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Red blood cell AMP-deaminase: Levels of activity in hemolysates from twenty different vertebrate species
Corap. Biochera. Physiol., 1973, VoL 46B, pp. 653 to 660. Pergamon Press. Printed in Great Britain
RED BLOOD CELL AMP-DEAMINASE: LEVELS OF ACTIVITY IN HEMOLYSATES FROM TWENTY DIFFERENT VERTEBRATE SPECIES WALTER C. KRUCKEBERG and OSCAR P. CHILSON Department of Biology, Washington University, St. Louis, Missouri 63130, U.S.A. (Received 19 March 1973)
Abstract--1. The levels of adenylate deaminase activity have been measured in the erythrocyte hemolysates and hemolysate supernatants from twenty different vertebrate species. 2. The chicken erythrocyte hemolysates proved to have the highest level of AMP-deaminase activity, with birds in general showing more enzyme activity than the other nucleated red cell types and the non-nucleated red blood cell hemolysates showed low levels of the enzyme activity. 3. A developmental difference in red blood cell adenylate deaminase activity was discovered between 1-day old and adult ducks and was confirmed between 1-day old and adult chickens. INTRODUCTION ON A superficial level mature vertebrate red blood cells present a deceiving illusion of homogeneity in structure, function and molecular constitution. However, subtle and gross differences do exist. On the morphological level, erythrocytes from birds, amphibians, reptiles and fish (with one exception, Bell, 1971) have been shown to contain nuclei, while their mammalian counterparts are anucleate. On the metabolic level it has been well established that avian erythrocytes demonstrate a functional citric acid cycle whereas mammalian red cells are able to metabolize primarily, if not solely, anaerobically (Bishop, 1964). Apart from hemoglobin, red blood cells have been shown to contain a wide variety of catalytic and non-catalytic constituents (Rapoport & Guest, 1940; Bishop, 1964; Bartlett, 1970) whose quantities fluctuate (perhaps significantly) from species to species and whose individual and related functions are presently open to investigation. These non-hemoglobin constituents may function in a variety of ways; in controlled maintenance of red cell integrity (Williamson, 1970), in modification of the oxygen-binding capability of hemoglobin (Brewer & Eaton, 1971), in the regulation of erythrocyte development (Marks & Kovach, 1966) and in the transporting of molecules other than oxygen (Henderson & LePage, 1959; Murray, 1971 ; Elwyn et al., 1972). 653
654
WALTER C. I4muc~n~G ANDOSC~mP. CHIL$ON
Variations in purine nucleotide metabolism have been documented between red cells from two different species. Inosine-5'-monophosphate (IMP), the first complete purine nudeotide in the de novo pathway for purine synthesis, is the precursor for both guanine and adenine nucleotides. Lowy et al. (1962) have shown that rabbit erythrocytes readily convert IMP to adenosine-5'-monophosphate (AMP), while the degree of conversion of this compound to adenine nucleotides by human red cells is negligible. Mager et al. (1966,1967) further documented that guanosine and adenosine phosphates rapidly turn over in human and rabbit erythrocytes and also produced data consistent with the concept that the dynamic levels of these purine nucleotides are maintained by the reutilization of preformed purines rather than by the de novo synthesis of the purine ring from glycine and formate. Adenosine-5'-monophosphate deaminase, the enzyme of major interest in this report, catalyzes the effectively irreversible hydrolysis of the purine nucleotide 5'-AMP to IMP and ammonia. AMP-deaminase was first found in human and rabbit erythroeytes (Conway & Cooke, 1939) and has been subsequently identified and characterized in human, dog and cat blood cells (Askari, 1965, 1968). These current research efforts, done with mammalian cells, as well as our finding that nucleated adult chicken red blood cells contain twenty times more deaminase activity than either anucleate human or rabbit erythrocytes (Henry & Chilson, 1973), prompted us to begin the work reported here. The objective of this work is to quantitate and compare the levels of AMPdeaminase activity in the red blood cell lysates of twenty species of vertebrates. It is hoped that this information will aid in the design of experiments to more completely identify the role of deaminase in red cell metabolism, and thus provide part of the background necessary for a more comprehensive understanding of nucleotide metabolism, as a whole, in red blood cells. MATERIALS AND METHODS
Washed and chilled blood cells from duck (Arms sp.), goose (Anser domesticus), turkey (Meleagris gallopavo), sheep (Ovls aries), goat (Capra hircus) and cat (Fells domestica) were obtained in Modified Alsever's solution from Colorado Serum Company, Denver, Colorado. Whole blood from cattle (Bos taurus), horse (Equus cabaUus), dog (Canisfamillaris) and adult White Leghorn chickens (Gallus domesticus) was obtained from animals housed at the Ralston Purina Research Farm, Gray Summit, Missouri. Adult wild mallard ducks (Anas platyrhynchos) and duck eggs were obtained from the St. Louis Zoo. Turtles (Chrysemys sp.) and frogs (Rana catesbeiana) were bought from Nasco Corporation, Ann Arbor, Michigan. Chicken eggs (White Leghorn) were obtained from Ken-Roy Hatchery, Berger, Missouri. Rats (Mus norveglcus albinus) and rabbits (Lepus cuniculus) were bought from Isaacs Lab Stock, Litchfield, Illinois. Mice (Mus musculus), hamsters (Mesocricetus auratus) and gerbils were taken from stocks maintained in the Washington University Biology Department. Dog blood was obtained through Dr. P. Hartroft, Washington University Medical School. Human blood was taken from local laboratory personnel. The birds, turtles and most of the small mammals were bled by decapitation; mice were bled from the femoral vein after anesthetization. Dogs and humans were bled by venipuncture, frogs by heart puncture.
RED BLOOD CELL AMP-DEAMINASE
655
During collection, whole blood was combined with Alsever's solution containing heparin. Cells (buffy coat included) were washed in Alsever's three times by centrifugation at 1000g for 10 mln at 4°C; the final pellet of cells was then resuspended in Alsever's solution. After taking an aliquot for counting and packed cell volume, the bulk of the stock cell suspension was lysed by quick freezing at -70°C, subsequently to be thawed and the hemolysate assayed. Hemolysate supernatants were recovered from centrifuged (30,000 g, 35 rain, 4°C) hemolysates. All blood samples were routinely stored frozen. The enzyme activity levels were found to be stable by assaying selected cell hemolysates within 1.5 hr of collection and noting no increase or decrease in activity when compared to subsequent re-assaying over a 2-7 day period (in many cases longer). Where possible, blood was obtained from more than one source. Hence it was found that the deaminase activities of 1-day-old chick and adult duck blood cells received through the mail from Colorado Serum Company were within 20% of activity levels of cells from chicks hatched in our laboratory and with duck blood from animals obtained from the St. Louis Zoo. No effort was made to specifically exclude white blood cells or thrombocytes, nor to fractionate the red cells in any way during the washing process. Also, although healthy animals were used as donors, no consistent consideration was given to possible variables in deaminase activity levels such as diet, seasonal variation and/or specific age. Enzyme activity was assayed using the method previously described (Henry & Chilson, 1969). Each lysate type was assayed at saturating substrate concentrations as demonstrated by measurement at at least two substrate concentrations. Hemoglobin was quantitated in hemolysate supernatants using a method as described by Miale (1967). Freshly prepared solutions of bovine hemoglobin were used as a standard. Cells were counted in a Levy-Hauser hemocytometer after having been diluted with Alsever's solution (Kabat & Meyer, 1961). Packed cell volumes were done by centrifuging blood containing 75 m m x 1 "1-1'2 mm microhematocrit capillary tubes at 3000 g for 15 rain in a Clinical model International centrifuge. All chemicals used were of reagent grade and were obtained from sources as reported by Henry & Chilson (1973). RESULTS
T h e results of assaying A M P deaminase activity in uncentrifuged red blood cell hemolysates from twenty different animal species are shown in Fig. 1 and Table 1. Enzyme activity is initially expressed on a cell count (gM NH3/109 cells per rain) basis and on a packed cell volume (#M NHa/ml cells per rain) basis (columns A and B respectively in Fig. 1). T h e results of adult red cell enzyme activity expressed on a cell count basis (Fig. 1, column A) can perhaps best be stated as follows: frog and chicken show the highest levels of activity with the nucleated cells (i.e. bird, frog, turtle, carp) showing significantly higher overall activities than the non-nucleated (mammalian) cells. On the basis of packed cell volume (Fig. 1, column B) the chicken again showed the highest levels of enzyme activity with birds in general indicated to have higher activities than the other nucleated cells. With the exception of human, mouse, hamster and perhaps rabbit, the mammals have the lowest levels o f deaminase activity. T h e average percentage r e c o v e r y of deaminase activity in 30,000 g hemolysate supernatants from all cell types was 77 per cent or more (Table 1). In no individual
4
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128.2 + 16"1 19.2 _+ 5.8 70-2 _+11.0 9"3 35"7 44.4 71-0 19"1 13.9 _+ 9.5 24"2 _+ 2"7 3"7 _+ 0"8 7"3 + 5"0 10"3 + 4"8 0"9 + 0.1 0.8 _+ 0-3 0.9 1-2 _+ 0.2 2-1 _+ 0.5 2"1 + 0.5 7.25_+ 1.10 0"80 18.6 -+ 2.2
6 1 4 . 1 1 1 1 1 4 3 4 3 1 3 1 5 2 5 2 1 5
No. of samples
Abbreviation: S.D. = Standard Deviation. * Airman & Dittmer (1964). t T w o separate groups of 1-day chicks--total of twenty chicks. $ One group of 1-day ducks--total of three ducks. § T hr e e separate groups of adult m i c e - - t o t a l of twenty-eight mice.
Adult chicken 1-Day chick t Adult duck 1-Day ducks Goose Turkey Pigeon Carp Turtle Frog Rabbit Human Mouse§ Horse Cow Sheep Dog Cat Rat Gerbil Goat Hamster
No. of samples
Mean-+ S.D. (/~M N H a / m l cells per m.ln)
. 97 78 83 100 98 88 96 88 92 78 97 77 91 78 88 83 100 82
89 97 88 .
Per cent recovery in supernatant (average value)
.
--
0"23 -0"56 0"06 -0-04 0.06 0"08 0"22 -0.02 0.03 0-02 0.04 0-03 -0"04
1.0 ---
(~M N H a / m l cells per rain) + cell volume
Mean
--
160 -131 311 -670 61 97 49 -50 31 66 57 61 -19
127 ---
Cell volume (~8),
DEAMINASE ACTIVITIF-S I N RED BLOOD CELL HF.MOLYSATK~ AND HEMOLYSATR SUPI~,NATANTS FROM TWENTY D I ~ SPECIRS
No. of samples
AMP
Mean_+ S.D. (#M NHa/109 cells per re_in)
T A B L E 1 - - S u M M A R Y OF
o b Z
.~
z ©
.~
.a
:~
o~ Oi o~
-
RED BLOOD
I'1
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657
CELL AMP-DEAMINASE
C
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Ul ?
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3
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FIG. 1. Summary of the AMP deaminase activities in red blood cell hemolysates and hemolysate supernatants from twenty different vertebrate species. The data
are displayed in three ways: A, hemolysate enzyme activity on a cell count basis (#M NHs/10gcells per minx 10-1); B, hemolysate enzyme activity on a packed cell volume basis ~ M NHs/ml cells per rain); and C, hemolysate supernatant activityon a mg hemoglobin basis (~M NHs/mg Hb per minx 10-2). Each column defines the mean enzyme activity. The short vertical line present in most of the columns defines_+ S.D. The absence of the standard deviation indicates that only one sample was assayed. The numbers of samples used to compute the standard deviations are given in Table 1. It is important to note that enzyme activity is represented on the ordinate as a log plot. case was recovery less than 77 per cent indicating that with the conditions used here, the major part of the enzyme activity is not tightly bound to the cell stroma. Expressing supernatant deaminase activity on a "per mg hemoglobin" basis (Fig. 1, column C), a familiar pattern of relative enzyme activities emerges: the chicken supernatant shows the highest amounts, birds in general show more enzyme activity than the other nucleated cell type supernatants, and the non-nucleated cell type supernatants have 10w levels of deaminase. Henry & Chilson (1973) first reported a marked difference between the A M P deaminase levels in 1-day-old chick and adult chicken blood cell hemolysates (confirmed, Table 2). In order to determine if domesticated chickens were unique in showing this developmental difference in enzyme activity, blood cells from adult and 1-day-old wild mallard ducks were similarly assayed. A developmental difference was also observed with these ducks (Table 2). In order to test for the presence of possible endogenous dissociable inhibitors which could modulate enzyme activity in a red cell hemolysate and give an illusion of species to species differences, certain representative supematants were combined and additivity of deaminase activity monitored within 3 hr of combining and up to a week after, samples having been stored frozen. In all eases studied, namely, 1-dayold chick : adult chicken, rabbit : duck, and goat : goose mixtures, the mathematical
658
WALTERC. KaUCKEBERCAND OSCARP. CHILSON
T A B L E 2 - - E V I D E N C E FOR DEVELOPMENTAL DI~'~'~ENCE8 I N
AMP DRAMINASE ACTIVITY I N
RED BLOOD
CELL LYSATES FROM 1-DAY OLD AND ADULT CHICKENS AND DUCKS
Hemolysate No. of samples Chicken (adult) Chick (1 day old) Duck(adult) Duck (1 day old)
3 2* 4 3J"
Mean __S.D. (#M NHa/10* No. of cells per min) samples 22.2+5.1 2"0+0"31 9.46+2.0 1"13
8 4 4 3
Supernatant Mean _+S.D. (#MNHa/ml No. of cells per rain) samples
Mean +_S.D. (/zM NHa/mg Hb per min)
128.20+16.1 19"20+ 5"8 70.2+11.0 9"30
0.323 +0'045 0"064 0.171 +0.039
4 1 4 --
* Two separate groups of 1-day-old chicks--total of twenty chicks. t One group of 1-day-old ducklings--total of three ducklings. sum of the individual enzymatic activities equalled the actual assayed activity of the mixture (10-rain reaction period, aliquots taken every 2 min) with maximal variation of no more than 3 per cent. In a couple of cases the range of individual hemolysate activities proved to be highly variable. The turtle blood hemolysates showed the widest range of deaminase activities of all the animals tested. On a cell count basis the turtle hemolysate values ranged from 0.37 to 5.51--an approximate fifteenfold difference. For comparison, rabbit, dog, rat, adult chicken and duck range differences were 1.9, 1-8, 1.4, 1-5 and 1.4 fold respectively. Individual human red cell deaminase values also showed an interesting pattern of variation. Three human samples (two males, one female), on both a cell count and packed cell volume basis, proved to have enzyme activities within 20 per cent of one another. The fourth sample (male) had 3.5 times more enzyme activity than the first three. The presence of greater deaminase activity in nucleated erythrocytes raised the question of how much of the total cellular enzyme the nuclei of the cells actually contained. Using the nuclear isolation procedure of Hammel & Bessman (1964), pilot studies indicated no more than 10 per cent of the total cellular enzyme was associated with the nuclei in adult chicken red blood cells (Kruckeberg, unpublished results). In those animals where the sex of the donor was known and red blood cell deaminase activities compared (i.e. dog, human, rat, gerbil, duck, turtle and frog). no consistent variation in deaminase activity could be detected between sexes. DISCUSSION This study of AMP deaminase levels on red blood cells from various animal species was initiated, in part, in order to establish whether the chicken was unique in exhibiting high enzyme activity and whether correlations could be made between red cell deaminase levels and, for example, the presence or absence of
RED BLOOD CHLL A M P - D ] ~ I N P ~ E
659
erythrocyte nuclei and/or presence or absence of phytate or 2,3-diphosphoglycerate (2,3-DPG) in the red cell. Of the animals tested, adult chicken red blood cells indeed show the highest levels of deaminase activity. On a ceU count basis the frog also shows high levels of enzyme activity; however, considering the difference in cell volume between these two cell types (frog, 670/~8 and chicken, 127 ft*) (Altman & Dith1~er, 1964) it is not surprising that a cell five times larger than a chicken cell would show equal amounts of enzyme activity on a cell count basis. Expressing the enzyme activities on a packed (cell) volume basis gives perhaps a more realistic base for comparison of ceils of such different sizes. The morphologically most obvious correlation would be linking the presence or absence of an erythrocyte nucleus to a specific deaminase activity level All the nucleated erythrocytes assayed here (i.e. birds, turtle, frog and fish) contained relatively high levels of enzyme activity. However, the normal absence of a nucleus does not necessarily mean uniformly low enzyme activity levels (e.g. human, mouse and hamster). Using the data from Rapoport & Guest (1940) and Bartlett (1970) we were able to find no overaU correlation between deaminase levels and 2,3-diphosphoglycerate amounts in mammalian and amphibian ceUs or phytate quantities in bird and turtle cells. It is, however, interesting to note that goat, sheep and cow blood calls, which contain very little deaminase activity, also have the least amounts of 2,3-DPG and small amounts of ATP, compared to the other animal type red blood cells in this group. The different activity levels of deaminase in 1-day-old and adult chicken and duck blood cells (Table 2) indicate a potential research area dealing with questions of developmental isozyme form changes of the enzyme, changes in red call populations in maturing organisms and differential function of deaminase at different points in time. These and other questions are currently being investigated in this laboratory using this developmental system. Acknowledgements--This research was supported in part by the National Science
Foundation (NSF-GB-19397) and was done in partial fulfillment of the requirements for the Doctor of Philosophy Degree by Walter Kruckeberg. The authors gratefully acknowledge the technical assistance of Cordelia Rosenbloom, Roberta Oshinsky and Bruce Fine. REFERENCES ALTMANP. L. & DITTMEaD. S. (1964) Biology Data Book, p. 267. Fed. Am. soc. for exp. Biol., Washington, D.C. ASKARIA. ~ FRANKLINJ. E. (1965) Effects of monovalent cations and ATP on erythrocyte AMP deaminase. Biochim. biophys. Acta 110, 162-173. ASKAaZA. & RAOS. N. (1968) Regulation of AMP deaminase by 2,3-diphosphoglyceric acid: a possible mechanism for the control of adenine nucleotide metabolism in human erythrocytes. Biochim. biophys./1eta 151, 198-203. BARTLETT G. R. (1970) Patterns of phosphate compounds in red blood cells of man and animals. In Red Cell Metabolism and Function (Edited by Bg~T.a G. J.), pp. 245-256. Plenum Press, New York.
660
WALTI~ C. KRUCKEBEIRGAND OSCARP. CHILSON
BELL D. J. (1971) Metabolism of the erythrocyte. In Physiology and Biochemistry of the Domestic Fowl (Edited by BELLD. J. & FURMAN B. M.), Vol. 2, p. 863. Academic Press, New York. BISHOP C. (1960) Purine metabolism in human and chicken blood, in vitro, j~. biol. Chem. 235, 3228-3232. BISHOP C. (1964) Overall red cell metabolism. In The Red Blood Cell (Edited by BtSHOP C. & SURCENORD. M. ), pp. 148-181. Academic Press, New York. Bp~w~ G. J. & EATON J. W. (1971) Erythrocyte metabolism: interaction with oxygen transport. Sdence, Wash. 171, 1205-1211. CONWAY E. J. & COOKER. (1939) The deaminases of adenosine and adenylic acid in blood and tissues. Biochem..~. 33, 479-492. ELWCN D. H., LAtmDm~W. J., PARIKHH. C. & WISE E. M. (1972) Roles of plasma and erythrocytes in interorgan transport of amino acids in dogs. Am..7. Physiol. 222, 1333-1342. H~MF.L C. L. & B m S ~ N S. P. (1964) Hemoglobin synthesis in avian erythrocytes, j~. biol. Chem. 239, 2228-2238. HENDERSONJ. F. & LEPAoE G. A. (1959) Transport of adenine-8-C 1~ among mouse tissues by blood cells. ~t. biol. Chem. 234, 3219-3223. HENRY H. & CmLSON O. P. (1973) Chicken red blood cell adenylate deaminase: purification and comparison with the enzymes from chicken brain and muscle. Comp. Biochem. Physiol. 44B, 121-136. LowY B. A., WILLIAMSM. K. & LONDON I. M. (1962) Enzymatic deficiencies of purine nucleotide synthesis in the human erythrocyte. ~t. biol. Chem. 237, 1622-1625. MAGF.RJ., DVILANSKYA., RaZXNA., WIND E. & Iz~= G. (1966) Turnover of purine nucleotides in human red blood cells, lsraelJ, reed. Sci. 2, 297-301. MAGER J., Hm~SHKOA., ZEtTLIN-BEcK R., SHOSHANIT. & RAZIN A. (1967) Turnover of purine nueleotides in rabbit erythrocytes--I. Studies in vivo. Biochim. biophys. Acts 149, 50-58. MARKS P. A. & KOVACHJ. S. (1966) Development of mammalian erythyroid ceUs. In Current Topics in Developmental Biology (Edited by MOSCONAA. A. & MONROYA.), Vol. 1, pp. 213-245. Academic Press, New York. MIALE J. B. (1967) Laboratory Medicine--Hematology, p. 1147. C.V. Mosby, New York. MURRAY A. W. (1971) The biological significance of purine salvage. Arm. Rev. Biochem. 40, 811-826. RAPOPORT S. & GUEST G. M. (1940) Distribution of acid-soluble phosphorus in the blood cells of various vertebrates, j~. biol. Chem. 138, 269-285. WILLIAMSONJ. (1970) General features of metabolic control as applied to the erythrocyte. In Red Cell Metabolism and Function (Edited by BmswlsR G. J.), pp. 117-136. Plenum Press, New York.
Key Word Index--AMP-deaminase; red blood cell enzyme; vertebrate red blood cells; purine nucleotide metabolism; red blood cell AMP-deaminase.
RED BLOOD CELL AMP-DEAMINASE: LEVELS OF ACTIVITY IN HEMOLYSATES FROM TWENTY DIFFERENT VERTEBRATE SPECIES WALTER C. KRUCKEBERG and OSCAR P. CHILSON Department of Biology, Washington University, St. Louis, Missouri 63130, U.S.A. (Received 19 March 1973)
Abstract--1. The levels of adenylate deaminase activity have been measured in the erythrocyte hemolysates and hemolysate supernatants from twenty different vertebrate species. 2. The chicken erythrocyte hemolysates proved to have the highest level of AMP-deaminase activity, with birds in general showing more enzyme activity than the other nucleated red cell types and the non-nucleated red blood cell hemolysates showed low levels of the enzyme activity. 3. A developmental difference in red blood cell adenylate deaminase activity was discovered between 1-day old and adult ducks and was confirmed between 1-day old and adult chickens. INTRODUCTION ON A superficial level mature vertebrate red blood cells present a deceiving illusion of homogeneity in structure, function and molecular constitution. However, subtle and gross differences do exist. On the morphological level, erythrocytes from birds, amphibians, reptiles and fish (with one exception, Bell, 1971) have been shown to contain nuclei, while their mammalian counterparts are anucleate. On the metabolic level it has been well established that avian erythrocytes demonstrate a functional citric acid cycle whereas mammalian red cells are able to metabolize primarily, if not solely, anaerobically (Bishop, 1964). Apart from hemoglobin, red blood cells have been shown to contain a wide variety of catalytic and non-catalytic constituents (Rapoport & Guest, 1940; Bishop, 1964; Bartlett, 1970) whose quantities fluctuate (perhaps significantly) from species to species and whose individual and related functions are presently open to investigation. These non-hemoglobin constituents may function in a variety of ways; in controlled maintenance of red cell integrity (Williamson, 1970), in modification of the oxygen-binding capability of hemoglobin (Brewer & Eaton, 1971), in the regulation of erythrocyte development (Marks & Kovach, 1966) and in the transporting of molecules other than oxygen (Henderson & LePage, 1959; Murray, 1971 ; Elwyn et al., 1972). 653
654
WALTER C. I4muc~n~G ANDOSC~mP. CHIL$ON
Variations in purine nucleotide metabolism have been documented between red cells from two different species. Inosine-5'-monophosphate (IMP), the first complete purine nudeotide in the de novo pathway for purine synthesis, is the precursor for both guanine and adenine nucleotides. Lowy et al. (1962) have shown that rabbit erythrocytes readily convert IMP to adenosine-5'-monophosphate (AMP), while the degree of conversion of this compound to adenine nucleotides by human red cells is negligible. Mager et al. (1966,1967) further documented that guanosine and adenosine phosphates rapidly turn over in human and rabbit erythrocytes and also produced data consistent with the concept that the dynamic levels of these purine nucleotides are maintained by the reutilization of preformed purines rather than by the de novo synthesis of the purine ring from glycine and formate. Adenosine-5'-monophosphate deaminase, the enzyme of major interest in this report, catalyzes the effectively irreversible hydrolysis of the purine nucleotide 5'-AMP to IMP and ammonia. AMP-deaminase was first found in human and rabbit erythroeytes (Conway & Cooke, 1939) and has been subsequently identified and characterized in human, dog and cat blood cells (Askari, 1965, 1968). These current research efforts, done with mammalian cells, as well as our finding that nucleated adult chicken red blood cells contain twenty times more deaminase activity than either anucleate human or rabbit erythrocytes (Henry & Chilson, 1973), prompted us to begin the work reported here. The objective of this work is to quantitate and compare the levels of AMPdeaminase activity in the red blood cell lysates of twenty species of vertebrates. It is hoped that this information will aid in the design of experiments to more completely identify the role of deaminase in red cell metabolism, and thus provide part of the background necessary for a more comprehensive understanding of nucleotide metabolism, as a whole, in red blood cells. MATERIALS AND METHODS
Washed and chilled blood cells from duck (Arms sp.), goose (Anser domesticus), turkey (Meleagris gallopavo), sheep (Ovls aries), goat (Capra hircus) and cat (Fells domestica) were obtained in Modified Alsever's solution from Colorado Serum Company, Denver, Colorado. Whole blood from cattle (Bos taurus), horse (Equus cabaUus), dog (Canisfamillaris) and adult White Leghorn chickens (Gallus domesticus) was obtained from animals housed at the Ralston Purina Research Farm, Gray Summit, Missouri. Adult wild mallard ducks (Anas platyrhynchos) and duck eggs were obtained from the St. Louis Zoo. Turtles (Chrysemys sp.) and frogs (Rana catesbeiana) were bought from Nasco Corporation, Ann Arbor, Michigan. Chicken eggs (White Leghorn) were obtained from Ken-Roy Hatchery, Berger, Missouri. Rats (Mus norveglcus albinus) and rabbits (Lepus cuniculus) were bought from Isaacs Lab Stock, Litchfield, Illinois. Mice (Mus musculus), hamsters (Mesocricetus auratus) and gerbils were taken from stocks maintained in the Washington University Biology Department. Dog blood was obtained through Dr. P. Hartroft, Washington University Medical School. Human blood was taken from local laboratory personnel. The birds, turtles and most of the small mammals were bled by decapitation; mice were bled from the femoral vein after anesthetization. Dogs and humans were bled by venipuncture, frogs by heart puncture.
RED BLOOD CELL AMP-DEAMINASE
655
During collection, whole blood was combined with Alsever's solution containing heparin. Cells (buffy coat included) were washed in Alsever's three times by centrifugation at 1000g for 10 mln at 4°C; the final pellet of cells was then resuspended in Alsever's solution. After taking an aliquot for counting and packed cell volume, the bulk of the stock cell suspension was lysed by quick freezing at -70°C, subsequently to be thawed and the hemolysate assayed. Hemolysate supernatants were recovered from centrifuged (30,000 g, 35 rain, 4°C) hemolysates. All blood samples were routinely stored frozen. The enzyme activity levels were found to be stable by assaying selected cell hemolysates within 1.5 hr of collection and noting no increase or decrease in activity when compared to subsequent re-assaying over a 2-7 day period (in many cases longer). Where possible, blood was obtained from more than one source. Hence it was found that the deaminase activities of 1-day-old chick and adult duck blood cells received through the mail from Colorado Serum Company were within 20% of activity levels of cells from chicks hatched in our laboratory and with duck blood from animals obtained from the St. Louis Zoo. No effort was made to specifically exclude white blood cells or thrombocytes, nor to fractionate the red cells in any way during the washing process. Also, although healthy animals were used as donors, no consistent consideration was given to possible variables in deaminase activity levels such as diet, seasonal variation and/or specific age. Enzyme activity was assayed using the method previously described (Henry & Chilson, 1969). Each lysate type was assayed at saturating substrate concentrations as demonstrated by measurement at at least two substrate concentrations. Hemoglobin was quantitated in hemolysate supernatants using a method as described by Miale (1967). Freshly prepared solutions of bovine hemoglobin were used as a standard. Cells were counted in a Levy-Hauser hemocytometer after having been diluted with Alsever's solution (Kabat & Meyer, 1961). Packed cell volumes were done by centrifuging blood containing 75 m m x 1 "1-1'2 mm microhematocrit capillary tubes at 3000 g for 15 rain in a Clinical model International centrifuge. All chemicals used were of reagent grade and were obtained from sources as reported by Henry & Chilson (1973). RESULTS
T h e results of assaying A M P deaminase activity in uncentrifuged red blood cell hemolysates from twenty different animal species are shown in Fig. 1 and Table 1. Enzyme activity is initially expressed on a cell count (gM NH3/109 cells per rain) basis and on a packed cell volume (#M NHa/ml cells per rain) basis (columns A and B respectively in Fig. 1). T h e results of adult red cell enzyme activity expressed on a cell count basis (Fig. 1, column A) can perhaps best be stated as follows: frog and chicken show the highest levels of activity with the nucleated cells (i.e. bird, frog, turtle, carp) showing significantly higher overall activities than the non-nucleated (mammalian) cells. On the basis of packed cell volume (Fig. 1, column B) the chicken again showed the highest levels of enzyme activity with birds in general indicated to have higher activities than the other nucleated cells. With the exception of human, mouse, hamster and perhaps rabbit, the mammals have the lowest levels o f deaminase activity. T h e average percentage r e c o v e r y of deaminase activity in 30,000 g hemolysate supernatants from all cell types was 77 per cent or more (Table 1). In no individual
4
1 1 1 1 7 4 6 4 3 2 3 1 6 2 6 2 1
1
1.7
-+0"2
3-9 6"8 11-4 4-5 5.8 _+3"3 19"2 +3"4 0"3 _+0"07 0"9 +0"50 0-7 +0"36 0.04-+ 0"02 0.04-+ 0.02 0.02 0.09 + 0"02 0.08 _+0.02 0.2 +0"02 0"6 _+0.20 0.02
1.1
22"2 + 5"1 2.0 _+0"3 9.5 _+2"0
3 2 4
4
1 1 1 I 7 4 6 4 3 2 3 1 6 2 6 2 1
1
8 4 4
128.2 + 16"1 19.2 _+ 5.8 70-2 _+11.0 9"3 35"7 44.4 71-0 19"1 13.9 _+ 9.5 24"2 _+ 2"7 3"7 _+ 0"8 7"3 + 5"0 10"3 + 4"8 0"9 + 0.1 0.8 _+ 0-3 0.9 1-2 _+ 0.2 2-1 _+ 0.5 2"1 + 0.5 7.25_+ 1.10 0"80 18.6 -+ 2.2
6 1 4 . 1 1 1 1 1 4 3 4 3 1 3 1 5 2 5 2 1 5
No. of samples
Abbreviation: S.D. = Standard Deviation. * Airman & Dittmer (1964). t T w o separate groups of 1-day chicks--total of twenty chicks. $ One group of 1-day ducks--total of three ducks. § T hr e e separate groups of adult m i c e - - t o t a l of twenty-eight mice.
Adult chicken 1-Day chick t Adult duck 1-Day ducks Goose Turkey Pigeon Carp Turtle Frog Rabbit Human Mouse§ Horse Cow Sheep Dog Cat Rat Gerbil Goat Hamster
No. of samples
Mean-+ S.D. (/~M N H a / m l cells per m.ln)
. 97 78 83 100 98 88 96 88 92 78 97 77 91 78 88 83 100 82
89 97 88 .
Per cent recovery in supernatant (average value)
.
--
0"23 -0"56 0"06 -0-04 0.06 0"08 0"22 -0.02 0.03 0-02 0.04 0-03 -0"04
1.0 ---
(~M N H a / m l cells per rain) + cell volume
Mean
--
160 -131 311 -670 61 97 49 -50 31 66 57 61 -19
127 ---
Cell volume (~8),
DEAMINASE ACTIVITIF-S I N RED BLOOD CELL HF.MOLYSATK~ AND HEMOLYSATR SUPI~,NATANTS FROM TWENTY D I ~ SPECIRS
No. of samples
AMP
Mean_+ S.D. (#M NHa/109 cells per re_in)
T A B L E 1 - - S u M M A R Y OF
o b Z
.~
z ©
.~
.a
:~
o~ Oi o~
-
RED BLOOD
I'1
A
657
CELL AMP-DEAMINASE
C
B ÷
i~ ,+
c~ OA.
a~
+i
!
Ul ?
i,
3
,I,
FIG. 1. Summary of the AMP deaminase activities in red blood cell hemolysates and hemolysate supernatants from twenty different vertebrate species. The data
are displayed in three ways: A, hemolysate enzyme activity on a cell count basis (#M NHs/10gcells per minx 10-1); B, hemolysate enzyme activity on a packed cell volume basis ~ M NHs/ml cells per rain); and C, hemolysate supernatant activityon a mg hemoglobin basis (~M NHs/mg Hb per minx 10-2). Each column defines the mean enzyme activity. The short vertical line present in most of the columns defines_+ S.D. The absence of the standard deviation indicates that only one sample was assayed. The numbers of samples used to compute the standard deviations are given in Table 1. It is important to note that enzyme activity is represented on the ordinate as a log plot. case was recovery less than 77 per cent indicating that with the conditions used here, the major part of the enzyme activity is not tightly bound to the cell stroma. Expressing supernatant deaminase activity on a "per mg hemoglobin" basis (Fig. 1, column C), a familiar pattern of relative enzyme activities emerges: the chicken supernatant shows the highest amounts, birds in general show more enzyme activity than the other nucleated cell type supernatants, and the non-nucleated cell type supernatants have 10w levels of deaminase. Henry & Chilson (1973) first reported a marked difference between the A M P deaminase levels in 1-day-old chick and adult chicken blood cell hemolysates (confirmed, Table 2). In order to determine if domesticated chickens were unique in showing this developmental difference in enzyme activity, blood cells from adult and 1-day-old wild mallard ducks were similarly assayed. A developmental difference was also observed with these ducks (Table 2). In order to test for the presence of possible endogenous dissociable inhibitors which could modulate enzyme activity in a red cell hemolysate and give an illusion of species to species differences, certain representative supematants were combined and additivity of deaminase activity monitored within 3 hr of combining and up to a week after, samples having been stored frozen. In all eases studied, namely, 1-dayold chick : adult chicken, rabbit : duck, and goat : goose mixtures, the mathematical
658
WALTERC. KaUCKEBERCAND OSCARP. CHILSON
T A B L E 2 - - E V I D E N C E FOR DEVELOPMENTAL DI~'~'~ENCE8 I N
AMP DRAMINASE ACTIVITY I N
RED BLOOD
CELL LYSATES FROM 1-DAY OLD AND ADULT CHICKENS AND DUCKS
Hemolysate No. of samples Chicken (adult) Chick (1 day old) Duck(adult) Duck (1 day old)
3 2* 4 3J"
Mean __S.D. (#M NHa/10* No. of cells per min) samples 22.2+5.1 2"0+0"31 9.46+2.0 1"13
8 4 4 3
Supernatant Mean _+S.D. (#MNHa/ml No. of cells per rain) samples
Mean +_S.D. (/zM NHa/mg Hb per min)
128.20+16.1 19"20+ 5"8 70.2+11.0 9"30
0.323 +0'045 0"064 0.171 +0.039
4 1 4 --
* Two separate groups of 1-day-old chicks--total of twenty chicks. t One group of 1-day-old ducklings--total of three ducklings. sum of the individual enzymatic activities equalled the actual assayed activity of the mixture (10-rain reaction period, aliquots taken every 2 min) with maximal variation of no more than 3 per cent. In a couple of cases the range of individual hemolysate activities proved to be highly variable. The turtle blood hemolysates showed the widest range of deaminase activities of all the animals tested. On a cell count basis the turtle hemolysate values ranged from 0.37 to 5.51--an approximate fifteenfold difference. For comparison, rabbit, dog, rat, adult chicken and duck range differences were 1.9, 1-8, 1.4, 1-5 and 1.4 fold respectively. Individual human red cell deaminase values also showed an interesting pattern of variation. Three human samples (two males, one female), on both a cell count and packed cell volume basis, proved to have enzyme activities within 20 per cent of one another. The fourth sample (male) had 3.5 times more enzyme activity than the first three. The presence of greater deaminase activity in nucleated erythrocytes raised the question of how much of the total cellular enzyme the nuclei of the cells actually contained. Using the nuclear isolation procedure of Hammel & Bessman (1964), pilot studies indicated no more than 10 per cent of the total cellular enzyme was associated with the nuclei in adult chicken red blood cells (Kruckeberg, unpublished results). In those animals where the sex of the donor was known and red blood cell deaminase activities compared (i.e. dog, human, rat, gerbil, duck, turtle and frog). no consistent variation in deaminase activity could be detected between sexes. DISCUSSION This study of AMP deaminase levels on red blood cells from various animal species was initiated, in part, in order to establish whether the chicken was unique in exhibiting high enzyme activity and whether correlations could be made between red cell deaminase levels and, for example, the presence or absence of
RED BLOOD CHLL A M P - D ] ~ I N P ~ E
659
erythrocyte nuclei and/or presence or absence of phytate or 2,3-diphosphoglycerate (2,3-DPG) in the red cell. Of the animals tested, adult chicken red blood cells indeed show the highest levels of deaminase activity. On a ceU count basis the frog also shows high levels of enzyme activity; however, considering the difference in cell volume between these two cell types (frog, 670/~8 and chicken, 127 ft*) (Altman & Dith1~er, 1964) it is not surprising that a cell five times larger than a chicken cell would show equal amounts of enzyme activity on a cell count basis. Expressing the enzyme activities on a packed (cell) volume basis gives perhaps a more realistic base for comparison of ceils of such different sizes. The morphologically most obvious correlation would be linking the presence or absence of an erythrocyte nucleus to a specific deaminase activity level All the nucleated erythrocytes assayed here (i.e. birds, turtle, frog and fish) contained relatively high levels of enzyme activity. However, the normal absence of a nucleus does not necessarily mean uniformly low enzyme activity levels (e.g. human, mouse and hamster). Using the data from Rapoport & Guest (1940) and Bartlett (1970) we were able to find no overaU correlation between deaminase levels and 2,3-diphosphoglycerate amounts in mammalian and amphibian ceUs or phytate quantities in bird and turtle cells. It is, however, interesting to note that goat, sheep and cow blood calls, which contain very little deaminase activity, also have the least amounts of 2,3-DPG and small amounts of ATP, compared to the other animal type red blood cells in this group. The different activity levels of deaminase in 1-day-old and adult chicken and duck blood cells (Table 2) indicate a potential research area dealing with questions of developmental isozyme form changes of the enzyme, changes in red call populations in maturing organisms and differential function of deaminase at different points in time. These and other questions are currently being investigated in this laboratory using this developmental system. Acknowledgements--This research was supported in part by the National Science
Foundation (NSF-GB-19397) and was done in partial fulfillment of the requirements for the Doctor of Philosophy Degree by Walter Kruckeberg. The authors gratefully acknowledge the technical assistance of Cordelia Rosenbloom, Roberta Oshinsky and Bruce Fine. REFERENCES ALTMANP. L. & DITTMEaD. S. (1964) Biology Data Book, p. 267. Fed. Am. soc. for exp. Biol., Washington, D.C. ASKARIA. ~ FRANKLINJ. E. (1965) Effects of monovalent cations and ATP on erythrocyte AMP deaminase. Biochim. biophys. Acta 110, 162-173. ASKAaZA. & RAOS. N. (1968) Regulation of AMP deaminase by 2,3-diphosphoglyceric acid: a possible mechanism for the control of adenine nucleotide metabolism in human erythrocytes. Biochim. biophys./1eta 151, 198-203. BARTLETT G. R. (1970) Patterns of phosphate compounds in red blood cells of man and animals. In Red Cell Metabolism and Function (Edited by Bg~T.a G. J.), pp. 245-256. Plenum Press, New York.
660
WALTI~ C. KRUCKEBEIRGAND OSCARP. CHILSON
BELL D. J. (1971) Metabolism of the erythrocyte. In Physiology and Biochemistry of the Domestic Fowl (Edited by BELLD. J. & FURMAN B. M.), Vol. 2, p. 863. Academic Press, New York. BISHOP C. (1960) Purine metabolism in human and chicken blood, in vitro, j~. biol. Chem. 235, 3228-3232. BISHOP C. (1964) Overall red cell metabolism. In The Red Blood Cell (Edited by BtSHOP C. & SURCENORD. M. ), pp. 148-181. Academic Press, New York. Bp~w~ G. J. & EATON J. W. (1971) Erythrocyte metabolism: interaction with oxygen transport. Sdence, Wash. 171, 1205-1211. CONWAY E. J. & COOKER. (1939) The deaminases of adenosine and adenylic acid in blood and tissues. Biochem..~. 33, 479-492. ELWCN D. H., LAtmDm~W. J., PARIKHH. C. & WISE E. M. (1972) Roles of plasma and erythrocytes in interorgan transport of amino acids in dogs. Am..7. Physiol. 222, 1333-1342. H~MF.L C. L. & B m S ~ N S. P. (1964) Hemoglobin synthesis in avian erythrocytes, j~. biol. Chem. 239, 2228-2238. HENDERSONJ. F. & LEPAoE G. A. (1959) Transport of adenine-8-C 1~ among mouse tissues by blood cells. ~t. biol. Chem. 234, 3219-3223. HENRY H. & CmLSON O. P. (1973) Chicken red blood cell adenylate deaminase: purification and comparison with the enzymes from chicken brain and muscle. Comp. Biochem. Physiol. 44B, 121-136. LowY B. A., WILLIAMSM. K. & LONDON I. M. (1962) Enzymatic deficiencies of purine nucleotide synthesis in the human erythrocyte. ~t. biol. Chem. 237, 1622-1625. MAGF.RJ., DVILANSKYA., RaZXNA., WIND E. & Iz~= G. (1966) Turnover of purine nucleotides in human red blood cells, lsraelJ, reed. Sci. 2, 297-301. MAGER J., Hm~SHKOA., ZEtTLIN-BEcK R., SHOSHANIT. & RAZIN A. (1967) Turnover of purine nueleotides in rabbit erythrocytes--I. Studies in vivo. Biochim. biophys. Acts 149, 50-58. MARKS P. A. & KOVACHJ. S. (1966) Development of mammalian erythyroid ceUs. In Current Topics in Developmental Biology (Edited by MOSCONAA. A. & MONROYA.), Vol. 1, pp. 213-245. Academic Press, New York. MIALE J. B. (1967) Laboratory Medicine--Hematology, p. 1147. C.V. Mosby, New York. MURRAY A. W. (1971) The biological significance of purine salvage. Arm. Rev. Biochem. 40, 811-826. RAPOPORT S. & GUEST G. M. (1940) Distribution of acid-soluble phosphorus in the blood cells of various vertebrates, j~. biol. Chem. 138, 269-285. WILLIAMSONJ. (1970) General features of metabolic control as applied to the erythrocyte. In Red Cell Metabolism and Function (Edited by BmswlsR G. J.), pp. 117-136. Plenum Press, New York.
Key Word Index--AMP-deaminase; red blood cell enzyme; vertebrate red blood cells; purine nucleotide metabolism; red blood cell AMP-deaminase.