From: KrNINS-II: Biochemistry, Pathophysiology, and Clinical Aspects
Edited by Setsuro Fujii, Hiroshi Moriya, Tomoji Suzuki
(Plenum Publishing Corporation, 1979)
HUMAN URINARY KALLIKREIN - BIOCHEMICAL AND PHYSIOLOGICAL ASPECTS
R. G e i g e r , U. S t u c k s t e d t e , B. F o r g - B r e y and E. F i n k
A b t e i l u n g für K l i n i s c h e Chemie und K l i n i s c h e B i o c h e m i e
i n d e r C h i r u r g i s c h e n K l i n i k d e r Universität München
Nussbaumstrasse 20, D-8000 München 2, Germany
The h i s t o r y o f k a l l i k r e i n b e g i n s w i t h t h e d i s c o v e r y o f u r i n a r y
k a l l i k r e i n more than f i f t y y e a r s ago. The r e n a l o r i g i n o f u r i n a r y
k a l l i k r e i n , though n o t f i n a l l y p r o v e d , i s l a r g e l y a c c e p t e d .
A
p o s s i b l e r o l e of k a l l i k r e i n i n the r e g u l a t i o n of kidney f u n c t i o n
and b l o o d p r e s s u r e has been debated f o r a l o n g time ( P i s a n o and
A u s t e r , 1976). However, n e i t h e r t h e q u e s t i o n f o r i t s o r i g i n , n o r
for i t s p h y s i o l o g i c a l r o l e i s f i n a l l y s e t t l e d .
I s o l a t i o n and C h a r a c t e r i z a t i o n
As a f i r s t s t e p i n t h e development o f a radioimmunoassay and
of e n z y m a t i c a s s a y s f o r human u r i n a r y k a l l i k r e i n an i s o l a t i o n
method f o r t h e enzyme was s e t up ( G e i g e r e t a l . 197 7).
The method
i s summarized i n T a b l e 1 (a s l i g h t l y d i f f e r e n t method was r e p o r t e d
e a r l i e r , Mann and G e i g e r , 1977).
P r e p a r a t i o n s o f human u r i n a r y k a l l i k r e i n o b t a i n e d from d i f f e r e n t
u r i n e p o o l s were s u b j e c t e d t o P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s
at pH 6 and 8. Two d i f f e r e n t p a t t e r n s o f p r o t e i n bands were obt a i n e d . For some p r e p a r a t i o n s o n l y one band was v i s i b l e a f t e r
s t a i n i n g , whereas o t h e r p r e p a r a t i o n s were r e s o l v e d i n t o t h r e e
bands as d e s c r i b e d a l s o by Matsuda e t a l . (1976). A l l bands c o n t a i n e d a c t i v e k a l l i k r e i n as w'as demonstrated by a c t i v e enzyme
s t a i n i n g u s i n g Z-Ser-Pro-Phe-Arg-MNA and c o u p l i n g w i t h F a s t B l u e B
S a l t (Smith e t a l . 1975). A f t e r i n c u b a t i o n w i t h T r a s y l o l , however,
o n l y a s i n g l e band o f l o w e r e l e c t r o p h o r e t i c m o b i l i t y was d e t e c t e d
( F i g . 1) f o r a l l p r e p a r a t i o n s .
235
R. GEIGER E T A L .
236
T a b l e 1.
I s o l a t i o n o f human u r i n a r y
Steps
kallikrein
Purification
C o l l e c t i o n o f human male u r i n e
Dialysis
Lyophilization
E x t r a c t i o n o f t h e crude u r i n e powder
S e p h a c r y l S-200
T r a s y l o l Sepharose
DEAE-Sepharose
1
89
240
Amino a c i d a n a l y s e s o f t h e human u r i n a r y k a l l i k r e i n were
done by common methods, c a r b o h y d r a t e c o n t e n t was d e t e r m i n e d as
d e s c r i b e d by K r y s t a l and Graham (1976). The r e s u l t s a r e shown i n
T a b l e 2 and 3. A c c o r d i n g t o our p r e l i m i n a r y r e s u l t s , t h e amino
a c i d c o m p o s i t i o n s o f human u r i n a r y k a l l i k r e i n i s v e r y s i m i l a r t o
t h a t o f p i g p a n c r e a t i c ß-kallikrein. The v a l u e s f o r S e r , G l y ,
A l a , Cys and Met a r e i d e n t i c a l , whereas minor d i f f e r e n c e s e x i s t f o r
the o t h e r amino a c i d s . Some d i f f e r e n c e s a l s o e x i s t i n t h e amino
a c i d c o m p o s i t i o n s o f p r e p a r a t i o n s i s o l a t e d from d i f f e r e n t u r i n e
pools.
I
B
F i g . 1.
A c r y l a m i d e g e l e l e c t r o p h o r e s i s (7.58% g e l , pH 8) o f
human k a l l i k r e i n .
A: p u r i f i e d enzyme
B: p u r i f i e d enzyme
complexed w i t h T r a s y l o l
HUMAN URINARY KALLIKREIN
237
T a b l e 2.
Amino a c i d c o m p o s i t i o n o f k a l l i k r e i n s from human
u r i n e and p o r c i n e p a n c r e a s .
Tryptophan was n o t d e t e r m i n e d , c y s t e i n a f t e r p e r f o r m i c a c i d o x i d a t i o n ( H i r s 1976).
Human u r i n a r y
kallikrein
Asp
Thr
Ser
Glu
Pro
Gly
Ala
Cys
Val
Met
He
Leu
Tyr
Phe
Lys
His
Arg
Trp
Porcine pancreatic
3-kallikrein
( F i e d l e r e t a l . 1977)
28
15
14
23
16
22
13
10
10
4
12
20
7
10
10
8
3
7
20-23
14
14
28-30
14
22
12-14
10
13-16
4
8-9
18
9
8-12
8-12
8-9
6
?
T a b l e 3.
C a r b o h y d r a t e c o n t e n t o f human u r i n a r y k a l l i k r e i n
and p o r c i n e p a n c r e a t i c k a l l i k r e i n
Kallikrein
from
Carbohydrate content
g/100 g p r o t e i n
P o r c i n e pancreas
form A
form B.
Human u r i n e
(Fiedler et a l .
1975)
5.6
11.5
10.5 - 15.6
R. GEIGER E T A L .
238
The d i f f e r e n c e s i n b o t h amino a c i d c o m p o s i t i o n and e l e c t r o p h o r e t i c p a t t e r n s s u g g e s t t h a t our p r e p a r a t i o n s o f human u r i n a r y
k a l l i k r e i n c o n t a i n m u l t i p l e forms as a r e s u l t o f l i m i t e d p r o t e o lysis.
L i m i t e d p r o t e o l y t i c a t t a c k causes c l e a v a g e s w i t h i n a
p r o t e i n c h a i n , e v e n t u a l l y r e s u l t i n g i n t h e r e l e a s e o f s i n g l e amino
acids or peptides.
These c l e a v a g e s and t h e l o s s o f p a r t s o f t h e
p r o t e i n m o l e c u l e c a n g i v e r i s e t o changes i n e l e c t r o p h o r e t i c
m o b i l i t y and amino a c i d c o m p o s i t i o n .
M u l t i p l e forms caused by
l i m i t e d p r o t e o l y s i s have a l s o been observed f o r p i g p a n c r e a t i c
k a l l i k r e i n ( F i e d l e r e t a l . 1977).
A s s a y Methods
I t has been o b s e r v e d i n our l a b o r a t o r y t h a t Ac-Phe-ArgOMe i s
a much b e t t e r s u b s t r a t e f o r p o r c i n e p a n c r e a t i c ( F i e d l e r 1 9 7 6 ) ,
submandibular and u r i n a r y k a l l i k r e i n s ( F r i t z e t a l . 1977) t h a n a N - a c e t y l a t e d a r g i n i n e e s t e r s . The c o r r e s p o n d i n g e t h y l e s t e r , A c Phe-ArgOEt , i s t h e most r a p i d l y h y d r o l y z e d s u b s t r a t e f o r human
u r i n a r y k a l l i k r e i n d e s c r i b e d as y e t ( F i e d l e r e t a l . 1978). T h e r e f o r e , a h i g h l y s e n s i t i v e a s s a y f o r human u r i n a r y k a l l i k r e i n c o u l d
be d e v e l o p e d employing Ac-Phe-ArgOEt as s u b s t r a t e . The a s s a y
(Table 4) i s analogous t o t h a t w i t h Bz-ArgOEt developed by
T r a u s c h o l d and Werle (1961).
The r e a c t i o n sequence i s shown i n
F i g . 2. The s e n s i t i v i t i e s o f t h i s and o t h e r a s s a y s f o r human
u r i n a r y k a l l i k r e i n a r e c o m p i l e d i n T a b l e 5. The s e n s i t i v i t y o f
the a s s a y a l l o w s t h e c o n v e n i e n t measurement o f t h e e s t e r a s e
a c t i v i t y o f human u r i n e . U r i n e samples 20 20 - 100 y l cause a
l i n e a r absorbance i n c r e a s e o f 0.04 t o 0.2 p e r 10 m i n . Known
amounts o f human u r i n a r y k a l l i k r e i n added t o u r i n e samples r a i s e d
the e s t e r a s e a c t i v i t y t o t h e expected e x t e n t .
I f t h e u r i n e samples
c o n t a i n e t h a n o l i t has t o be removed by d i a l y s i s .
Therefore,
e t h a n o l i n t a k e s h o u l d be a v o i d e d d u r i n g t h e u r i n e c o l l e c t i o n p e r i o d .
T a b l e 4.
Assay o f human u r i n a r y k a l l i k r e i n u s i n g t h e s u b s t r a t e
Ac-Phe-ArgOEt
2.00 m l
0.10
0.10
0.02
(0.28+x)
The
ml
ml
ml
ml
0.15 M sodium d i p h o s p h a t e b u f f e r , pH 8.7
c o n t a i n i n g 0.15 M s e m i c a r b a z i d i u m
c h l o r i d e and 0.0375 M g l y c i n e
0.03 M NAD
0.015 M AcPheArgOEt a c e t a t e
a l c o h o l dehydrogenase (100 mg/3.4 ml)
water
5 min p r e i n c u b a t i o n a t 25°C
(0.5-x) m l enzyme s o l u t i o n
F i n a l volume: 3 m l
change i n absorbance i s m o n i t o r e d f o r 10 m i n . a t 366 nm.
HUMAN URINARY KALLIKREIN
239
KALLIKREIN
AC-PHE-ARGOET
C L9 He
OH + NAD
3
Fig.
2.
A
>
N
AC-PHE-ARG
ALKOHOLDEHYDROGENASE
( «
)
R
+ C2
M
H5
.„
OH
^
CH? - C . , + NADHo
^
H
R e a c t i o n scheme o f k a l l i k r e i n assay u s i n g Ac-Phe-ArgOEt
as S u b s t r a t e .
A number o f experiments were undertaken t o v e r i f y t h a t t h e
esterase a c t i v i t y r e f l e c t s the k a l l i k r e i n content of u r i n e . T r a s y l o l completely i n h i b i t e d the e s t e r a s e a c t i v i t y .
Dialysed urine
samples o f 10 d i f f e r e n t persons were a s s a y e d .
The r e s u l t s were
compared w i t h those o b t a i n e d by t h e dog b l o o d p r e s s u r e a s s a y , by
a radioimmunoassay f o r human u r i n a r y k a l l i k r e i n (Mann and G e i g e r
1977) and by t h e assay w i t h D-Val-Leu-ArgNHNp as s u b s t r a t e ( F i g .
3).
Though the c o r r e l a t i o n c o e f f i c i e n t o f t h e r e s u l t s o f t h e
Ac-Phe-ArgOEt assay and t h e dog b l o o d p r e s s u r e a s s a y ( F i g . 3a)
T a b l e 5.
Absorbance changes i n a s s a y s o f human u r i n a r y
k a l l i k r e i n with various substrates.
( R e a c t i o n volume 3 m l , c u v e t t e l i g h t path 1 cm)
REACTION
BLANK
(AA.10
3
1
X MIN" )
(AA.10
3
1
1 100
AC-PHE-ARGOET
0.6
D-VAL-LEU-ARGOET
0.6
430
B Z -ARGOET
0.5
24
15
Z-TYRONP
D-VAL-LEU-ARG-P-NITRANILIDE
"
29
1
0.3
BZ-PHE-VAL-ARG-PGLU-GLY-ARG-P-
460
3
D-PRO-PHE-ARG-PTOS-GLY-PRO-ARG-P-
0.0
"
1
X MIN' X IT )
0.3
R E L A T I VE
SENSITIVITIES
R. GEIGER E T A L .
240
i s r a t h e r c l o s e t o 1, a c o n s i d e r a b l e s c a t t e r i n g o f t h e d a t a i s
observed. This i s not too s u r p r i s i n g , s i n c e the c o e f f i c i e n t o f
v a r i a t i o n o f t h e b l o o d p r e s s u r e a s s a y f o r k a l l i k r e i n amounts t o
20% (Arens and H a b e r l a n d 1973). The c o r r e l a t i o n between t h e
Ac-Phe-ArgOEt a s s a y and b o t h t h e radioimmunoassay ( F i g . 3b) and
the D - V a l - L e u - A r g - p - n i t r o a n i l i d e a s s a y i s even more s a t i s f a c t o r y .
The e x c e l l e n t c o r r e l a t i o n between t h e two e n z y m a t i c a s s a y s ( F i g .
3c) s t r o n g l y s u g g e s t s t h a t i n b o t h a s s a y s t h e same enzyme i s
d e t e r m i n e d . F o r a m i x t u r e o f enzymes a s i m i l a r l y good c o r r e l a t i o n
would o n l y be expected i f t h e s e enzymes were always e x c r e t e d i n
identical ratios.
D - V a l - L e u - A r g - p - n i t r o a n i l i d e has been suggested q u i t e r e c e n t l y
by KABI (Amundsen e t a l . 1978) as a s u b s t r a t e f o r t h e d e t e r m i n a t i o n
ng/ml Kallikrein
D-ValLeuArgNHNp [u/l]
RIA
F i g . 3.
C o m p a r i s i o n o f t h e Ac-Phe-ArgOEt a s s a y f o r human u r i n a r y
k a l l i k r e i n w i t h the blood p r e s s u r e assay ( a ) , the r a d i o immunoassay (b) and t h e D-Val-Leu-ArgNHNp a s s a y ( c ) .
10 samples o f human u r i n e were measured.
HUMAN URINARY KALLIKREIN
241
of human u r i n a r y k a l l i k r e i n . H y d r o l y s i s o f p - n i t r o a n i l i d e s can
be m o n i t o r e d a t 405 nm. N e g l i g i b l e spontaneous h y d r o l y s i s o f
t h e s e compounds a l l o w s w o r k i n g a t 37°C. To o b t a i n abosrbance
changes o f s u f f i c i e n t magnitude, an i n c u b a t i o n time o f 30 min was
n e c e s s a r y f o r u r i n e samples o f 50 t o 500 u l . T h i s l o n g i n c u b a t i o n
time p r e c l u d e d c o n t i n u o u s m o n i t o r i n g o f t h e r e a c t i o n . The r e s u l t s
had t o be c o r r e c t e d f o r t h e i n h e r e n t absorbance o f t h e u r i n e
samples t h a t was determined i n p a r a l l e l ( b l a n k s c o n t a i n w a t e r
i n s t e a d o f s u b s t r a t e ) . The r e a c t i o n c o n d i t i o n s a r e g i v e n i n
T a b l e 6.
E v i d e n t l y , b o t h Ac-Phe-ArgOEt and D-Val-Leu-ArgNHNp a r e u s e f u l
s u b s t r a t e s f o r k a l l i k r e i n d e t e r m i n a t i o n i n human u r i n e . The
advantage o f t h e Ac-Phe-ArgOEt assay i s t h e p o s s i b i l i t y o f c o n t i n u o u s m o n i t o r i n g d u r i n g t h e t e s t , a drawback i s t h e a l c o h o l
s e n s i t i v i t y o f t h e r e a c t i o n . T h i s i s n o t found i n t h e a s s a y w i t h
D-Val-Leu-ArgNHNp, b u t due t o i t s low s e n s i t i v i t y , t h i s method
s u f f e r s from t h e d i s a d v a n t a g e s o f a two p o i n t a s s a y .
The radioimmunoassay f o r human u r i n a r y k a l l i k r e i n was a l s o
a p p l i e d t o c l a r i f y , whether endogenous g l a n d u l a r k a l l i k r e i n i s
p r e s e n t i n t h e b l o o d . We found g l a n d u l a r k a l l i k r e i n i n human
serum i n c o n c e n t r a t i o n s o f 10-15 ng/ml. I n o r d e r t o a s c e r t a i n
t h a t t h e r a d i o i m m u n o a s s a y a b l e s u b s t a n c e was n o t a low m o l e c u l a r
d e g r a d a t i o n p r o d u c t , serum samples were s u b j e c t e d t o g e l f i l t r a t i o n
and t h e f r a c t i o n s t e s t e d by radioimmunoassay ( F i g . 4 ) . A f t e r g e l
f i l t r a t i o n one peak was d e t e c t e d i n t h e p o s i t i o n o f a m o l e c u l a r
w e i g h t o f about 80,000 whereas o u r k a l l i k r e i n p r e p a r a t i o n was
e l u t e d i n t h e p o s i t i o n o f about 50,000. The e x a c t o r i g i n o f t h e
g l a n d u l a r k a l l i k r e i n i n b l o o d i s unknown, a d i s c r i m i n a t i o n b y
radioimmunoassay i s i m p o s s i b l e because o f t h e i m m u n o l o g i c a l c r o s s -
T a b l e 6.
A s s a y o f human u r i n a r y k a l l i k r e i n u s i n g t h e s u b s t r a t e
D-Val-Leu-ArgNHNp
0.4 ml
X ml
(0.5-x) m l
0.4 M TRIS/HC1, pH 8.2
urine
water
5 m i n p r e i n c u b a t i o n a t 37°C
0.1 m l
0.001
M D-Val-Leu-ArgNHNp
I n c u b a t i o n a t 37°C
The absorbance i n c r e a s e (405 nm) a f t e r 30 m i n . i s r e a d .
R. GEIGER E T A L .
242
r e a c t i v i t y of thevarious t i s s u e k a l l i k r e i n s .
The p r e s e n c e o f
glandular k a l l i k r e i n i n blood leads to the assumption that r e n a l
f i l t r a t i o n might c o n t r i b u t e t o some e x t e n t t o t h e amount o f k a l l i k r e i n found i n u r i n e .
Gelfiltration
of
human
serum
0.20
Fraction
F i g . 4.
number
G e l f i l t r a t i o n o f human serum t h r o u g h S e p h a c r y l
S-200.
The f r a c t i o n s were a s s a y e d by a radioimmunoassay f o r
human u r i n a r y k a l l i k r e i n .
ABBREVIATIONS
Oi
Bz-ArgOEt: N - B e n z o y l - a r g i n i n e e t h y l e s t e r , Z-TyrONp: Carbobenzox y t y r o s i n e n i t r o p h e n y l e s t e r , Ac-Phe-ArgOMe: A c e t y l - p h e n y l a l a n y l a r g i n i n e m e t h y l e s t e r , Ac-Phe-ArgOEt: A c e t y l - p h e n y l a l a n y l - a r g i n i n e
e t h y l e s t e r , Z-Ser-Pro-Phe-Arg-MNA: C a r b o b e n z o x y - s e r y l - p r o l y l p h e n y l a l a n y l - a r g i n i n e - p - n i t r o a n i l i d e , D-Val-Leu-ArgOEt: D - v a l y l leucyl-arginine ethyl ester.
ACKNOWLEDGEMENT
T h i s work was s u p p o r t e d by Deutsche F o r s c h u n g s g e m e i n s c h a f t ,
S o n d e r f o r s c h u n g s b e r e i c h 5 1 , München. We a r e i n d e b t e d t o Dr. C.
K u t z b a c h , Bayer AG, f o r p r o v i d i n g p r e p r o c e s s e d s t a r t i n g m a t e r i a l
f o r t h e i s o l a t i o n o f human u r i n a r y k a l l i k r e i n . We thank v e r y much
P r o f . H. F r i t z f o r h i s i n t e r e s t i n t h i s work and h i s s t i m u l a t i n g
d i s c u s s i o n s and comments.
HUMAN URINARY KALLIKREIN
243
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