not only soluble, but shows a much greater stability. Our results with respect to the release of enzymes are similar to those of Burger et al. (6). They found that lipids, acetylcholinesterase, aldolase, and glucose 6-phosphate dehydrogenase are bound to a variable extent to the bovine red cell membrane. In our case, we found that glucosephosphate isomerase and glucose 6-phosphate dehydrogenase are scarcely retained in the cell membrane during the preparative procedure. Aldolase and aspartate aminotransferase, however, are partly released and partly retained in the membrane. Isocitrate dehydrogenase is completely retained in the membrane. The behavior of this enzyme is different also to what has been found in the human and, as mentioned above, goose erythrocyte, where it is released from the cell in large amounts during haemolysis (1). Evidence for the variable behavior of membrane-bound enzymes with respect to their firmness of binding to red cell membrane has been given by the work of Wins and Schoffeniels (23) on pig red cell oxidoreductases. Of the enzymes studied, they found the highest intensity of association for glyoxylate reductase and the lowest for triosephosphate dehydrogenase. Enzymes like NADH demalate dehydrogenase, hydrogenase, NADPH dehydrogenase, and lactate dehydrogenase showed an intermediate behavior. It is apparent also that significant differences exist between the bovine and human red cell. In addition to what has been mentioned with respect to isocitrate dehydrogenase, other facts should be added.

BOVINE

RED

CELL

MEMBRANE

Burger et al. (6) demonstrated that they show different divalent-cation requirements for membrane stability, different solubilization characteristics of acetycholinesterase, and different release characteristics of haemoglobin. Therefore, besides some similarities that exist between red cell membranes from the two species, they show important differences that must be considered when the erythrocyte membrane from a particular species is studied. From data of Table III it is apparent that, under the experimental conditions used in this work, enzymes distribute unevenly between the cell membrane and the cell interior. The expression “cell interior” is used here to refer to that part of the cell volume that remains after excluding the cell membrane. The membrane, in turn, can be isolated by centrifugation after haemolysis. It is apparent then that glucose 6-phosphate dehydrogenase and glucose phosphate isomerase have in the membrane a much lower concentration than in the intact cell. This necessarily requires that they be preferently distributed in the cell interior and very little in the membrane. Aldolase and aspartate aminotransferase seem, on the basis of the same criterion, to be evenly distributed between the cell membrane and the cell interior. And isocitrate dehydrogenase seems to be present only in the membrane and none inside the cell. These data suggest that some sort of arrangement might exist for these enzymes in the cell, which with the available data can only be grossly described in terms of a preferred location either in the cell membrane or in its interior. This distribution would be in agreement with the idea that has been, since long, postulated by some authors (24) about a vectorial assembly of enzymes and its role in defining the direction of substrate utilization within the cell. A question that arises is whether or not this enzyme distribution represents a true distribution, i.e., one found in the intact cell. This question cannot at present be answered with the experimental evidence here available. The possibility exists that these enzymes could become attached to the membrane on lowering the ionic

ENZYMES

19

strength of the cell interior during hypoosmotic haemolysis. However, due to the fact that we have used an exhaustive washing procedure, in which no traces of haemoglobin are left in the membranes, the membranes must have been depleted to a large extent from their components (25), particularly those loosely bound. Therefore, the remaining components might exist attached to the membrane because they do really exist as such in the intact membrane. The alternate possibility should also be considered: that the enzymes found in the haemolyzate do exist attached to the cell membrane in the intact cell and that their release occurs as a consequence of the ionic strength decrease caused by haemolysis. This possibility cannot be excluded and it is supported by the work of Green et al. (26). Another point that seems important to discuss here is the contribution that these enzymes can make to the cell membrane structure. This question is relevant since, even when for the activity of many membrane-bound enzymes has been found necessary the presence of membrane lipids (6, 9), no definite proof exists yet of a specific protein associated to the lipids that contributes to form the membrane structure as revealed for instance by X-ray diffraction data. On the other hand, since no definite physical limit has yet been proposed for the cell membrane, the role played by the so-called loosely bound enzymes remains to be defined. The work of Hoogeveen et al. (10) shows that an important part of membrane proteins are loosely bound watersoluble proteins. Due to the similarity of the washing procedures used in this work and the work of Hoogeveen et al., it is likely that some of the components of the soluble protein fractions I and II they described might correspond to some of the enzymes activities here found. It remains to be shown, however, whether or not these enzymes play an essential role in the maintenance of the membrane structure. They might just be enzymes ionically bounded to the membrane skeleton but not associated to the membrane lipids. This, however, would not

20

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HEIN

AND

exclude their physiological significance, for instance, in the utilization of particular metabolites or in their transport inside the cell. ACKNOWLEDGMENTS This work was partially supported with a grant from FORGE (Fund for Overseas Research Grants and Education), U.S.A. We thank Dr. Alfred0 Zamora, from the Institute of Morphology, for his valuable help in getting the electron micrographs, to the Faculty of Agronomy and to the Faculty of Veterinary Science, Universidad Austral de Chile, for the supply of blood used in this study, and to Mrs. Aurora Kucera-Poo for some aldolase determinations.

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SIEGMUND-MONTEFUSCO 10. HOOGEVEEN, J. TH., JULIANO, R., COLEMAN, R., AND ROTHSTEIN, A. (1970) J. Membrane Biol. 3, 156. 11. HELLER, M., AND HANAHAN, D. (1972) Biochim. Biophys. Acta 255, 239. 12. ORNSTEIN, L. (1964) Ann. N.Y. Acad. Sci. 121, 321. 13. DAVIES, B. J. (1964) Ann. N. Y. Acad. Sci. 121,350. 14. LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L., AND RANDALL, R. J. (1951) J. Biol. Chem. 193, 466. 15. L~~HR, G. W., AND WALLER, H. D. (1968) in Methods of Enzymatic Analysis (Bergmeyer, H.-U., ed.), second printing, p. 744, Verlag Chemie, Weinheim. 16. BRUNS, F. H., AND BERGMEYER, H.-U. (1968) in Methods of Enzymatic Analysis (Bergmeyer, H.-U., ed.), second printing, p. 724, Verlag Chemie, Weinheim. 17. SIBLEY, J. A., AND LEHNINGER, A. L. (1949) J. Biol. Chem. 177, 859. 18. RAPPAPORT, F., FISCHL. J., AND PINTO, N. (1959) Clin. Chim. Acta 4, 227. 19. HOHORST, H.-J. (1968) in Methods of Enzymatic Analysis (Bergmeyer, H.-U., ed.), second printing, p. 328, Verlag Chemie, Weinheim. 20. BESSEY, 0. A., LOWRY, 0. H., AND BROCK, M. J. 1946) J. Biol. Chem. 164,321. 21. BERNT, E., AND BERGMEYER, H.-U. (1968) in Methods of Enzymatic Analysis (Bergmeyer, H.-U., ed.), second printing, p. 859, Verlag Chemie, Weinheim. 22. LENARD, J. (1970) Biochemistry 9, 5037. 23. WINS, P., AND SCHOFFENIELS, E. (1969) Biochim. Biophys. Acta 185, 287. 24. MITCHELL, P. (1963) Biochem. Sot. Symp. Cambridge Engl. 22, 142. 25. TISHKOFF, G. H., ROBSCHEIT-ROBBINS, F. S., AND WHIPPLE, G. H. (1953) Blood 8, 459. 26. GREEN, D. E., MURER, E., HULTIN, H. O., RICHARDSON, S. H., SALMON, B., BRIERLEY, G. P., AND BAUM, H. (1965) Arch. Biochem. Biophys. 112, 635.