2. Methods and Materials. Assays of phosphorylase and phosphorylase kinase activity were ..... these two enzymes in the different fractions (Fig. 10) ⢠.. -.-----~--.
Hoerl,W.H., Jennissen,H.P., Groeschel-Stewart,U., & Heilmeyer,L.M.G. (1975) Calcium and cyclic AMP dependent phosphorylation of enzymes and contractile proteins. In Calcium Transport in Contraction and Secretion (E.Carafoli et al., ed), pp. 535-546. North Holland Publishing Company Co., Amsterdam.
Calcium TrtllUporl in Contraction and Secretion. E. CARAFOLI. (Ed. et aI) North-Holland Publishing Company (/975)
CALCIUM AND CYCLIC AMP DEPENDENT PHOSPHORYLATION OF ENZYMES AND CONTRACTILE PROTEINS W.H. Herl, H.P. Jennissen, U. Greschel-Stewart, L.M.G. Heilmeyer,Jr. Institut fUr Physiologische Chemie, Ruhr-Universitat, Bochum and Universitats-Frauenklinik, Wurzburg. 1. Introduction Ca 2 + and 3'5' cAMP are two intracellular second messengers of nervous impulses and hormonal signals respectively. Nervous stimulation controls muscle contraction and relaxation and seems to act by the release and uptake of Ca 2 + from the sarcoplasmic reticulum (for review 1). A calcium receptor protein of the fibrillar system, troponin, is located in the thin filament. Together with tropomyosin troponin blocks myosin actin interaction at calcium concentrations lower than 10- 8 M. This inhibition is released at calcium concentrations of > 10- 6 M. Synchronously with muscle contraction conversion of inactive phosphorylase
~
to the active
~
form occurs
thereby leading to glycogen breackdown and reformation of ATP. Phosphorylase a is formed by phosphorylation of the enzyme, which is catalyzed by a Ca 2 + dependent protein kinase, phosphorylase kinase, a large oligomer (MW 1.25 x 10 6 ) of the composition a4 (MW 'X. 135.000,
P
120.000,
Jt
P4
t4
42.000) (for review 2). This kinase 3
also catalyzes the phosphorylation of troponin, (Stull et al. , Pratje et al. 4, Perry et al. 5 ) and one of its own subunits, as will be shown below (see also Hayakawa et al. 6). In addition, this enzyme seems to be localized in membranes (W.H. Horl et al. Phosphorylation of proteins is also catalyzed by 3'5'
7).
cA~W
de-
pendent protein kinases. Among others,substrates for these enzymes are phosphorylase kinase (Hayakawa et al.
6 ; P. Cohen 8 ) and
troponin (Pratje and Heilmeyer 9), 2. Methods and Materials Assays of phosphorylase and phosphorylase kinase activity were performed on an Auto-Analyzer according to Haschke and Heilmeyer 10 and Jennissen and Heilmeyer 11 respectively. Proteins were isolated and phosphorylated as described previously (Pratje and Heilmeyer
4).
Thin sections of muscle were stained with FITC labelled antihodies 12 as described by Reder Vesicles of the sarcoplasmic reticulum were isolated and assayed for ATP activity according to de Meis and Hasselbach 13. Polyacrylamide electrophoresis in presence of SDS was carried out as described by Weber and Osborn 14
535
536
3. Results and Discussion A short electrical stimulation of the diaphragm of normal results in a complete conversion of phosphorylase
~
to
~
mice
as shown
in Table 1. Conversion of phosphorylase b to a in diaphragm of normal I-strain mice upon stimulation
and
total phosphorylase activity at rest U/mg
phosphorylase a activity after-stimulation U/mg
normal mice
0.425
0.429
I-strain mice
0.365
0.025
Table 1: Mouse diaphragm was divided into two parts and separately incubated in Krebs-Ringer solution in an atmosphere of 95~ 02, 5~ CO 2 , One part was stimulated indirectly at 50 cycles/sec for 20 sec (stimulus 30V 5msec duration), the other part was not stimulated. Both muscles were then frozen in freon, homogenized in a vibrator, extracted and assayed for phosphorylase ~ and ~ activity. In I-strain mice, deficient in phosphorylase kinase (Lyon et al. 15) no
~
to
~
conversion of phosphorylase can be detected. A
positive inotropic effect on the contraction can be demonstrated I-strain mice diaphragm upon addition of epinephrine (Fig. w z
a::
I
Q..
w
z
ii: w
I
......·R!!!.!!!-. -.
.
II _\
~iil!I···lI-ao;;· ,.
ii:
w
.~
~
-
10m sec
'",51
.
.
1).
.
ii
~
E
11.
Vi Z
5000
::J
w
4QOO
U1
< z
;-
11. 1/1 "'0 0 l: :z: -11. 11. 1/1 If) !:: 0 III
0.6
...J
0.2
~
E
z :z: w ::J11. 0 :l:
04
lhosphoryl Volum@n Prot~in ATPas~ Fraction Kinaw Phosphat. Phaspharyl. Kinaw Synt~tas" ./, ./. ml ./. U ./, mg/ml U ./. U 'I. U U U
~81 100
900
14.2
322 100
2.44000 xg wdi_nl
190
13.2
183
568
50942
72 251
25.6
20330 17.5 0.263
118
4104
29.8
3.ATP wash
410
5.7
147
45.6
51220
12 291
29.7
20910 18.0 0.262
17.8 4182
304
4. Kel wash
250
3.4
179
55.4
11304
1.6
362
36.9
6000
5.1 0.120
8.1
14.75
10.7
5.
215
2.9
154
47.6
4300
06 286
292
4515
3.9 0.095
64
8.39
6.1
38
16.1
136
421
2621
0.4 155 15.8
3002
2.6 0011
0.8
3.95
2.9
1.crud~
II
extract
"
6.crude vesicles
Fig~
709239 100
116100 100
1.476 100
137.70 100
8. Distribution of glycogen metabolizing enzymes among various fractions during preparation of vesicles 13 of the sarcoplasmic reticulum. Enzymes were assayed as referred to in Methods.
542
W
