- Email: [email protected]
Developmental alterations in 5′-nucleotidase kinetics and lipid composition of rat heart sarcolemma
Mechanisms of Ageing and Development, 22 (1983) 151-158 Elsevier Scientific Publishers Ireland Ltd.
151
D E V E L O P M E N T A L ALTERATIONS IN 5'-NUCLEOTIDASE KINETICS AND LIPID C O M P O S I T I O N OF RAT H E A R T S A R C O L E M M A
A T I F B. A W A D * and J Y O T I P. C H A T I ' O P A D H Y A Y Kirksville College of Osteopathic Medicine, Kirksville, MO 63501 (U.S.A.)
(Received October 25th, 1982) (Revision received January 14th, 1983) SUMMARY The objective of these studies was to examine the mechanism by which the specific activity of heart sarcolemma 5'-nucleotidase decreases as function of age. We examined the kinetic properties and the lipid composition of the sarcolemma from animals with different ages. The age groups used were 1 month, 6-8 months and 13-15 months. It was found that the Km of this enzyme increases as the animal develops from 1 month to 6-15 months. The opposite was true with 5'-nucleotidase Vmax. There was no significant difference between the middle age and the older age groups in those parameters. The results of these experiments suggest that the increase in Km in sarcolemma 5'-nucleotidase could be due to the reduction of the sarcolemmal polyunsaturated fatty acid concentration, the only lipid alteration observed. K e y words: 5'-Nucleotidase; Heart sarcolemma; Kinetics; D e v e l o p m e n t ; Lipid composition
INTRODUCTION 5'-Nucleotidase, a sarcolemmai m e m b r a n e - b o u n d enzyme, has a role in myocardial function [1,2]. The product of its action on adenosine m o n o p h o s p h a t e (AMP), i.e. adenosine, has been shown to have a vasodilatory effect and thus plays an important role in modulating the rate of blood flow to the heart [3,4]. Recent published work from our laboratory [5] indicated that the specific activity of this enzyme in purified heart sarcolemma is influenced by age. As the animal gets older the specific activity of sarcolemmal 5'-nucleotidase is reduced. The mechanism by which 5'-nucleotidase is influenced by age is not known. In an attempt to elucidate this mechanism we examined the kinetic properties of this enzyme in heart sarcolemma from animals with different ages. As m e m b r a n e *To whom correspondence should be addressed. 0tl47-6374/83/$3.1XJ Printed and Published in Ireland
© 1983 Elsevier Scientific Publishers Ireland Ltd.
152
lipids have been shown to influence the activities of several m e m b r a n e - b o u n d enzymes [6-8], we followed the alterations in the lipid composition of these fractions as the animal develops. MATERIALS AND METHODS
Animals Male Sprague-Dawley rats of three age groups, l month, 6-8 months and 13-15 months, were used in these studies. Animals were fed Purina Chow, had free access to water and were housed in facilities that are t e m p e r a t u r e controlled. Animals were exposed to a 12-12 hour light-dark cycle. Preparation of plasma m em branes To prepare heart sarcolemma, animals were killed by cervical dislocation and hearts were excised. The cardiac sarcolemma fractions were isolated from heart by the method of Bers [9] with some modification as described in our previous communication [5]. Protein was assayed in the sarcolemma preparation by the method of Lees and Paxman ]1(11. A s s a y for marker e n z y m e s 5'-Nucleotidase (EC 3.1.3.5) activity was measured according to the method described by Touster et al. [ I 1]. The incubation medium (0.5 ml) contained 5 mM A M P (pH 7.0), 100mM glycine (pH 9.1), and 1 0 m M MgCI2. Incubation was p e r f o r m e d at 37°C for 20 min. The inorganic phosphorus released was determined in the medium [12] after adding 0.5 ml of 1.2M perchloric acid. p-Nitrophenyl phosphatase activity (EC 3.1.3.1) was assayed at pH 7.6 in 1.0ml of a medium containing 50 mM Tris-HCl, 1 m M E D T A , 5 m M MgClz and 5 m M p-nitrophenyl phosphate. The reaction mixture was incubated at 37°C for 2[)min. The reaction was stopped with 2.0 ml of 1 M N a O H . The amount of p-nitrophenol released was quantitated by measuring the absorbance at 4111 nm and c o m p a r e d to the 50/xM p-nitrophenol standard [9]. Succinic dehydrogenase (EC 1.3.99.1) and retononeinsensitive N A D H cytochrome c reductase (EC 1.6.99.3) activities were measured according to the methods of Green et al. [13] and Sottocasa et al. [14], respectively. E n z y m e kinetics of 5'-nucleotidase were studied using A M P as substrate. Km and Vma x values were calculated from Woolf's plot [15]. Lipid analysis Lipids were extracted from the cardiac sarcolemma with c h l o r o f o r m - m e t h a n o l (2:1, v/v) according to the method of Folch et al. [16]. These extracts were assayed for cholesterol and phospholipids according to the method of Pollet et al. [17] and Raheja et al. [18], respectively. Lipid extracts were saponified and the released free fatty acids were methylated according to the method of Morrison and Smith [19]. Methylated fatty acids were then analyzed by gas-liquid chromatography using a 6-ft column packed with 10% SP-2330 on 100-200mesh
153
Chromosorb WAW (Supelco, Bellefonte, PA, U.S.A.). The oven temperature was maintained at 220°C. Detector and injection port temperatures were 235°C and 240°C, respectively. N2 served as the carrier gas at a flow rate of 50 ml/min. Peaks were identified using authentic fatty acid standards. RESULTS
Purity of sarcolemma preparations Th e purity of sarcolemma membrane preparations was examined by assaying the activities of marker enzymes, 5'-nucleotidase, p-nitrophenyl phosphatase, succinic dehydrogenase and rotenone-insensitive N A D H cytochrome c reductase. The enrichment of sarcolemma membranes prepared from animals of ditierent ages with plasma membrane enzymes is demonstrated in Table I. Our sarcolemma preparations were enriched in 5'-nucleotidase and p-nitrophenyl phosphatase activities to the extent of 24-27-fold and 7-8-fold, respectively, as compared to their corresponding homogenates. The difference between the purification factors of 5'-nucleotidase and p-nitrophenyl phosphatase has been recently reported by Hosey [20] in isolating sarcolemma from newborn chick hearts using mild low-salt isotonic so!ution for isolation. The author reported purification values of 34-fold and 9-fold for theSe two enzymes, respectively, which are very close to our present data. When Hosey examined his membranes for autophosphorylation, it was found that they were physiologically active [20]. We could not detect any activity for succinic dehydrogenase in the isolated sarcolemma, which indicates the absence of mitochondrial contamination in these fractions. There was about 5-fold enrichment in the rotenone-insensitive N A D H cytochrome c reductase activity in these membrane preparations over the homogenate, indicating the presence of microsomal contamination. The degree of this contamination, however, did not
TABLE
I
SPECIFIC ACTIVITIES
OF SARCOLEMMAL
MARKER
ENZYMES
Each value is the mean ± S.E. o f 4 samples.
Specific activity (l~mol per mg protein per h) Age (mSnths)
Sarcolemma
Homogenate
Pudficaaon
5'-Nucleotidase
1 6-8 13-15
6 7 . 9 3 ± 2.46 4 5 . 5 9 ± 1.91 ~ 4 0 . 8 7 ± 5.63 a
2.79 ± 0 . 1 8 1.83 - 0 . 0 4 a 1.62 ± 0.01 ab
2 4 . 8 0 ± 1.99 2 6 . 2 9 - 0.73 25.11 +--3.23
p-Nitrophenyl phosphatase
I 6-8 13-15
1.99 ± 0.13 1.79 ± 0.18 1.80 ± 0 . 1 6
0.24 ± 0.01 0.22 ± 0.02 0.23 ± 0.02
aSignificantly ditierent (p -< 0.05) from those of 1-month-old animals. bSignificantly ditterent (p - 0.05) from those of 6-8-month-old animals.
8.26 ± 0.92 8.02---0.17 7.31 ± 1.2
154
differ
significantly
from
somal
contamination
dicated
by several
activities
one
age group
in highly workers
of 5’-nucleotidase
to another.
purified
using
The presence
sarcolemmal
different
of membrane
methods
of this micro-
preparations and animal
and homogenate
animals were significantly different from those derived and 13-15-month-old animals. On the other hand,
has been species
derived
in-
121). The
from younger
from those of 6-&monththe specific activities of
p-nitrophenyl phosphatase in both membranes and homogenates were not influenced by age. This confirms our earlier observation that S’-nucleotidase activity activity
decreases remains
with
the
unchanged
increase
in age
while
p-nitrophenyl
phosphatase
[S].
Efiect of age on the kinetics of 5’-nucleotidase The results of the experiments on the kinetic properties (K, and V,,,,,) of sarcolemma-bound 5’-nucleotidase from rats of different age groups are shown in Table II and Fig. 1. The K, value for sarcolemmal 5’-nucleotidase from l-monthold animals
was significantly
lower
than
those
from
&8-
and
13-15-month-old
5 6-
4 .8 -
4 .o -
3 [Sl v
2. 4-
I .6-
0 I .’ I
_-L -0.8
0.8
I
1.6
I
2.4
I
I
3.2
4.0
I 4.8
IS1 Fig. 1. Woolf plot of 5’-nucleotidase from cardiac sarcolemma of rats of three different ages. The concentrations of AMP used ranged from 100 to 4000 PM. The velocity of the enzymatic reaction was measured in nmol substrate per mg protein per min. Each point in the graph represents the average of 3 separate experiments.
155
TABLE
It
Km A N D Vmax V A L U E S O F 5 ' - N U C L E O T I D A S E OF DIFFERENT AGES
OF CARDIAC
SARCOLEMMA
FROM
RATS
Each value is the mean -+ S.E. o f 3 separate experiments.
Age (months)
1 6-8 13-15
Km (IzM)
Vma~ (nmol per mg protein per rain)
493.21 - 14.49 735.99 ± 4 2 . 9 9 ~ 791.10 -+ 90.71 a
1446.3 ± 130.0 994.66 ± 26.69~ 9 2 5 . 3 4 -+ 17.56"
aSignificantly different (p --< 0.05) from those of l-month-old animals.
animals. But there were no significant differences in Km and V~.ax between 6-8-month- and 13-15-month-old animals. The values of Km we obtained for 5'-nucleotidase in sarcolemma from 6-8- and 13-15-month-old animals are close to those reported by other workers [22].
Lipid composition of cardiac sarcolemma The lipid contents of heart sarcolemma derived from rats of different ages are given in Table III. The concentrations of cholesterol and phospholipid in these membranes did not change with age. Accordingly, the cholesterol/phospholipid ratio also was not influenced by the age of the animal. It was found also that the total lipids (cholesterol + phospholipid) per mg of protein was not influenced by age, which implies that the protein concentration of these membranes was maintained constant. The fatty acid compositions of the sarcolemma lipids from the three age groups are shown in Table IV. The concentration of saturated fatty acids in the cardiac sarcolemma consistently increased with the increase in age whereas the concentration of polyunsaturated fatty acid decreased with the increase in age. The concentration of monounsaturated fatty acids in the membranes of l-month-old animals was significantly higher than those of 6-8-monthold animals but reached the same level by 13-15-month-old animals. Alterations TABLE
III
EFFECT OF DEVELOPMENT CARDIAC SARCOLEMMA
ON
THE
LIPID
CONTENT
(#g/rag
PROTEIN)
Each value is the mean +- S.E. of 4 experiments.
Age (months)
Cholesterol
Phospholipid
Cholesterol/phospholipid
1 6-8 13-15
2 3 2 . 5 4 _+ 17.71 187.53 - 9 . 3 8 206.08-+0.95
8 0 8 . 6 4 _+ 73.41 775.73 _ 6 2 . 8 8 846.55-+87.90
0.31 -+ 0.02 0 . 2 4 - 0.05 0.25±0.04
OF
RAT
156 TABLE IV FATTY ACID COMPOSITION OF CARDIAC SARCOLEMMA FROM RATS OF DIFFERENT AGES Values (in per cent) are the mean ± S.E. of 3 samples.
Age (months) Fatty acids
1
6-8
13--15
14: 0 14 : 1 16 : 0 16: 1 18 : 0 18 : 1 18 : 2 20 : 4 22 : 6 Others Saturated Monounsaturated Polyunsaturated
tr~ tr 13.36 ± 0.87 tr 20.93 +- 0.06 18.99 +_0.70 17.09 ± 0.88 20.16 -+ 0.50 3.73 _+O. 15 4.26 -+ 0.80 34.64 _+0.84 20.12 ± 0.73 40.98 +_0.66
0.7 ± 0.1 tr 14.2 4- 0.85 0.61) -+ 0.35 .34.60 _+ 1.05b 13.50 -+ 0.35 b 13.30 4- 0.25 ° 17.80 -+ 0.70 4.11 ± 0.25 0.51 -+ 0.01 49.51) + 0,65 b 14.78 ± 0.32° 35.20 + 0,69°
1.25 ± 0.54 0.61) -+ (}.3{I 18.16 -+ 1.35b~ 0.66 _+0.05 29.65 +_ 1.52b 14.49 + 1.22b 12.29 +- 0.62b 16.40 +_ 1.52° 3.74 ± 0.61) 2.42 +_0.72 49.06 _+0.23° 16.09 ± 1.54 32.93 _+2.2(P
a < 0.5% of total fatty acids. bSignificantly different (p --<0.05) from those of l-month-old animals. cSignificantly different (p -< 0.05) from those of 6-8-month-old animals.
in s i n g l e f a t t y a c i d s w e r e concentrations The
concentration months
during development.
o f 1 6 : 0 a n d 1 8 : 0 in t h e c a r d i a c s a r c o l e m m a
concentration
decreased
also observed
of oleic acid, on the other
of t w o p o l y u n s a t u r a t e d
with development.
w h i l e it w a s d e l a y e d
hand,
fatty acids,
The decrease
For example,
i n c r e a s e d w i t h age.
decreased
i.e. 1 8 : 2
the
w i t h age. T h e
and 20:4,
steadily
in 1 8 : 2 w a s n o t i c e d as e a r l y as 6--8
to 13-15 months
with respect to arachidonic
acid
concentration. DISCUSSION The results of these experiments sarcolemmal
5'-nucleotidase
are
animal develops, 5'-nucleotidase in Km s u g g e s t s a d e c r e a s e enzyme.
On
the other
(6-15 months) membranes. extractability enzymes
indicate that both
influenced
in t h e b i n d i n g affinity o f t h e s u b s t r a t e
hand,
of
could
not
the decrease
sarcolemma
be
explained
from
the
in 5 ' - n u c l e o t i d a s e in t h e e n z y m e on
the
As the
o f t h e a n i m a l ( T a b l e I).
(AMP)
Vma x
to the
in o l d e r a g e
concentration
in t h e s e
basis of differences
myofilaments
since the purification factors were maintained
development
Vmax o f c a r d i a c
Km i n c r e a s e s w h i l e Vmax d e c r e a s e s . T h e i n c r e a s e
could indicate a reduction
This
K m and
by the age of the animal.
and
its e f f e c t
in t h e
on
constant throughout
these the
157
The information regarding the effect of age on 5'-nucleotidase kinetics is very limited. The only studies that we are aware of are those of Agnisola et al. [22]. These authors reported an increase in Km value for liver plasma membrane 5'-nucleotidase, which is in agreement with our observation. On the other hand, with the exception of one of their age groups, they found that 5'-nucleotidase Vm,x was not influenced by age. However, one should take their results with caution since their liver membranes were crude whei'eas ours are purified membranes. The observed alterations in sarcolemmal fatty acid composition as the animal develops may throw some light on the mechanism by which development influences the Km of the 5'-nucleotidase. The decrease in polyunsaturated fatty acid content of these membranes in the older age could alter the fluidity of these membranes. Several studies indicated that the fluidity of the membranes influences the kinetic properties of several enzymes [23-25]. Our earlier work with Ehrlich ascites tumor cells indicated that the enrichment of their plasma membranes with polyunsaturated fatty acids in vivo resulted in a decrease in the K~ of the Na+-dependent a-aminoisobutyric acid uptake [26]. This uptake system has similar kinetic properties to those of enzymes [27]. The decrease in Vmaxof sarcolemmal 5'-nucleotidase in older ages (6-8 and 13-15 months) could be interpreted as a reduction in the rate of enzyme synthesis by the myocardial cells. Several studies have indicated similar observations with other enzymes [28, 29]. ACKNOWLEDGMENT
This work was supported by grant 81-04-002 from the National Osteopathic Foundation. REFERENCES 1 H. Fleit, M. Conklyn, R.D. Stebbius and R. Silber, Function of 5'-nucleotidase in the uptake of adenosine from AMP by human lymphocytes. J. Biol. Chem., 250 (1975) 8889-8892. 2 G.P. Frick and J.M. Lowenstein, Vectorial production of adenosine by 5'-nucleotidase in the perfused rat heart. J. Biol. Chem., 253 (1978) 1240-1244. 3 R. Rubio and R.M. Berne, Release of adenosine by the normal myocardium in dogs and its relationship to the regulation of coronary resistance. Circ. Res., 25 (1969) 407---415. 4 R.M. Berne, Cardiac nucleotides in hypoxia: Possible role in regulation of coronary blood flow. Am. J. Physiol., 204 (1963) 317-322. 5 A.B. Awad and S.W. Clay, Age-dependent alterations in lipids and function of rat heart sarcolemma. Mech. Ageing Dev., 19 (1982) 333-342. 6 H. Sandermann, Jr., Regulation of membrane enzymes by lipids. Biochim. Biophys. Acta, 515 (1978) 2~--237. 7 G.Y. Sun and A.Y. Sun, Synaptosomal plasma membranes: Acyl group composition of phosphoglycerides and (Nat + K+)-ATPase activity during fatty acid deficiency. J, Neurochem., 22 (1974) 15--18. 8 J. Bernsohn and F.J. Spitz, Linoleic and linolenic acid dependency of some brain membrane-bound enzymes after lipid deprivation in rats. Biochem. Biophys. Res. Commun., 57 (1974) 293--298. 9 D.M. Bers, Isolation and characterization of cardiac sarcolemma. Biochim. Biophys. Acra, 555 (1979) 131-146.
158
10 M.B. Lees and S. Paxman, Modification of the Lowry procedure for the analysis of proteolipid protein. Anal. Biochem., 47 (1972) 184-192. 11 O. Touster, N.N. Aronson, Jr., J.T. Dulaney and H. Hendrickson, Isolation of rat liver plasma membranes. Use of nucleotide pyrophosphatase and phosphodiesterase-I as marker enzymes. J. Cell Biol., 47 (1970) 604-618. 12 C.H. Fiske and Y. Subbarow, The colorimetric determination of phosphorus. J. Biol. Chem., 66 (1925) 375--400. 13 D.E. Green, S. Mii and P.M. Kolut, Studies on the terminal electron transport system, i-Succinic dehydrogenase. J. Biol. Chem., 217 (1955) 551-567. 14 G.L. Sottocasa, B. Kuylenstierna, L. Ernster and A. Bergstrand, An electron transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J. Cell Biol., 32 (1967) 415-437. 15 H.N. Christensen and G.A. Palmer, Enzyme Kinetics, W.B. Saunders Company, Philadelphia, London, 1967, pp. 101-103. 16 J. Foleh, M. Lees and G.H. Sloane-Stanley, A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226 (1957) 497-509. 17 S. Pollet, S. Ermidou, F.L. Saux, M. Monge and N. Baumann, Microanalysis of brain lipids: Multiple two dimensional thin layer chromatography. J. Lipid Res., 19 (1978) 916--921. 18 R.K. Raheja, C. Kaur, A. Singh and I.S. Bhatia, New colorimetric method for the quantitative estimation of phospholipids without acid digestion. J. Lipid Res., 14 (1973) 695~697. 19 W.R. Morrison and L.M. Smith, Preparation of fatty acid methyl esters and dimethylacetate from lipids with boron fluoride-methanol. J. Lipid Res., 5 (1964) ~XI-~8. 20 M.M. Hosey, Chick heart membranes: isolation and analysis of autophosphorylation. Biochim. Biophys. Acta, 690 (1982) 106-116. 21 W.B. Wegticki, K. Owens, F.F. Kennel, A. Kessner, L. Harris, R.M. Wise and G.V. Vahouny, Preparation and properties of highly enriched cardiac sarcolemma from isolated adult myocytes. J. Biol. Chem., 255 (1980) 3605--3609. 22 C. Agnisola, L. Foti and I.T. Genoino, Influence of age on 5'-nucleotidase in plasma membranes isolated from rat liver. Mech. Ageing Dev., 13 (1980) 227-239. 23 A. Solyom and E.G. Trams, Enzyme markers in characterization of isolated plasma membranes. Enzyme, 13 (1972) 329-372. 24 R.P. Brivio-Haugland, S.L. Louis, K. Musch, N, Waldeck and M.A. Willian~s, Liver plasma membranes from essential fatty acid deficient rats: isolation, fatty acid composition and activities of 5'-nucleotidase, ATPase and adenylate cyclase. Biochim. Biophys. Acta, 43,? (1976~150-163. 25 B. Bloj, R.D. Morero, R.N. Farias and R.E. Trucco, Membrane lipid fatty acids and regulation of membrane-bound enzymes: Allosteric behavior of erythrocyte Mg2+-ATPase, (Na÷+ K+)-ATPase and acetylcholinesterase from rats fed different fat-supplemented diets. Biochim. Biophys. Acta, 311 (1973) 67-79. 26 T.L. Kaduce, A.B. Awad, L.J. Fontenelle and A.A. Spector, Effect of fatty acid saturation on a-aminoisobutyric acid transport in Ehrlich ascites cells. J. Biol. Chem., 19 (1977) 6624--6630. 27 D.L. Oxender and H.N. Christensen, Distinct mediating systems for transport of neutral amlno acids by the Ehrlich cell. J. Biol. Chem., 238 (1963) 3686-3699. 28 R.G. Hansford and F. Castro, Age linked changes in the activity of enzymes of the tricarboxylate cycle and lipid oxidation, and of carnitine content, in muscles of the rat. Mech. Ageing Dev., 19 (1982) 191-201. 29 S.W. O'Connor, P.J. Scarpace and I.B. Abrass, Age-associated decrease of adenylate cyclase activity in rat myocardium. Mech. Ageing Dev., 16 (1981) 91-95.
151
D E V E L O P M E N T A L ALTERATIONS IN 5'-NUCLEOTIDASE KINETICS AND LIPID C O M P O S I T I O N OF RAT H E A R T S A R C O L E M M A
A T I F B. A W A D * and J Y O T I P. C H A T I ' O P A D H Y A Y Kirksville College of Osteopathic Medicine, Kirksville, MO 63501 (U.S.A.)
(Received October 25th, 1982) (Revision received January 14th, 1983) SUMMARY The objective of these studies was to examine the mechanism by which the specific activity of heart sarcolemma 5'-nucleotidase decreases as function of age. We examined the kinetic properties and the lipid composition of the sarcolemma from animals with different ages. The age groups used were 1 month, 6-8 months and 13-15 months. It was found that the Km of this enzyme increases as the animal develops from 1 month to 6-15 months. The opposite was true with 5'-nucleotidase Vmax. There was no significant difference between the middle age and the older age groups in those parameters. The results of these experiments suggest that the increase in Km in sarcolemma 5'-nucleotidase could be due to the reduction of the sarcolemmal polyunsaturated fatty acid concentration, the only lipid alteration observed. K e y words: 5'-Nucleotidase; Heart sarcolemma; Kinetics; D e v e l o p m e n t ; Lipid composition
INTRODUCTION 5'-Nucleotidase, a sarcolemmai m e m b r a n e - b o u n d enzyme, has a role in myocardial function [1,2]. The product of its action on adenosine m o n o p h o s p h a t e (AMP), i.e. adenosine, has been shown to have a vasodilatory effect and thus plays an important role in modulating the rate of blood flow to the heart [3,4]. Recent published work from our laboratory [5] indicated that the specific activity of this enzyme in purified heart sarcolemma is influenced by age. As the animal gets older the specific activity of sarcolemmal 5'-nucleotidase is reduced. The mechanism by which 5'-nucleotidase is influenced by age is not known. In an attempt to elucidate this mechanism we examined the kinetic properties of this enzyme in heart sarcolemma from animals with different ages. As m e m b r a n e *To whom correspondence should be addressed. 0tl47-6374/83/$3.1XJ Printed and Published in Ireland
© 1983 Elsevier Scientific Publishers Ireland Ltd.
152
lipids have been shown to influence the activities of several m e m b r a n e - b o u n d enzymes [6-8], we followed the alterations in the lipid composition of these fractions as the animal develops. MATERIALS AND METHODS
Animals Male Sprague-Dawley rats of three age groups, l month, 6-8 months and 13-15 months, were used in these studies. Animals were fed Purina Chow, had free access to water and were housed in facilities that are t e m p e r a t u r e controlled. Animals were exposed to a 12-12 hour light-dark cycle. Preparation of plasma m em branes To prepare heart sarcolemma, animals were killed by cervical dislocation and hearts were excised. The cardiac sarcolemma fractions were isolated from heart by the method of Bers [9] with some modification as described in our previous communication [5]. Protein was assayed in the sarcolemma preparation by the method of Lees and Paxman ]1(11. A s s a y for marker e n z y m e s 5'-Nucleotidase (EC 3.1.3.5) activity was measured according to the method described by Touster et al. [ I 1]. The incubation medium (0.5 ml) contained 5 mM A M P (pH 7.0), 100mM glycine (pH 9.1), and 1 0 m M MgCI2. Incubation was p e r f o r m e d at 37°C for 20 min. The inorganic phosphorus released was determined in the medium [12] after adding 0.5 ml of 1.2M perchloric acid. p-Nitrophenyl phosphatase activity (EC 3.1.3.1) was assayed at pH 7.6 in 1.0ml of a medium containing 50 mM Tris-HCl, 1 m M E D T A , 5 m M MgClz and 5 m M p-nitrophenyl phosphate. The reaction mixture was incubated at 37°C for 2[)min. The reaction was stopped with 2.0 ml of 1 M N a O H . The amount of p-nitrophenol released was quantitated by measuring the absorbance at 4111 nm and c o m p a r e d to the 50/xM p-nitrophenol standard [9]. Succinic dehydrogenase (EC 1.3.99.1) and retononeinsensitive N A D H cytochrome c reductase (EC 1.6.99.3) activities were measured according to the methods of Green et al. [13] and Sottocasa et al. [14], respectively. E n z y m e kinetics of 5'-nucleotidase were studied using A M P as substrate. Km and Vma x values were calculated from Woolf's plot [15]. Lipid analysis Lipids were extracted from the cardiac sarcolemma with c h l o r o f o r m - m e t h a n o l (2:1, v/v) according to the method of Folch et al. [16]. These extracts were assayed for cholesterol and phospholipids according to the method of Pollet et al. [17] and Raheja et al. [18], respectively. Lipid extracts were saponified and the released free fatty acids were methylated according to the method of Morrison and Smith [19]. Methylated fatty acids were then analyzed by gas-liquid chromatography using a 6-ft column packed with 10% SP-2330 on 100-200mesh
153
Chromosorb WAW (Supelco, Bellefonte, PA, U.S.A.). The oven temperature was maintained at 220°C. Detector and injection port temperatures were 235°C and 240°C, respectively. N2 served as the carrier gas at a flow rate of 50 ml/min. Peaks were identified using authentic fatty acid standards. RESULTS
Purity of sarcolemma preparations Th e purity of sarcolemma membrane preparations was examined by assaying the activities of marker enzymes, 5'-nucleotidase, p-nitrophenyl phosphatase, succinic dehydrogenase and rotenone-insensitive N A D H cytochrome c reductase. The enrichment of sarcolemma membranes prepared from animals of ditierent ages with plasma membrane enzymes is demonstrated in Table I. Our sarcolemma preparations were enriched in 5'-nucleotidase and p-nitrophenyl phosphatase activities to the extent of 24-27-fold and 7-8-fold, respectively, as compared to their corresponding homogenates. The difference between the purification factors of 5'-nucleotidase and p-nitrophenyl phosphatase has been recently reported by Hosey [20] in isolating sarcolemma from newborn chick hearts using mild low-salt isotonic so!ution for isolation. The author reported purification values of 34-fold and 9-fold for theSe two enzymes, respectively, which are very close to our present data. When Hosey examined his membranes for autophosphorylation, it was found that they were physiologically active [20]. We could not detect any activity for succinic dehydrogenase in the isolated sarcolemma, which indicates the absence of mitochondrial contamination in these fractions. There was about 5-fold enrichment in the rotenone-insensitive N A D H cytochrome c reductase activity in these membrane preparations over the homogenate, indicating the presence of microsomal contamination. The degree of this contamination, however, did not
TABLE
I
SPECIFIC ACTIVITIES
OF SARCOLEMMAL
MARKER
ENZYMES
Each value is the mean ± S.E. o f 4 samples.
Specific activity (l~mol per mg protein per h) Age (mSnths)
Sarcolemma
Homogenate
Pudficaaon
5'-Nucleotidase
1 6-8 13-15
6 7 . 9 3 ± 2.46 4 5 . 5 9 ± 1.91 ~ 4 0 . 8 7 ± 5.63 a
2.79 ± 0 . 1 8 1.83 - 0 . 0 4 a 1.62 ± 0.01 ab
2 4 . 8 0 ± 1.99 2 6 . 2 9 - 0.73 25.11 +--3.23
p-Nitrophenyl phosphatase
I 6-8 13-15
1.99 ± 0.13 1.79 ± 0.18 1.80 ± 0 . 1 6
0.24 ± 0.01 0.22 ± 0.02 0.23 ± 0.02
aSignificantly ditierent (p -< 0.05) from those of 1-month-old animals. bSignificantly ditterent (p - 0.05) from those of 6-8-month-old animals.
8.26 ± 0.92 8.02---0.17 7.31 ± 1.2
154
differ
significantly
from
somal
contamination
dicated
by several
activities
one
age group
in highly workers
of 5’-nucleotidase
to another.
purified
using
The presence
sarcolemmal
different
of membrane
methods
of this micro-
preparations and animal
and homogenate
animals were significantly different from those derived and 13-15-month-old animals. On the other hand,
has been species
derived
in-
121). The
from younger
from those of 6-&monththe specific activities of
p-nitrophenyl phosphatase in both membranes and homogenates were not influenced by age. This confirms our earlier observation that S’-nucleotidase activity activity
decreases remains
with
the
unchanged
increase
in age
while
p-nitrophenyl
phosphatase
[S].
Efiect of age on the kinetics of 5’-nucleotidase The results of the experiments on the kinetic properties (K, and V,,,,,) of sarcolemma-bound 5’-nucleotidase from rats of different age groups are shown in Table II and Fig. 1. The K, value for sarcolemmal 5’-nucleotidase from l-monthold animals
was significantly
lower
than
those
from
&8-
and
13-15-month-old
5 6-
4 .8 -
4 .o -
3 [Sl v
2. 4-
I .6-
0 I .’ I
_-L -0.8
0.8
I
1.6
I
2.4
I
I
3.2
4.0
I 4.8
IS1 Fig. 1. Woolf plot of 5’-nucleotidase from cardiac sarcolemma of rats of three different ages. The concentrations of AMP used ranged from 100 to 4000 PM. The velocity of the enzymatic reaction was measured in nmol substrate per mg protein per min. Each point in the graph represents the average of 3 separate experiments.
155
TABLE
It
Km A N D Vmax V A L U E S O F 5 ' - N U C L E O T I D A S E OF DIFFERENT AGES
OF CARDIAC
SARCOLEMMA
FROM
RATS
Each value is the mean -+ S.E. o f 3 separate experiments.
Age (months)
1 6-8 13-15
Km (IzM)
Vma~ (nmol per mg protein per rain)
493.21 - 14.49 735.99 ± 4 2 . 9 9 ~ 791.10 -+ 90.71 a
1446.3 ± 130.0 994.66 ± 26.69~ 9 2 5 . 3 4 -+ 17.56"
aSignificantly different (p --< 0.05) from those of l-month-old animals.
animals. But there were no significant differences in Km and V~.ax between 6-8-month- and 13-15-month-old animals. The values of Km we obtained for 5'-nucleotidase in sarcolemma from 6-8- and 13-15-month-old animals are close to those reported by other workers [22].
Lipid composition of cardiac sarcolemma The lipid contents of heart sarcolemma derived from rats of different ages are given in Table III. The concentrations of cholesterol and phospholipid in these membranes did not change with age. Accordingly, the cholesterol/phospholipid ratio also was not influenced by the age of the animal. It was found also that the total lipids (cholesterol + phospholipid) per mg of protein was not influenced by age, which implies that the protein concentration of these membranes was maintained constant. The fatty acid compositions of the sarcolemma lipids from the three age groups are shown in Table IV. The concentration of saturated fatty acids in the cardiac sarcolemma consistently increased with the increase in age whereas the concentration of polyunsaturated fatty acid decreased with the increase in age. The concentration of monounsaturated fatty acids in the membranes of l-month-old animals was significantly higher than those of 6-8-monthold animals but reached the same level by 13-15-month-old animals. Alterations TABLE
III
EFFECT OF DEVELOPMENT CARDIAC SARCOLEMMA
ON
THE
LIPID
CONTENT
(#g/rag
PROTEIN)
Each value is the mean +- S.E. of 4 experiments.
Age (months)
Cholesterol
Phospholipid
Cholesterol/phospholipid
1 6-8 13-15
2 3 2 . 5 4 _+ 17.71 187.53 - 9 . 3 8 206.08-+0.95
8 0 8 . 6 4 _+ 73.41 775.73 _ 6 2 . 8 8 846.55-+87.90
0.31 -+ 0.02 0 . 2 4 - 0.05 0.25±0.04
OF
RAT
156 TABLE IV FATTY ACID COMPOSITION OF CARDIAC SARCOLEMMA FROM RATS OF DIFFERENT AGES Values (in per cent) are the mean ± S.E. of 3 samples.
Age (months) Fatty acids
1
6-8
13--15
14: 0 14 : 1 16 : 0 16: 1 18 : 0 18 : 1 18 : 2 20 : 4 22 : 6 Others Saturated Monounsaturated Polyunsaturated
tr~ tr 13.36 ± 0.87 tr 20.93 +- 0.06 18.99 +_0.70 17.09 ± 0.88 20.16 -+ 0.50 3.73 _+O. 15 4.26 -+ 0.80 34.64 _+0.84 20.12 ± 0.73 40.98 +_0.66
0.7 ± 0.1 tr 14.2 4- 0.85 0.61) -+ 0.35 .34.60 _+ 1.05b 13.50 -+ 0.35 b 13.30 4- 0.25 ° 17.80 -+ 0.70 4.11 ± 0.25 0.51 -+ 0.01 49.51) + 0,65 b 14.78 ± 0.32° 35.20 + 0,69°
1.25 ± 0.54 0.61) -+ (}.3{I 18.16 -+ 1.35b~ 0.66 _+0.05 29.65 +_ 1.52b 14.49 + 1.22b 12.29 +- 0.62b 16.40 +_ 1.52° 3.74 ± 0.61) 2.42 +_0.72 49.06 _+0.23° 16.09 ± 1.54 32.93 _+2.2(P
a < 0.5% of total fatty acids. bSignificantly different (p --<0.05) from those of l-month-old animals. cSignificantly different (p -< 0.05) from those of 6-8-month-old animals.
in s i n g l e f a t t y a c i d s w e r e concentrations The
concentration months
during development.
o f 1 6 : 0 a n d 1 8 : 0 in t h e c a r d i a c s a r c o l e m m a
concentration
decreased
also observed
of oleic acid, on the other
of t w o p o l y u n s a t u r a t e d
with development.
w h i l e it w a s d e l a y e d
hand,
fatty acids,
The decrease
For example,
i n c r e a s e d w i t h age.
decreased
i.e. 1 8 : 2
the
w i t h age. T h e
and 20:4,
steadily
in 1 8 : 2 w a s n o t i c e d as e a r l y as 6--8
to 13-15 months
with respect to arachidonic
acid
concentration. DISCUSSION The results of these experiments sarcolemmal
5'-nucleotidase
are
animal develops, 5'-nucleotidase in Km s u g g e s t s a d e c r e a s e enzyme.
On
the other
(6-15 months) membranes. extractability enzymes
indicate that both
influenced
in t h e b i n d i n g affinity o f t h e s u b s t r a t e
hand,
of
could
not
the decrease
sarcolemma
be
explained
from
the
in 5 ' - n u c l e o t i d a s e in t h e e n z y m e on
the
As the
o f t h e a n i m a l ( T a b l e I).
(AMP)
Vma x
to the
in o l d e r a g e
concentration
in t h e s e
basis of differences
myofilaments
since the purification factors were maintained
development
Vmax o f c a r d i a c
Km i n c r e a s e s w h i l e Vmax d e c r e a s e s . T h e i n c r e a s e
could indicate a reduction
This
K m and
by the age of the animal.
and
its e f f e c t
in t h e
on
constant throughout
these the
157
The information regarding the effect of age on 5'-nucleotidase kinetics is very limited. The only studies that we are aware of are those of Agnisola et al. [22]. These authors reported an increase in Km value for liver plasma membrane 5'-nucleotidase, which is in agreement with our observation. On the other hand, with the exception of one of their age groups, they found that 5'-nucleotidase Vm,x was not influenced by age. However, one should take their results with caution since their liver membranes were crude whei'eas ours are purified membranes. The observed alterations in sarcolemmal fatty acid composition as the animal develops may throw some light on the mechanism by which development influences the Km of the 5'-nucleotidase. The decrease in polyunsaturated fatty acid content of these membranes in the older age could alter the fluidity of these membranes. Several studies indicated that the fluidity of the membranes influences the kinetic properties of several enzymes [23-25]. Our earlier work with Ehrlich ascites tumor cells indicated that the enrichment of their plasma membranes with polyunsaturated fatty acids in vivo resulted in a decrease in the K~ of the Na+-dependent a-aminoisobutyric acid uptake [26]. This uptake system has similar kinetic properties to those of enzymes [27]. The decrease in Vmaxof sarcolemmal 5'-nucleotidase in older ages (6-8 and 13-15 months) could be interpreted as a reduction in the rate of enzyme synthesis by the myocardial cells. Several studies have indicated similar observations with other enzymes [28, 29]. ACKNOWLEDGMENT
This work was supported by grant 81-04-002 from the National Osteopathic Foundation. REFERENCES 1 H. Fleit, M. Conklyn, R.D. Stebbius and R. Silber, Function of 5'-nucleotidase in the uptake of adenosine from AMP by human lymphocytes. J. Biol. Chem., 250 (1975) 8889-8892. 2 G.P. Frick and J.M. Lowenstein, Vectorial production of adenosine by 5'-nucleotidase in the perfused rat heart. J. Biol. Chem., 253 (1978) 1240-1244. 3 R. Rubio and R.M. Berne, Release of adenosine by the normal myocardium in dogs and its relationship to the regulation of coronary resistance. Circ. Res., 25 (1969) 407---415. 4 R.M. Berne, Cardiac nucleotides in hypoxia: Possible role in regulation of coronary blood flow. Am. J. Physiol., 204 (1963) 317-322. 5 A.B. Awad and S.W. Clay, Age-dependent alterations in lipids and function of rat heart sarcolemma. Mech. Ageing Dev., 19 (1982) 333-342. 6 H. Sandermann, Jr., Regulation of membrane enzymes by lipids. Biochim. Biophys. Acta, 515 (1978) 2~--237. 7 G.Y. Sun and A.Y. Sun, Synaptosomal plasma membranes: Acyl group composition of phosphoglycerides and (Nat + K+)-ATPase activity during fatty acid deficiency. J, Neurochem., 22 (1974) 15--18. 8 J. Bernsohn and F.J. Spitz, Linoleic and linolenic acid dependency of some brain membrane-bound enzymes after lipid deprivation in rats. Biochem. Biophys. Res. Commun., 57 (1974) 293--298. 9 D.M. Bers, Isolation and characterization of cardiac sarcolemma. Biochim. Biophys. Acra, 555 (1979) 131-146.
158
10 M.B. Lees and S. Paxman, Modification of the Lowry procedure for the analysis of proteolipid protein. Anal. Biochem., 47 (1972) 184-192. 11 O. Touster, N.N. Aronson, Jr., J.T. Dulaney and H. Hendrickson, Isolation of rat liver plasma membranes. Use of nucleotide pyrophosphatase and phosphodiesterase-I as marker enzymes. J. Cell Biol., 47 (1970) 604-618. 12 C.H. Fiske and Y. Subbarow, The colorimetric determination of phosphorus. J. Biol. Chem., 66 (1925) 375--400. 13 D.E. Green, S. Mii and P.M. Kolut, Studies on the terminal electron transport system, i-Succinic dehydrogenase. J. Biol. Chem., 217 (1955) 551-567. 14 G.L. Sottocasa, B. Kuylenstierna, L. Ernster and A. Bergstrand, An electron transport system associated with the outer membrane of liver mitochondria. A biochemical and morphological study. J. Cell Biol., 32 (1967) 415-437. 15 H.N. Christensen and G.A. Palmer, Enzyme Kinetics, W.B. Saunders Company, Philadelphia, London, 1967, pp. 101-103. 16 J. Foleh, M. Lees and G.H. Sloane-Stanley, A simple method for the isolation and purification of total lipids from animal tissues. J. Biol. Chem., 226 (1957) 497-509. 17 S. Pollet, S. Ermidou, F.L. Saux, M. Monge and N. Baumann, Microanalysis of brain lipids: Multiple two dimensional thin layer chromatography. J. Lipid Res., 19 (1978) 916--921. 18 R.K. Raheja, C. Kaur, A. Singh and I.S. Bhatia, New colorimetric method for the quantitative estimation of phospholipids without acid digestion. J. Lipid Res., 14 (1973) 695~697. 19 W.R. Morrison and L.M. Smith, Preparation of fatty acid methyl esters and dimethylacetate from lipids with boron fluoride-methanol. J. Lipid Res., 5 (1964) ~XI-~8. 20 M.M. Hosey, Chick heart membranes: isolation and analysis of autophosphorylation. Biochim. Biophys. Acta, 690 (1982) 106-116. 21 W.B. Wegticki, K. Owens, F.F. Kennel, A. Kessner, L. Harris, R.M. Wise and G.V. Vahouny, Preparation and properties of highly enriched cardiac sarcolemma from isolated adult myocytes. J. Biol. Chem., 255 (1980) 3605--3609. 22 C. Agnisola, L. Foti and I.T. Genoino, Influence of age on 5'-nucleotidase in plasma membranes isolated from rat liver. Mech. Ageing Dev., 13 (1980) 227-239. 23 A. Solyom and E.G. Trams, Enzyme markers in characterization of isolated plasma membranes. Enzyme, 13 (1972) 329-372. 24 R.P. Brivio-Haugland, S.L. Louis, K. Musch, N, Waldeck and M.A. Willian~s, Liver plasma membranes from essential fatty acid deficient rats: isolation, fatty acid composition and activities of 5'-nucleotidase, ATPase and adenylate cyclase. Biochim. Biophys. Acta, 43,? (1976~150-163. 25 B. Bloj, R.D. Morero, R.N. Farias and R.E. Trucco, Membrane lipid fatty acids and regulation of membrane-bound enzymes: Allosteric behavior of erythrocyte Mg2+-ATPase, (Na÷+ K+)-ATPase and acetylcholinesterase from rats fed different fat-supplemented diets. Biochim. Biophys. Acta, 311 (1973) 67-79. 26 T.L. Kaduce, A.B. Awad, L.J. Fontenelle and A.A. Spector, Effect of fatty acid saturation on a-aminoisobutyric acid transport in Ehrlich ascites cells. J. Biol. Chem., 19 (1977) 6624--6630. 27 D.L. Oxender and H.N. Christensen, Distinct mediating systems for transport of neutral amlno acids by the Ehrlich cell. J. Biol. Chem., 238 (1963) 3686-3699. 28 R.G. Hansford and F. Castro, Age linked changes in the activity of enzymes of the tricarboxylate cycle and lipid oxidation, and of carnitine content, in muscles of the rat. Mech. Ageing Dev., 19 (1982) 191-201. 29 S.W. O'Connor, P.J. Scarpace and I.B. Abrass, Age-associated decrease of adenylate cyclase activity in rat myocardium. Mech. Ageing Dev., 16 (1981) 91-95.