R. A. Harkness, R. J. Simmonds, and M. C. O'Connor. Urate Metabolism i n a Mongrel .... K. Hugh-Jones, and D. P e r r e t t. Complete Adenosine Deaminase ...
PURINE METABOLISM IN M A N - I I I Biochemical, Immunological, and Cancer Research
E d i t e d by
Aurelio Rapado Fundacion Jimenez Diaz Madrid, Spain
R.W.E. Watts M.R.C. Clinical Research Centre Harrow, England
and
Chris H.M.M. De Bruyn Department of Human Genetics University of Nijmegen Faculty o f Medicine Nijmegen, The Netherlands
PLENUM PRESS · NEW YORK AND LONDON
Contents of Part Β
I.
PURINE METABOLISM PATHWAYS AND REGULATION A. De Novo S y n t h e s i s ; P r e c u r s o r s a n d R e g u l a t i o n
De Novo P u r i n e S y n t h e s i s i n C u l t u r e d Human Fibroblasts R.B. G o r d o n , L. Thompson, L.A. J o h n s o n , a n d B.T. Emmerson C o m p a r a t i v e M e t a b o l i s m o f a New A n t i l e i s h m a n i a l Agent, A l l o p u r i n o l Riboside, i n t h e P a r a s i t e and t h e Host C e l l D. J . N e l s o n , S.W. L a F o n , G.B. E l i o n , J . J . M a r r , and R.L. B e r e n s Purine Metabolism i n Rat S k e l e t a l Muscle E. R. T u l l y a n d T.G. S h e e h a n A l t e r a t i o n s i n Purine Metabolism i n Cultured F i b r o b l a s t s w i t h HGPRT D e f i c i e n c y a n d w i t h PRPPP S y n t h e t a s e S u p e r a c t i v i t y E. Z o r e f - S h a n i a n d 0. S p e r l i n g Purine Metabolism i n Cultured Endothelial Cells S. N e e s , A.L. G e r b e s , B. Willershausen-Zönnchen, and E. G e r l a c h Determinants o f 5-Phosphoribosyl-l-Pyrophosphate (PRPP) S y n t h e s i s i n Human F i b r o b l a s t s K.0, R a i v i o , Ch. L a z a r , H. K r u m h o l z , a n d M.A. B e c k e r X a n t h i n e O x i d o r e d u c t a s e I n h i b i t i o n b y NADH as a Regulatory Factor o f Purine Metabolism M.M. J e z e w s k a a n d Z.W. K a m i n s k i
vii
1
7
13
19
25
31
35
viii
CONTENTS OF PART Β
Β. N u c l e o t i d e M e t a b o l i s m Human P l a c e n t a l A d e n o s i n e K i n a s e : P u r i f i c a t i o n and C h a r a c t e r i z a t i o n C M . A n d r e s , T.D. P a l e l l a , a n d I . H . F o x
41
Long-Term E f f e c t s o f Ribose on Adenine N u c l e o t i d e Metabolism i n Isoproterenol-Stimulated Hearts H.-G. Z i m m e r , Η. I b e l , G. S t e i n k o p f f , a n d H. K o s c h i n e C. S a l v a g e
. .
45
Pathways
P u r i n e S a l v a g e Enzymes i n Man a n d L e i s h m a n i a d o n o v a n i T.A. K r e n i t s k y , G.W. K o s z a l k a , J.V. T u t t l e , D. L. A d a m c z y k , G.B. E l i o n , a n d J . J . M a r r R e g u l a t i o n o f P u r i n e S a l v a g e Enzymes i n Έ. R.A. L e v i n e a n d M.W.
coli
. . .
51
57
Taylor
D. C a t a b o l i s m P u r i n e T r a n s p o r t and t h e C e l l C y c l e M.P. R i v e r a , M.R. G r a u , J . R i g a u , and A. Goday
61
H y p o x a n t h i n e T r a n s p o r t i n Human E r y t h r o c y t e s C. S a l e r n o a n d G i a c o m e l l o
69
U p t a k e o f A d e n o s i n e i n Human E r y t h r o c y t e s M. K r a u p p , P. C h i b a , a n d M.M. Müller
73
Effect
o f A c t i n o m y c i n D on i n v i v o P u r i n e B i o s y n t h e s i s i n Hamster C e l l s M.W. T a y l o r , K.C. G u p t a , a n d L. Z a w i s t o w i c h
Purine Catabolism i n I s o l a t e d Hepatocytes: Influence o f Coformycin G. V a n d e n B e r g h e , F. B o n t e m p s , a n d H.G.
II.
79
85 Hers
ENZYMOLOGY AND PURINE METABOLISM A. P h o s p h o r i b o s y l t r a n s f e r a s e s
I n a c t i v a t i o n o f H y p o s a n t h i n e Guanine Phosphor i b o s y l t r a n s f e r a s e by Guanosine D i a l d e h y d e : An A c t i v e S i t e D i r e c t e d I n h i b i t o r L.A, J o h n s o n , R.B, G o r d o n , a n d B.T. Emmerson
87
CONTENTS OF PART Β
ix
Role
o f Human H y p o x a n t h i n e G u a n i n e P h o s p h o r i b o s y l transferase i nNucleotide Interconversion . . . . A. G i a c o m e l l o a n d C. S a l e r n o
93
P u r i f i c a t i o n and C h a r a c t e r i z a t i o n o f Mammalian Adenine P h o s p h o r i b o s y l t r a n s f e r a s e M.W. T a y l o r and H.V. H e r s h e y The
103
Effect
W.
o f Phosphoribosylpyrophosphate on S t a b i l i t y and C o n f i g u r a t i o n o f Hypoxant h i n e g u a n i n e p h o s p h o r i b o s y l t r a n s f e r a s e and Adeninephosphoribosyltransferase from Human E r y t h r o c y t e s Gröbner a n d N. Zöllner
I
Chemical M o d i f i c a t i o n o f Hypoxanthine-phosphor i b o s y l t r a n s f e r a s e and I t s P r o t e c t i o n by S u b s t r a t e s and P r o d u c t s W.. G u t e n s o h n a n d H. J a h n Β.. N u c l e o s i d e
Phosphoribosylating
l
l
117
Enzymes
Structural
J,A.
S t u d i e s o f Human A d e n i n e P h o s p h o r i b o s y l t r a n s f e r a s e P u r i f i e d by A f f i n i t y Chromatography H o l d e n , G.S. M e r e d i t h , a n d W.N. K e l l e y
123
Phosphoribosylpyrophosphate (PRPP) S y n t h e t a s e Mutant i n Salmonella typhimurium B,. J o c h i m s e n , Β. G a r b e r , a n d J.S. G o t s C. N u c l e o s i d e
Cleaving
131
Enzymes
Methylmereaptopurine Ribonucleoside T o x i c i t y i n Human F i b r o b l a s t s : I n h i b i t i o n o f P h o s p h o r i b o s y l p y r o p h o s p h a t e S y n t h e t a s e as w e l l as A m i d o p h o s p h o r i b o s y l t r a n s f e r a s e R..C.K. Yen a n d M.A. B e c k e r
137
A d e n o s i n e Kinase: R e g u l a t i o n by S u b s t r a t e s , M a g n e s i u m , a n d pH R..L. M i l l e r and D.L. Adamczyk A d e n o s i n e and D e o x y a d e n o s i n e K i n a s e f r o m Rat L i v e r N.. O g a s a w a r a , Y. Yamada, a n d H. G o t o
145
. . . .
151
D- D e a m i n a t i n g Enzymes R a d i o i m m u n o c h e m i c a l A n a l y s i s o f Human E r y t h r o c y t e Adenosine Deaminase P..E. Daddona, M.A. F r o h m a n , a n d W.N. K e l l e y
157
χ
CONTENTS OF PART Β
A d e n o s i n e Deaminase C o n v e r s i o n P r o t e i n s : A P o t e n t i a l Role P.P. T r o t t a a n d M.E. B a l i s R e g u l a t o r y P r o p e r t i e s o f AMP D e a m i n a s e I s o z y m e s N. O g a s a w a r a , H. G o t o , a n d Y. Yamada
163
169
Human A d e n o s i n e D e a m i n a s e : S t o i c h i o m e t r y o f t h e L a r g e F o r m Complex P.E. Daddona a n d W.N. K e l l e y
177
Guanase f r o m Human L i v e r - P u r i f i c a t i o n a n d Characterization R. K u z m i t s , H. S t e m b e r g e r , and M.M. Müller
183
Ε. O x i d a t i n g
Enzymes
N i c o t i n a m i d e and L i v e r X a n t h i n e Oxidase A. D i S t e f a n o , Μ. P i z z i c h i n i , a n d Ε. M a r i n e l l o X a n t h i n e O x i d a s e A c t i v i t y i n Human I n t e s t i n e s . H i s t o c h e m i c a l and R a d i o c h e m i c a l Study C. A u s e h e r , Ν. Amory, P. v a n d e r Kemp, and F. D e l b a r r e
III.
189
197
PYRIMIDINE METABOLISM
P u r i n e and P y r i m i d i n e M e t a b o l i s m i n H e r e d i t a r y O r o t i c a c i d u r i a D u r i n g a 15-Year Follow-Up Study D. R. W e b s t e r , H.A. Simmonds, C.F. P o t t e r , a n d D.M.O. B e c r o f t o f A l l o p u r i n o l on P y r i m i d i n e M e t a b o l i s m i n Human W h i t e B l o o d C e l l s : R o l e o f t h e Salvage Pathway P. B a n h o l z e r , W. Gröbner, a n d N. Zöllner
203
Effect
Kinetics E. H.
and Compartmentation o f E r y t h r o c y t e Pyrimidine Metabolism H a r l e y , P. Z e t l e r , a n d S. N e a l
S i m u l t a n e o u s D e t e r m i n a t i o n o f Rates o f P u r i n e and P y r i m i d i n e S y n t h e s i s i n C u l t u r e d Human Lymphoblasts and F i b r o b l a s t s W.H. H u i s m a n , K.O. R a i v i o , a n d M.A. B e c k e r o f Enzymes o f P u r i n e a n d P y r i m i d i n e M e t a b o l i s m i n Nine Mycoplasma Species M. Hamet, C. B o n i s s o l , a n d P. C a r t i e r
209
217
223
Activities
231
CONTENTS OF PART Β
IV.
x«
LYMPHOCYTE PURINE METABOLISM RESEARCH
I n c r e a s e s i n P u r i n e E x c r e t i o n and Rate o f S y n t h e s i s b y D r u g s I n h i b i t i n g IMP D e h y d r o g e n a s e o r Adenylosuccinate Synthetase A c t i v i t i e s R.C. W i l l i s a n d J.E. S e e g m i l l e r
237
T
P o s s i b l e Role f o r 5 - N u c l e o t i d a s e i n Deoxyadenosine S e l e c t i v e T o x i c i t y t o C u l t u r e d Human Lymphoblasts R.L. W o r t m a n n , B.S. M i t c h e l l , N.L. E d w a r d s , and I . H . F o x
243
C y c l i c N u c l e o t i d e L e v e l s and Mechanism o f I n h i b i t i o n of Leucocyte F u n c t i o n by Adenosine Deaminase I n h i b i t i o n A.D. M e i s e l , Ch. N a t a r a j a n , G. S t e r b a , a n d H.S. D i a m o n d
251
Purine R i b o n u c l e o s i d e and D e o x y r i b o n u c l e o s i d e M e t a b o l i s m i n Thymocytes F.F. S n y d e r a n d T. L u k e y
259
Molecular Mechanism(s) o f Deoxyribonucleoside i n T-Lymphoblasts J.M. W i l s o n , B.S. M i t c h e l l , a n d W.N. K e l l e y
265
I n h i b i t i o n o f Immune C e l l F u n c t i o n b y A d e n o s i n e : Biochemical Studies T.P. Zimmerman, G. W o I b e r g , G.S. D u n c a n , R.D. D e e p r o s e , a n d R.J. H a r v e y I n t e r a c t i o n s Between Energy M e t a b o l i s m and Adenine N u c l e o t i d e M e t a b o l i s m i n Human Lymphoblasts S.S. M a t s u m o t o , K.O. R a i v i o , R.C. W i l l i s , and J.E. S e e g m i l l e r Enzymes o f P u r i n e I n t e r c o n v e r s i o n s i n S u b f r a c t i o n s of Lymphocytes J.P.R.M. v a n L a a r h o v e n , G.Th. S p i e r e n b u r g , C.H.M.M. De B r u y n , a n d E.D.A.M. S c h r e t l e n Measurement o f t h e R a t e s o f S y n t h e s i s and D e g r a d a t i o n of Hypoxanthine-guanine phosphoribosylt r a n s f e r a s e i n Human L y m p h o b l a s t s P. Moore M a t t e s a n d W.N. K e l l e y
271
277
283
289
xü
CONTENTS O F PART Β 1
Human 5 - N u c l e o t i d a s e . P r o p e r t i e s a n d C h a r a c t e r i z a t i o n o f t h e Enzyme f r o m P l a c e n t a . L y m p h o c y t e s and L y m p h o b l a s t o i d C e l l s i n C u l t u r e W. G u t e n s o h n Metabolism
D. A.
V.
and T o x i c i t y o f 9-Beta-D-Arabinof u r a n o s y l a d e n i n e i n Human M a l i g n a n t Τ C e l l s and Β C e l l s i n T i s s u e C u l t u r e C a r s o n , J . K a y e , a n d J.E. S e e g m i l l e r
295
299
IMMUNE SYSTEM AND PURINE METABOLISM
S u p p r e s s i o n o f C e l l u l a r I m m u n i t y Due t o I n h i b i t i o n o f P u r i n e N u c l e o s i d e Phosphorylase by Allopurinol-Riboside Y. N i s h i d a , N. K a m a t a n i , K. T a n i m o t o , and I . Akaoka
309
T
Lymphocyte 5 - N u c l e o t i d a s e D e f i c i e n c y : C l i n i c a l and M e t a b o l i c C h a r a c t e r i s t i c s o f t h e A s s o c i a t e d Hypogammaglobulinemia N.L. E d w a r d s , J.T. C a s s i d y , a n d I . H . Fox I m m u n o l o g i c a l S t u d i e s on Lesch-Nyhan P a t i e n t s C.H.M.M. d e B r u y n , Ph. G a u s s e t , J . D u c h a t e a u , E. Vamos, S. K u l a k o w s k i a n d G. D e l e s p e s s e Activity
L.F. J.E.
VI.
315
321
T
o f Ecto-5 -Nucleotidase i n Lymphoblastoid C e l l Lines Derived from C a r r i e r s o f C o n g e n i t a l X-Linked Agammaglobulinemia Thompson, G.R. B o s s , A. B i a n c h i n o , a n d Seegmiller
327
CANCER RESEARCH
A d e n o s i n e Deaminase and P u r i n e N u c l e o s i d e P h o s p h o r y l a s e i n Acute and C h r o n i c Lymphatic Leukemia H. L u d w i g , Η. W i n t e r l e i t n e r , R. K u z m i t s , a n d M.M. Müller P u r i n e S a l v a g e Enzymes i n L y m p h o c y t e s a n d Granulocytes from Patients w i t h S m a l l - C e l l Carcinoma o f t h e Lung P. N y g a a r d a n d J . M e j e r
333
339
CONTENTS OF PART Β
xiii
Treatment o f Acute L y m p h o b l a s t i c Leukemia w i t h t h e A d e n o s i n e Deaminase I n h i b i t o r 2 -Deoxycoformycin B.S. M i t c h e l l , Ch.A. K o l l e r , a n d W.N. K e l l e y r
347
Increase of Phosphoribosylpyrophosphate Levels i n C u l t u r e d L1210 Leukemia C e l l s Exposed t o Methotrexate J.M. B u e s a , A. L e y v a , a n d H.M. P i n e d o
351
P u r i n e Salvage Pathway i n Leukemic C e l l s A. Goday, M.R. G r a u , I . J a d r a q u e , a n d M.P. R i v e r a
357
B i o c h e m i c a l Consequences o f T r e a t m e n t w i t h t h e A d e n o s i n e Deaminase I n h i b i t o r 2'-Deoxycoformycin R.M. P a i n e , J . F . S m y t h , a n d K.R. H a r r a p
365
In vitro
and i n v i v o E f f e c t o f D e o x y c o f o r m y c i n i n Human Τ C e l l L e u k e m i a A.L. Y u , F.H. K u n g , B. B a k a y , a n d W.L. Nyhan
373
Uniqueness
o f Deoxyribonucleotide Metabolism i n Human M a l i g n a n t Τ C e l l L i n e s D.A. C a r s o n , J . K a y e , S. M a t s u m o t o , J.E. S e e g m i l l e r , a n d L . Thompson
VII.
381
METHODOLOGY
H i g h Performance L i q u i d Chromatography o f Plasma P y r i m i d i n e s and P u r i n e s and I t s A p p l i c a t i o n i n Cancer Chemotherapy A. L e y v a , J . S c h o r n a g e l , a n d H.M. P i n e d o U r a t e - B i n d i n g P r o t e i n s i n Plasma S t u d i e d by A f f i n i t y Chromatography M.L. C i o m p i , A. L u c a c c h i n i , D. S e g n i n i , and M.R. M a z z o n i
395
Chromatographic D e t e r m i n a t i o n o f PRPP-Synthetase A c t i v i t y i n Human B l o o d C e l l s P. N y g a a r d a n d K.F. J e n s e n P u r i f i c a t i o n o f M y o c a r d i a l Adenosine Kinase Using A f f i n i t y and Ion-Exchange Chromatography M.P. U i t e n d a a l , J.W. De J o n g , E. H a r m s e n , and E. K e i j z e r
389
401
. . - .
409
xiv
CONTENTS OF PART Β
M i c r o m e t h o d s f o r t h e Measurement o f P u r i n e Enzymes i n L y m p h o c y t e s J.P.R.M. v a n L a a r h o v e n , G.Th. Spierenburg, F . T . J . J . O e r l e m a n s , and C.H.M.M. De B r u y n A R a p i d S c r e e n i n g Method f o r I n b o r n E r r o r s o f P u r i n e and P y r i m i d i n e M e t a b o l i s m U s i n g Isotachophoresis H.A. Simmonds, A. S a h o t a , and R. Payne A n a l y s i s o f Serum P u r i n e s and P y r i m i d i n e s b y Isotachophoresis F. O e r l e m a n s , Th. V e r h e g g e n , F. M i k k e r s , F. E v e r a e r t s , a n d C.H.M.M. De B r u y n D e t e r m i n a t i o n o f U r i c A c i d i n Serum: C o m p a r i s o n o f a S t a n d a r d E n z y m a t i c M e t h o d and I s o t a c h o phoresis F. O e r l e m a n s , Th. V e r h e g g e n , F. M i k k e r s , F. E v e r a e r t s , and C.H.M.M. De B r u y n A d d i t i o n P r o d u c t s o f U r i c A c i d and P.A. S i m k i n and Q.P. L e e Automated R e t r i e v a l of Purine L. F e r r e i r o and A. Rey Author Subject
Index Index
Formaldehyde
Literature
415
421
429
435
441
445
449 453
Contents of Part A
I. The
The
CLINICAL GOUT N a t u r a l H i s t o r y o f H y p e r u r i c e m i a Among Asymptotic Relatives of Patients W i t h Gout T.-F. Y i i and C. Kaung C l i n i c a l D i f f e r e n t i a t i o n o f P r i m a r y Gout From P r i m a r y R e n a l D i s e a s e i n P a t i e n t s W i t h B o t h Gout and R e n a l D i s e a s e Β. T. Emmerson, P. J . S t r i d e a n d G. W i l l i a m s
R e n a l F a i l u r e i n Young S u b j e c t s W i t h F a m i l i a l Gout H. A. Simmonds, J . S. Cameron, C. F. P o t t e r , D. W a r r e n , T. G i b s o n , a n d D. F a r e b r o t h e r S t u d y o f L i p i d and P u r i n e L e v e l s i n G o u t P a t i e n t s and A n a l y s i s o f M o r t a l i t y L. G. D a r l i n g t o n , J . S l a c k , and J . T. S c o t t
.
1
9
15
Family
U r i c A c i d T u r n o v e r i n N o r m a l s , i n G o u t and i n Chronic Renal F a i l u r e Using C-Uric Acid C. V i t a l i , G. P a s e r o , A, C l e r i c o , L. R i e n t e , N. M o l e a , A. P i l o , G. M a r i a n i , a n d R. B i a n c h i
21
1 4
E r y t h r o c y t e Adenosine-Deaminase A c t i v i t y i n Gout a n d H y p e r u r i c e m i a A. C a r c a s s i , P. M a c r i , G. C h i a r o n i , and S. B o s c h i C l i n i c a l V a r i a b i l i t y of t h e Gouty D i a t h e s i s W. J . A r n o l d and R. A. Simmons
xv
27
33
39
CONTENTS O F PART A
xvi
C l i n i c a l F e a t u r e s o f 4,000 G o u t y i n Japan N. N i s h i o k a a n d K. M i k a n a g i
Subjects 47
F r e q u e n c y o f C h o n d r o c a l c i n o s i s o f t h e Knees a n d A v a s c u l a r N e c r o s i s o f t h e F e m o r a l Heads i n Gout, a C o n t r o l l e d Study A. S t o c k m a n , L. G. D a r l i n g t o n , a n d J . T. S c o t t Gaschromatographic E v a l u a t i o n o f U r i n a r y 1 7 - K e t o s t e r o i d s , E t i o c h o l a n o l o n e and Dehydroepiandrosterone i n P r i m a r y Gout and H y p e r u r i c e m i a A. C a r c a s s i , F. L o r e , G. M a n a s s e , P. M a c r i , and M. P i s a n o Hormonal Aspects o f Gouty P a t i e n t s U. V a l e n t i n i , G. R i a r i o - S f o r z a , R. and E. M a r i n e l l o
59
65 Marcolongo,
Determination o f Tubular Secretion o f Urate i n H e a l t h y a n d G o u t y Men L. B. S o r e n s e n a n d D. J . L e v i n s o n Ribose T o l e r a n c e i n Gouty P a t i e n t s M. P i z z i c h i n i , R. M a r c o l o n g o , a n d E. M a r i n e l l o
II.
55
73
81
URIC ACID AND RENAL STONES
U r i n a r y Urate and U r i c A c i d R e l a t i v e S a t u r a t i o n i n N o r m o u r i c u r i c C a l c i u m O x a l a t e Stone Formers W i t h Normal U r i n a r y Calcium Oxalate Saturation M. Labeeuw, C. G e r b a u l e t , N. P o z e t , P. Z e c h , and J . T r a e g e r C o r r e l a t i o n Between t h e U r i c A c i d and C a l c i u m C o n c e n t r a t i o n i n U r i n e . R e s u l t s o f a Long Term Study on R e c u r r e n t Stone-Formers and Healthy Controls P. L e s k o v a r , R. Härtung, a n d M. K r a t z e r I n t e r a c t i o n o f H y p e r u r i c u r i a and H y p e r o x a l u r i a on R e n a l C a l c i u m O x a l a t e Stone F o r m a t i o n F. H e r i n g , Κ.-Η. B i g a l k e , a n d W. L u t z e y e r Uric Acid/Calcium Oxalate N e p h r o l i t h i a s i s . C l i n i c a l and Biochemical F i n d i n g s i n 86 P a t i e n t s A. Rapado, J.M. C a s t r i l l o , M. D i a z - C u r i e l , M. L. T r a b a , M. S a n t o s , L . C i f u e n t e s - D e l a t t e
87
93
99
109
CONTENTS OF PART Α
^,.
The U r i c A c i d : C y s t i n e C o r r e l a t i o n i n t h e U r i n e of R e c u r r e n t Calcium Oxalate Stone-Formers and H e a l t h y C o n t r o l s P. L e s k o v a r , R. Härtung, a n d M. K r a t z e r The R o l e
of Urate i n Idiopathic Urolithiasis S. R. S i l c o c k
115
Calcium 121
M i n e r a l o g i e C o m p o s i t i o n o f 66 M i x e d U r i n a r y C a l c u l i o f C a l c i u m O x a l a t e and U r i c Acid J . R. M i n o n - C i f u e n t e s , M. S a n t o s , a n d L. C i f u e n t e s - D e l a t t e
129
H y p e r u r i c e m i a and C y s t i n u r i a F. L i n a r i , M. M a r a n g e l l a , B. M a l f i , G. V a c h a , Μ. B r u n o , G. G i o r c e l l i , a n d B. F r u t t e r o
135
M o n o s o d i u m U r a t e M o n o h y d r a t e as S p h e r u l i t e s J . J . F i e c h t n e r a n d P. A. S i m k i n
141
III.
CLINICAL AND
PHYSIOLOGICAL ASPECTS OF PURINE METABOLISM
Tumoural Hypouricemia A. Lesmes, M. D i a z - C u r i e l , a n d J . M.
145 Castrillo
H e r e d i t a r y Renal Hypouricemia With H y p e r u r i c o s u r i a and V a r i a b l y A b s o r p t i v e H y p e r c a l c i u r i a a n d U r o l i t h i a s i s - A New Syndrome 0. S p e r l i n g a n d A. de V r i e s H e r e d i t a r y and E n v i r o n m e n t a l F a c t o r s I n f l u e n c i n g o n t h e Serum U r i c A c i d T h r o u g h o u t Ten Years P o p u l a t i o n Study i n Japan K. N i s h i o k a a n d K. M i k a n a g i The N a t u r a l H i s t o r y o f U r a t e O v e r p r o d u c t i o n i n S i c k l e C e l l Anemia H. S. D i a m o n d , A. D. M e i s e l , a n d D. H o l d e n
155
161
Salvage G.
Pathway i n E r y t h r o c y t e s o f P a t i e n t s With Psoriasis P a r t s c h , F. M a y e r , R. E b e r l , a n d A. L u g e r
149
Serum 5 - N u c l e o t i d a s e i n P r o g r e s s i v e Dystrophy F. Lähoda a n d K. B a i e r
167
Muscular 173
CONTENTS OF PART A
xviü
P u r i n e M e t a b o l i s m i n Duchenne M u s c u l a r Dystrophy C. Η. Μ. M. De B r u y n , S. K u l a k o w s k i , C. A. v a n Bennekom, P. R e n o i r t e , a n d Μ. M. Müller o f Adenine and Adenosine i n E r y t h r o c y t e s of P a t i e n t s w i t h Myotonic Muscular D y s t r o p h y (MMD) Μ. M. M ü l l e r , Μ. F r a s s , a n d B. M a m o l i
177
Metabolism
and E n z y m o l o g i c a l S t u d i e s i n a C h i l d W i t h Type I Glycogen Storage D i s e a s e A s s o c i a t e d W i t h P a r t i a l D e f i c i e n c y o f H e p a t i c Glucose6-Phosphatase G. N u k i a n d J . P a r k e r
183
Clinical
D i f f e r e n t i a l Absorption of Purine Nucleotides, N u c l e o s i d e s a n d Bases C. F. P o t t e r , A. C a d e n h e a d , H. A. Simmonds, a n d J . S. Cameron
189
203
I n f l u e n c e o f D i e t a r y P r o t e i n o n Serum a n d Urinary Uric Acid W. Löffler, W. Gröbner, a n d N. Zöllner
209
E f f e c t o f H y p o x a n t h i n e i n M e a t o n Serum U r i c A c i d and U r i n a r y U r i c A c i d E x c r e t i o n W. K. S p a n n , W. Gröbner, a n d N. Zöllner
215
On t h e M e c h a n i s m o f t h e P a r a d o x i c a l E f f e c t o f S a l i c y l a t e on Urate E x c r e t i o n H. S. D i a m o n d , G. S t e r b a , K. J a y a d e v e n , a n d A. D. M e i s e l The U r i c o s u r i c A c t i o n o f P r o t e i n i n Man F. M a t z k i e s , G. B e r g , a n d H. Mädl E f f e c t s on P u r i n e M e t a b o l i s m S t u d i e d W i t h High Pressure L i q u i d Chromatography R. A. H a r k n e s s , R. J . Simmonds, a n d M. C. O'Connor
221
227
Hypoxic
Urate Metabolism P. A. S i m k i n
i n a Mongrel
Dog
233
237
COINTENTS OF P A R T A
IV.
STUDIES ON
x i x
XANTHINURIA
Thie E f f e c t
C. A.
o f W e i g h t R e d u c t i o n o n P l a s m a and U r i n a r y L e v e l s o f O x y p u r i n e s i n a n Obese Xanthinuric Patient A u s c h e r , C. P a s q u i e r , N. A m o r y , G. Gay, A i s e n e , and G. D e b r y
X a m t h i n u r i a : The Cause o f H y p o u r i c e m i a Hepatic Disease H. J . C a s t r o - M e n d o z a , A. Rapado, C. and J . M. C a s t r i l l o
in 247 De
La
Piedra,
Xainthine-Coproporphyrin I I I H. J . C a s t r o - M e n d o z a
V. A
THERAPEUTICAL ASPECTS OF
241
251
DISORDERS I N PURINE METABOLISM
C o n t r o l l e d Study o f t h e E f f e c t o f Long Term A l l o p u r i n o l T r e a t m e n t on R e n a l F u n c t i o n i n Gout T. G i b s o n , H. A. Simmonds, C. P o t t e r , and V. R o g e r s
St:udies W i t h A l l o p u r i n o l i n P a t i e n t s W i t h I m p a i r e d Renal F u n c t i o n G. B. E l i o n , F. M. B e n e z r a , T. D. B e a r d m o r e , and W. N. K e l l e y P h i a r m a c o l o g i c a l E f f e c t s o f 1 , 3 , 5 - T r i a z i n e s and E x c r e t i o n C h a r a c t e r i s t i c s i n t h e Rat M. H r o p o t , F. Sörgel, Β. v . Kerekjärto, H. J . L a n g , a n d R. Muschaweck T i i e n i l i c A c i d i n the Treatment Hypertension T. G i b s o n , C. P o t t e r , H. A. and R. I . G l e a d l e
o f Gout
257
263
Their 269
and 277
Simmonds, V.
Rogers,
B e n z b r o m a r o n e as a L o n g - T e r m U r i c o s u r i c A g e n t R. B l u e s t o n e , J . K l i n e n b e r g , a n d I . K. Lee Thie A c t i o n o f B e n z b r o m a r o n e i n R e l a t i o n t o Sex a n d A c c o m p a n y i n g D i s e a s e s H. F e r b e r , U. B a d e r , and F. M a t z k i e s Covalitin®: A New D r u g f o r t h e T r e a t m e n t Uric Lithiasis T. C o v a l i u
283
Age, 287
of 295
CONTENTS O F PART A
XX
VI.
MUTATIONS AFFECTING PURINE METABOLISM A.
Phosphoribosyltransferases
H y p o x a n t h i n e S a l v a g e i n Man: I t s I m p o r t a n c e i n U r a t e O v e r p r o d u c t i o n i n t h e Lesch-Nyhan Syndrome N. L. E d w a r d s , D. P. R e c k e r , a n d I . H. F o x
301
Aspect o f Purine Metabolic A b e r r a t i o n Associated W i t h U r i c A c i d O v e r p r o d u c t i o n a n d Gout L. C. Y i p , T.-F. Y ü , a n d Μ. E. B a l i s
307
P r o p e r t i e s o f a Mutant Hypoxanthine-Phosphoribosylt r a n s f e r a s e i n a P a t i e n t W i t h Gout W. Gröbner a n d W. G u t e n s o h n
313
V a r i a t i o n i n Human HPRT a n d I t s R e l a t i o n s h i p t o N e u r o l o g i c and B e h a v i o r a l M a n i f e s t a t i o n s B. B a k a y , E. N i s s i n e n , L. Sweetman, U. F r a n e k e , and W. L. N y h a n
317
H i g h HPRT A c t i v i t y i n F i b r o b l a s t s f r o m P a t i e n t s W i t h L e s c h - N y h a n Syndrome d u e t o B a c t e r i a l "L-Form" C o n t a m i n a t i o n I . W i l l e r s , S, S i n g h , K. R. H e l d , a n d H. W. Goedde
327
K i n e t i c s o f a HGPRT M u t a n t S h o w i n g Substrate I n h i b i t i o n Ε. H. H a r l e y , C. M. Adnams, a n d L. M. S t e y n
333
B.
Nucleoside
P h o s p h o r i b o s y l a t i n g Enzymes
Spectrum o f 2,8-Dihydroxyadenine U r o l i t h i a s i s i n C o m p l e t e APRT D e f i c i e n c y H. A. Simmonds, T. M. B a r r a t t , D. R. W e b s t e r , A. S a h o t a , K. J . V a n A c k e r , J . S. Cameron, a n d M. D i l l o n C o m p l e t e A d e n i n e P h o s p h o r i b o s y l t r a n s f e r a s e (APRT) D e f i c i e n c y i n Two S i b l i n g s : R e p o r t o f a New Case P. C a r t i e r , M. Hamet, A. V i n c e n s , J . L. P e r i g n o n I n h e r i t a n c e o f Adenine P h o s p h o r i b o s y l t r a n s f e r a s e (APRT) D e f i c i e n c y K. J . V a n A c k e r , H. A. Simmonds, C. F. P o t t e r , a n d A. S a h o t a
337
343
349
xxi
CCHNTENTS OF P A R T A
Iimmunological E v a l u a t i o n of a Family D e f i c i e n t i n A d e n i n e P h o s p h o r i b o s y l T r a n s f e r a s e (APRT) W. J . S t e v e n s , Μ. E. P e e t e r m a n s , and K. J . V a n A c k e r
355
Activities
o f A m i d o p h o s p h o r i b o s y l t r a n s f e r a s e and Purine Phosphoribosyltransferases i n D e v e l o p i n g Rat B r a i n J . A l l s o p a n d R. W. E. W a t t s
361
Puirine Nucleoside Phosphorylase D e f i c i e n c y ; Genetic S t u d i e s i n a Dutch Family G. E. J . S t a a l , M. J . M. v a n d e r V l i s t , R. Geerdink, J . M. J a n s e n - S c h i l l h o r n v a n V e e n , B. J . M. Zegers, and J . W. S t o o p C.
Nucleoside
C l e a v i n g Enzymes
Abnormal Regulation of Purine Metabolism i n a C u l t u r e d Mouse T - C e l l Lymphoma M u t a n t P a r t i a l l y Deficient i n Adenylosuccinate Synthetase B. U l l m a n , M. A. W o r m s t e d , B. B. L e v i n s o n , L. J . A. Cohen, S. M. C l i f t , a n d D. W. M a r t i n , J r .
375 Gudas,
Suiperactive Phosphoribosylpyrophosphate Synthetase W i t h A l t e r e d R e g u l a t o r y and C a t a l y t i c Properties M. A. B e c k e r , K. 0. R a i v i o , B. B a k a y , W. B. Adams, and W. L. N y h a n AM1P
P h o s p h a t a s e A c t i v i t y i n Human T e r m P l a c e n t a : S t u d i e s on P l a c e n t a l 5 - N u c l e o t i d a s e Μ. H. M a g u i r e a n d T. P. K r i s h n a k a n t h a f
D.
Deaminating
367
387
393
Enzymes
Adlenosine and D e o x y a d e n o s i n e M e t a b o l i s m i n t h e E r y t h r o c y t e s o f a P a t i e n t With Adenosine Deaminase D e f i c i e n c y A. S a h o t a , H. A. Simmonds, C. F. P o t t e r , J . G. W a t s o n , K. H u g h - J o n e s , and D. P e r r e t t
397
C o m p l e t e A d e n o s i n e D e a m i n a s e (ADA) D e f i c i e n c y W i t h o u t I m m u n o d e f i c i e n c y , and P r i m a r y H y p e r o x a l u r i a , i n a 1 2 - Y e a r - 0 1 d Boy J . L. P e r i g n o n , M. Hamet, P. C a r t i e r , and C. G r i s c e l l i
403
C O N T E N T S O F PART A
xxii
M e t a b o l i s m o f Adenosine and Deoxyadenosine b y S t o r e d Human Red C e l l s G. R. B a r t l e t t A d e n o s i n e Deaminase and P u r i n e N u c l e o s i d e Phosphorylase A c t i v i t i e s During Culturing of Fibroblasts M. P. U i t e n d a a l , F. T. J . J . O e r l e m a n s , De B r u y n , T . L. O e i , a n d P. Hösli
409
415 C. Η. Μ. M.
S-Adenosylhomocysteine Metabolism i n Adenosine Deaminase D e f i c i e n t C e l l s M. S. H e r s h f i e l d a n d Ν. M. K r e d i c h A l t e r e d Deoxynucleoside Triphosphate Levels P a r a l l e l i n g Deoxyadenosine T o x i c i t y i n A d e n o s i n e D e a m i n a s e I n h i b i t e d Human Lymphocytes H. G. B l u e s t e i n , L. F. Thompson, D. A. A l b e r t , and J . E. S e e g m i l l e r
421
427
Author Index
433
Subject Index
437
PURINE METABOLISM I N CULTURED CORONARY ENDOTHELIAL CELLS
S. N e e s , A.L. G e r b e s , B. Willershausen-Zönnchen a n d E. G e r l a c h Physiologisches
Institut
d e r Universität München
Pettenkoferstr.
12, D-8000 München 2
A l t h o u g h i t i s w e l l known t h a t e n d o t h e l i a l c e l l s a r e i n v o l v e d i n s e v e r a l b i o l o g i c a l p r o c e s s e s s u c h as t r a n s p o r t * ! , h e m o s t a s i s ^ , s y n t h e s i s o f c o l l a g e n - ^ , h i s t a m i n e ^ a n d p r o s t a g l a n d i n s ^ , o u r knowledge concerning i n t e r m e d i a r y metabolism o f t h e endothelium i s r a t h e r l i m i t e d . I n t h e course o f s t u d i e s on i n t e r r e l a t i o n s h i p s b e t ween h e a r t f u n c t i o n a n d c a r d i a c m e t a b o l i s m ^ 7 became i n t e r e s t e d i n some f e a t u r e s o f p u r i n e m e t a b o l i s m o f c o r o n a r y e n d o t h e l i a l c e l l s . O u r i n t e r e s t was i n i t i a t e d b y t h e a s s u m p t i o n t h a t t h e s e c e l l s might c o n t r i b u t e t o t h e p r o d u c t i o n o f v a s o a c t i v e adenosine w h i c h i s c o n s i d e r e d t o p l a y an i m p o r t a n t r o l e i n t h e m e t a b o l i c r e g u l a t i o n o f c o r o n a r y b l o o d flow^»^. T h e s t u d i e s - n o t p o s s i b l e o f c o u r s e t o be p e r f o r m e d under i n v i v o c o n d i t i o n s - were c a r r i e d o u t on c u l t u r e d e n d o t h e l i a l c e l l s i s o l a t e d f r o m c o r o n a r y v e s s e l s o f g u i n e a p i g h e a r t s as r e c e n t l y d e s c r i b e d ^ . w
e
MATERIALS AND METHODS C u l t u r e M e d i u m 199 ( S e r o m e d , München) c o n t a i n i n g p e n i c i l l i n e ( 2 0 0 TJ/ml) a n d s t r e p t o m y c i n e ( 2 0 0 u g / m l ) was s u p p l e m e n t e d w i t h f e t a l c a l f s e r u m ( 2 0 % ) a n d L - g l u t a m i n e ( 2 mM). C o l u m n p a c k i n g s ( t o t a l l y p o r o u s s i l i c a ) f o r H i g h P r e s s u r e L i q u i d C h r o m a t o g r a p h y (HPLC) w e r e o b t a i n e d f r o m M a c h e r e y & N a g e l , Düren. N u c l e o t i d e s , n u c l e o sides and bases f o r c a l i b r a t i o n were purchased from Boehringer Mannheim, a l l o t h e r m a t e r i a l s o f h i g h e s t a v a i l a b l e p u r i t y f r o m Merck, Darmstadt. P r e p a r a t i o n o f c e l l s and c e l l c u l t u r e : Guinea p i g h e a r t s were c a n n u l a t e d t h r o u g h t h e a o r t a , and t h e i r c o r o n a r y s y s t e m , washed 25
26
S. N E E S E T A L .
f r e e o f b l o o d , was f i l l e d w i t h a n i s o t o n i c b u f f e r s o l u t i o n c o n t a i n i n g c o l l a g e n a s e and t r y p s i n ( 0 . 1 % e a c h ) . A f t e r an e x p o s u r e o f 20 m i n p e r f u s i o n was s t a r t e d a g a i n a n d a l l e n d o t h e l i a l c e l l s w h i c h had been d e t a c h e d , were c o l l e c t e d f r o m t h e p e r f u s a t e by c e n t r i f u g a t i o n . S u b s e q u e n t l y , t h e c e l l s w e r e washed w i t h c u l t u r e medium and s e e d e d i n c u l t u r e d i s h e s . C u l t i v a t i o n was p e r f o r m e d a t 3 7 ° C i n a h u m i d i f i e d a i r a t m o s p h e r e c o n t a i n i n g 3% C O 2 . D e p e n d i n g o n t h e i n o c u l u m c o n f l u e n c y was r e a c h e d a f t e r 2 t o 4 w e e k s . C o n t a m i n a t i o n s w i t h f i b r o b l a s t s a n d s m o o t h m u s c l e c e l l s w e r e u s u a l l y l e s s t h a n 2% As j u d g e d b y e l e c t r o n m i c r o s c o p y t h e c u l t i v a t e d e n d o t h e l i a l c e l l s revealed important morphological c r i t e r i a o f endothelial c e l l s i n v i v o ( c l u s t e r s o f f r e e r i b o s o m e s , smooth and r o u g h e n d o p l a s m i c r e t i c u l u m , clumps o f coarse f i l a m e n t s , f i n e f i l a m e n t s and p r o m i n e n t microtubules i n t h e cytoplasma). A n a l y s i s o f n u c l e o t i d e s , n u c l e o s i d e s and bases: C u l t u r e d e n d o t h e l i a l c e l l s w e r e e x t r a c t e d w i t h 0.4 Ν p e r c h l o r i c a c i d . Q u a n t i t a t i o n o f t h e d i f f e r e n t p u r i n e compounds i n t h e n e u t r a l i z e d c e l l e x t r a c t s was c a r r i e d o u t b y a p p l i c a t i o n o f s p e c i a l l y e l a b o r a t e d H P L C - t e c h n i q u e s u s i n g weak a n i o n e x c h a n g e c o l u m n s f o r t h e s e p a r a t i o n o f t h e n u c l e o t i d e s and r e v e r s e phase columns f o r n u c l e o s i d e s and b a s e s . D e t e r m i n a t i o n o f enzyme a c t i v i t i e s : S p e c i f i c a c t i v i t i e s o f enzymes i n v o l v e d i n n u c l e o t i d e m e t a b o l i s m w e r e m e a s u r e d i n a 20 0 0 0 g membrane p r e p a r a t i o n a s w e l l as i n a s o l u b l e 200 0 0 0 g s u p e r n a t a n t f r a c t i o n o f e n d o t h e l i a l c e l l s . Enzyme t e s t s w e r e p e r formed u s i n g s t a n d a r d p r o c e d u r e s , s u b s t r a t e s and p r o d u c t s were s e p a r a t e d b y HPLC.
RESULTS AND DISCUSSION I n T a b l e 1 mean v a l u e s f r o m t h r e e i n d i v i d u a l s e r i e s o f a n a l y ses c o n c e r n i n g c o n t e n t s o f p u r i n e n u c l e o t i d e s , n u c l e o s i d e s a n d b a ses i n n o n - g r o w i n g c o n f l u e n t e n d o t h e l i a l c e l l c u l t u r e s a r e l i s t e d . For reasons o f comparison r e s p e c t i v e d a t a f o r normoxic m y o c a r d i a l tissue are also given. Obviously, endothelial c e l l s contain extra o r d i n a r i l y h i g h a m o u n t s o f ATP, ADP a n d AMP. The sum o f t h e a d e n i n e n u c l e o t i d e s (£?ATP, ADP, AMP) r e a c h e s w i t h m o r e t h a n 15 u m o l e s / g a v a l u e w h i c h i s a b o u t t h r e e t i m e s h i g h e r t h a n t h e mean a d e n i n e n u c l e o t i d e content o f c a r d i a c t i s s u e . Another i n t e r e s t i n g f e a t u r e of e n d o t h e l i a l c e l l s concerns t h e i r h i g h l e v e l s o f adenine n u c l e o t i d e d e g r a d a t i v e s . The c o n t e n t s o f a d e n o s i n e , i n o s i n e , a d e n i n e and hypoxanthine a r e about 1 t o 2 o r d e r s o f magnitude h i g h e r t h a n t h e r e s p e c t i v e v a l u e s f o r the myocardium. I n c o n t r a s t t o the h i g h l e v e l s o f adenine n u c l e o t i d e s guanine n u c l e o t i d e s a r e present i n en d o t h e l i a l c e l l s only i n small q u a n t i t i e s , which a r e s i m i l a r t o those i n m y o c a r d i a l and o t h e r t i s s u e s .
P U R I N E M E T A B O L I S M IN C U L T U R E D E N D O T H E L I A L
27
CELLS
T a b l e 1: C o n t e n t o f a d e n i n e n u c l e o t i d e s a n d t h e i r dephosphorylated d e g r a d a t i v e s i n c o n f l u e n t c o r o n a r y e n d o t h e l i a l c e l l s and i n myocard i a l t i s s u e o f g u i n e a p i g s . Mean v a l u e s f r o m t h r e e i n d i v i d u a l s e r i e s o f a n a l y s e s o f 11 c u l t u r e d i s h e s e a c h . Endothelial
cells
Myocardium
nmoles/g
nmoles/g
ATP
11 960
4 280
ADP
2 760
1 050
AMP
630
160
^>
f
ATP, ADP,
AMP
Adenosine
1 5 350
5 490
2
87
1 .2
Inosine
100
Adenine
60
0.5
Hypoxanthine
50
0.9
GTP
233
200
GDP
157
100
GMP
37
23
Guanosine
*)
*)
Guanine
*)
*)
not
detectable
A d d i t i o n a l experiments revealed t h a t growth s t a t e o f t h e c u l t u r e s d i d n o t p r o f o u n d l y i n f l u e n c e t h e t o t a l content o f adenine nucleotides. Furthermore, incubation of confluent c e l l c u l t u r e s i n p u r i n e - f r e e media f o r t h r e e days d i d n o t r e s u l t i n any d e t e c t a b l e r e d u c t i o n o f t h e a d e n i n e n u c l e o t i d e c o n t e n t . On t h e o t h e r h a n d , e n d o t h e l i a l c e l l s p r o v e d t o be s e n s i t i v e t o l a c k o f o x y g e n . I t i s evident from t h e data i n F i g . 1 that b r i e f periods o f anoxic i n c u b a t i o n (1.5 and 3 m i n , r e s p e c t i v e l y ) cause a pronounced decrease o f ATP w i t h a c o r r e s p o n d i n g i n c r e a s e i n ADP a n d AMP l e v e l s . S i m u l t a n e o u s l y , r e m a r k a b l e amounts o f a d e n o s i n e a r e f o r m e d and r e l e a s e d f r o m t h e c e l l s i n t o t h e i n c u b a t i o n medium. I t appears from a l l these o b s e r v a t i o n s t h a t t h e extremely h i g h adenine n u c l e o t i d e l e v e l s are very l i k e l y a s p e c i f i c f e a t u r e of c u l t u r e d e n d o t h e l i a l c e l l s . T h i s view i s f u r t h e r supported by det e r m i n a t i o n s o f a c t i v i t y v a l u e s o f enzymes i n v o l v e d i n d e g r a d a t i o n and s y n t h e s i s o f a d e n i n e n u c l e o t i d e s ( T a b l e 2 ) . W h i l e 5 - n u c l e o t i dase a c t i v i t y i n e n d o t h e l i a l c e l l s exceeds by f a r t h a t o f myocar1
S. N E E S E T
28
AL
ANOXIA
NORMOXIA
1.5min
3min
10
l nmoles/g ]
F i g . 1: I n f l u e n c e o f a n o x i a on l e v e l s o f a d e n i n e n u c l e o t i d e s i n c o r o n a r y e n d o t h e l i a l c e l l s and on t h e r e l e a s e o f a d e n o s i n e i n t o t h e m e d i u m (mean v a l u e s f r o m 3 e x p e r i m e n t s )
d i a l t i s s u e , t h e o p p o s i t e h o l d s t r u e f o r adenosine deaminase, the a c t i v i t y o f w h i c h i s much h i g h e r i n t h e m y o c a r d i u m . T h e s e d i f f e r e n c e s i n t h e p a t t e r n o f enzyme a c t i v i t i e s may r e a s o n a b l y e x p l a i n t h a t e n d o t h e l i a l c e l l s c o n t a i n adenosine i n r a t h e r h i g h amounts compared w i t h t h e s m a l l q u a n t i t i e s o f t h i s n u c l e o s i d e f o u n d i n t h e m y o c a r d i u m . As i s f u r t h e r e v i d e n t f r o m t h e d a t a i n T a b l e 2, a c t i v i t i e s o f G-6-PDH and P R P P - s y n t h e t a s e , i n d i r e c t l y i n v o l v e d i n t h e b i o s y n t h e s i s o f n u c l e o t i d e s , p r o v e d t o be much h i g h e r i n e n d o t h e l i a l c e l l s t h a n i n c a r d i a c t i s s u e . These f i n d i n g s are i n a c c o r d a n c e w i t h r e s u l t s f r o m p r e l i m i n a r y s t u d i e s , i n w h i c h by use o f 1-^Cg l y c i n e and ^ C - l a b e l e d p u r i n e b a s e s p u r i n e n u c l e o t i d e s y n t h e s i s i n e n d o t h e l i a l c e l l s was shown t o p r o c e e d v i a s a l v a g e and de n o v o pathways.
SUMMARY E n d o t h e l i a l c e l l s from coronary vessels of guinea p i g h e a r t s w e r e i s o l a t e d , c u l t i v a t e d and m o r p h o l o g i c a l l y c h a r a c t e r i z e d . - C e l l s
P U R I N E M E T A B O L I S M IN C U L T U R E D E N D O T H E L I A L C E L L S
29
T a b l e 2: Enzyme a c t i v i t i e s i n c u l t u r e d c o r o n a r y e n d o t h e l i a l and i n c a r d i a c t i s s u e f r o m g u i n e a p i g s .
Specific
activity
Endothelial cells
[nmoles/min
95
13.6
Alkaline phosphatase [ E . C . 3.1.3.1]
14.6
24
1 .1
Adenosine deaminase [ E . C . 3.5.4.4]
3.4
Glu-6-P-dehydrogenase [E.C. 1.1.1.49]
12.7
*mg]
Cardiac tissue
5'-Nucleotidase [ E . C . 3.1.3.5]
AMP deaminase [E.C.3.5.4.6]
cells
2.2
28
4.2
PRPP-synthetase [ E . C . 2.7.6.6]
6.58
1 .9
APR-transferase [ E . C . 2.4.2.7]
0.7
0.2
GPR-transferase [ E . C . 2.4.2.8]
0.3
0.1
Adenylate cyclase [E.C. 4.6.1.1]
0.1
0.4
Phosphodiesterase [ E . C . 3.1.4.1]
2.1
15
f r o m c o n f l u e n t c u l t u r e s c o n t a i n e d a d e n i n e n u c l e o t i d e s and t h e i r dep h o s p h o r y l a t e d d e g r a d a t i v e s i n e x c e p t i o n a l l y h i g h amounts.- Adenine n u c l e o t i d e l e v e l s were o n l y s l i g h t l y i n f l u e n c e d by t h e g r o w t h s t a t e o f t h e c u l t u r e s and r e m a i n e d s t a b l e d u r i n g i n c u b a t i o n f o r t h r e e d a y s i n p u r i n e - f r e e medium. I n c o n t r a s t , b r i e f i n c u b a t i o n o f e n d o t h e l i a l c e l l s under anoxic c o n d i t i o n s r e s u l t e d i n a s u b s t a n t i a l b r e a k d o w n o f a d e n i n e n u c l e o t i d e s a s s o c i a t e d w i t h an enhanced f o r m a t i o n and r e l e a s e o f a d e n o s i n e . - Measurements o f s p e c i f i c a c t i v i -
S. N E E S E T
30
AL
t i e s o f enzymes i n v o l v e d i n a d e n i n e n u c l e o t i d e s y n t h e s i s and d e gradation lend a d d i t i o n a l support to the view that a very a c t i v e adenine n u c l e o t i d e metabolism i s a t y p i c a l f e a t u r e of c u l t u r e d coronary endothelial c e l l s .
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C l e m e n t i and G. E. P a l a d e , I n t e s t i n a l c a p i l l a r i e s . P e r m e a b i l i t y t o p e r o x i d a s e and f e r r i t i n , J . C e l l . B i o l . 41:33 (1969) A. J a f f e , L. W. H o y e r , and R. L. Nachman, S y n t h e s i s o f v o n W i l l e b r a n d f a c t o r b y c u l t u r e d human e n d o t h e l i a l c e l l s , P r o c . N a t l . A c a d . S e i . USA 71:1906 ( 1 9 7 4 ) V. H o w a r d , E. J . M a c a r a k , D. Günsen, and N. A. K e f a l i d e s , Characterizationof the collagen synthesized by e n d o t h e l i a l c e l l s i n c u l t u r e , P r o c . N a t l . A c a d . Sei. USA, 73:2361 ( 1 9 7 6 ) M. H o l l i s and L. A. R o s e n , H i s t i d i n e d e c a r b o x y l a s e a c t i v i t y o f b o v i n e a o r t i c e n d o t h e l i u m and i n t i m a - m e d i a , P r o c . Soc. Exp. B i o l . Med. 141:978 ( 1 9 7 2 ) A. G i m b r o n e J r . and R. W. A l e x a n d e r , A n g i o t e n s i n I I s t i m u l a t i o n of prostaglandin production i n c u l t u r e d human v a s c u l a r e n d o t h e l i u m , S c i e n c e 189:219 ( 1 9 7 5 ) Schräder and E. G e r l a c h , C o m p a r t m e n t a t i o n o f c a r d i a c a d e n i n e n u c l e o t i d e s and f o r m a t i o n o f a d e n o s i n e , Pflüg e r s A r c h . 367:129 (1976) Schräder, S. Nees, and E. G e r l a c h , E v i d e n c e f o r a c e l l s u r f a c e a d e n o s i n e r e c e p t o r on c o r o n a r y myocytes and a t r i a l m u s c l e c e l l s , Pflügers A r c h . 369:251 ( 1 9 7 7 ) M. B e r n e , C a r d i a c n u c l e o t i d e s i n h y p o x i a : a p o s s i b l e r o l e i n r e g u l a t i o n o f c o r o n a r y b l o o d f l o w , Am. J . P h y s i o l . 204:317 ( 1 9 6 3 ) G e r l a c h , B. D e u t i c k e and R. H. D r e i s b a c h , Der N u c l e o t i d - A b b a u im H e r z m u s k e l b e i S a u e r s t o f f m a n g e l und s e i ne mögliche B e d e u t u n g für d i e K o r o n a r d u r c h b l u t u n g , N a t u r w i s s e n s c h a f t e n 50:229 ( 1 9 6 3 ) Nees, A. L. G e r b e s , B. Willershausen-Zönnchen, a n d E. G e r l a c h , I s o l a t i o n , c u l t u r e and m o r p h o l o g i c c h a r a c t e r i z a t i o n of e n d o t h e l i a l c e l l s from coronary v e s s e l s , a b s t r a c t of the 51st Meeting (Spring Meeting) o f t h e Deutsche P h y s i o l o g i s c h e G e s e l l s c h a f t , K i e l (1979)
