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Species-determined differences in cardiac phosphorylase activity
Comp. Biochem. Physiol., 1966, Vol. 18, pp. 931 to 936. Pergamon Press Ltd. Pn'nted in Great Britain
SPECIES-DETERMINED DIFFERENCES IN CARDIAC PHOSPHORYLASE ACTIVITY* W I N I F R E D G. N A Y L E R t and N. C. R. M E R R I L L E E S ~ Baker Medical Research Institute, Melbourne, Victoria, Australia, and Department of Anatomy, University of Melbourne, Victoria, Australia (Received 28 February 1966)
Abstract--1. The activity of the enzyme phosphorylase in muscle excised from a variety of animals was determined. 2. Total phosphorylase activity in ventricular muscle excised from trout, carp and tench fish was significantly greater than that in ventricular muscle excised from other animals, including rabbits, lampreys, toads and tortoises, provided that activity was expressed on a wet weight basis. 3. The percentage of the enzyme phosphorylase in the a form in toad microsomal preparation was significantly greater than that in microsomal preparations similarly separated from ventricular muscle derived from a variety of other animals. INTRODUCTION THE enzyme phosphorylase is present in a wide variety of tissues (Sutherland & Rall, 1960). This enzyme, which catalyses the reaction glycogen + inorganic phosphate -- glucose-l-phosphate + glycogen: glucose residue, occurs in both cardiac and skeletal muscle in two forms, the a form which is active in the absence of adenosine monophosphate and the b form which requires the presence of adenylic acid as a co-factor to elicit activity (Cori et al., 1943 ; Cori & Illingworth, 1956; Rail et al., 1956; Belford & Feinleib, 1959; Mayer & Moran, 1960). The addition of minute amounts of adrenaline to either skeletal or cardiac muscle promotes transformation of phosphorylase b to the a form, this transformation being an indirect effect involving increased synthesis of cyclic adenosine monophosphate (Rail & Sutherland, 1958; Sutherland &Rall, 1960). During a series of experiments planned to investigate the effect of inotropically active drugs on hearts isolated from a wide variety of animals the occurrence of a marked species-dependent difference in cardiac phosphorylase activity was noted. These findings are described in this paper. * This work was done during the tenure of Grants-in-Aid from the National Heart Foundation of Australia. f Postal address: Commercial Road, Prahran S.1, Victoria, Australia. ++Postal address: Department of Anatomy, University of Melbourne, Parkville, Victoria, Australia. 931
932
WINIFRED G. NAYLER AND N . C. R. MERRILLEES
MATERIALS AND METHODS Hearts were removed from lampreys (Mordacia), rainbow trout (Salmo gairdneri), Asiatic carp (Carassius auratus), European tench ( Tinca tinca), tortoises (Chelodina longicollis) and rabbits (Orictologus cuniculus), all of which had been stunned by a blow on the head, and from toads (Bufo marinus) which had been pithed. Atria and superficially located fat deposits were rapidly discarded and the ventricles either immediately covered with an icecold aqueous solution containing 0"25 M sucrose + 0"005 M histidine, pH 7"4, or frozen by means of forceps which previously had been chilled in a dry-ice isopentane mixture. T h e frozen ventricles were weighed, pulverized and assayed for phosphorylase activity as described below. Ventricles which had been immersed in 0'25 M sucrose+0.005 M histidine solution were weighed, homogenized and then used for the isolation of particular subcellular fractions as required.
Determination of phosphorylase activity in ventricular muscle Each 100 mg of frozen pulverized ventricle was extracted with 5 ml of ice-cold 0-02 M sodium fluoride containing 0'001 M disodium ethylenediaminetetraacetic acid, pH 7'0. After centrifugation at 1000 g for 30 min at 0°C the tissue extract was added to 0"5 ml of substrate mixture with and without adenosine-5-phosphate, to provide duplicate tissueextract concentrations of 1/150 as described previously (Nayler & Wright, 1963). T h e substrate mixture contained 1% glycogen (as rabbit liver glycogen, Nutritional Biochem. Lab. purified as described by Krebs et al., 1959), 0-016 glucose-l-phosphate and, for the determination of total phosphorylase activity, 0"001 M adenosine-5-phosphate. T h e pH was adjusted to 6.1. Inorganic phosphate was determined by the method of Lowry & Lopez (1946), before and after 5 rain incubation at either 25 or 30°C as required. T h e reaction was terminated by the addition of trichloroacetic acid. Phosphorylase activity was calculated as described by Belford & Feinleib (1959). Thus the ratio PO4 formed without AMP x 100 -PO4 formed with AMP is designated in the present paper as the percentage of active phosphorylase a. All determinations were made in duplicate.
Preparation of microsomal subcellular fractions Ventricles which had been immersed in ice-cold 0'25 M sucrose + 0"005 M histidine solution were homogenized, using a Potter-Elvejehm type homogenizer operating at 1425 rev/min. The internal diameter of the smooth glass tube of the particular homogenizer used was 2"545 cm and the outside diameter of the pestle 2"53 era. During homogenization a cooling jacket (2°C) was used to prevent excessive heating of the homogenate, the final volume of which was adjusted to provide a 10% suspension (w/v referred to wet weight of tissue) in 0"25 M sucrose+0"005 M histidine solution. T h e resultant suspension was centrifuged at 600 g for 30 rain at 2°C in a Serval refrigerated centrifuge. The sediment was discarded and the supernatant centrifuged at 10,000 g for 30 min at 2°C after which the resultant sediment was discarded and the supernatant centrifuged at 104,000 g for 60 rain in a No. 30 head of a Spinco preparative ultracentrifuge. T h e resultant pale pink sediment, the microsomalfraction, was resuspended in an aqueous solution containing 0-25 M sucrose, 0"005 M histidine, 0"02 M sodium fluoride and 0.001 M disodium ethylenediaminetetraacetic acid so that the suspension contained approximately 0"4 mg protein/ml. Protein estimations were made according to the method of Lowry et al. (1951) using bovine albumin as the standard. T h e presence of small membrane-limited vesicles in this fraction was confirmed by electron microscopy.
933
CARDIAC P H O S P H O R Y L A S E A C T I V I T Y
In some additional experiments ventricular muscle isolated from toads, trout and rabbits was frozen and pulverized before being homogenized in ice-cold sucrose--histidine solution as described above. Mierosomal fractions were then isolated as described above and used for the determination of phosphorylase activity.
Determination of phosphorylase activity in microsomal subcellular fraction A microsomal suspension containing approximately 0"2 mg protein in 0-5 ml of the suspending medium was added to the substrate solution containing glycogen and glucose-l-phosphate with and without adenosine-5-phosphate as described above. Inorganic phosphate liberated during 5 rain incubation at either 25 or 30°C was determined as previously described. RESULTS
Ventricular phosphorylase activity T h e total phosphorylase activity displayed by extracts prepared from h o m o genized ventricles of toads, tortoises, lampreys and rabbits was found to be
120
g
÷ 4÷÷÷ ÷~ ÷÷ ÷÷ ÷ ÷ ÷
ioo
> ~_ 80
o
÷
.++ +++
~ 4o
~
20
÷ 4. ÷ 4 4..F4-4. 4-
TOAD
÷ 4÷÷4.÷4 ÷ 4. ÷ 4-4.÷
LAMPREY
÷
4. ÷+ ÷ ÷ 44..
TORTOISE
RABBIt
FISH
FIG. 1. Total phosphorylase activity, as pmoles PO4 liberated per g wet weight of ventricular muscle, isolated from toads, lampreys, tortoises, rabbits and fish. Incubation was effected at 25°C in the presence of added adenylic acid. significantly* ( P > 0.001) lower, per g wet weight, than that of similar extracts prepared under identical conditions from h o m o g e n i z e d ventricles of the various fish hearts used in this study. T h e results of a series of experiments, in which phosphorylase activity was established by incubating the various tissue extracts with the substrate mixture at 25°C, are summarized in Fig. 1 and Table 1. * Tests of significance calculated according to the Student's t test.
934
W I N I F R E D G . N A Y L E R AND N . C . R . MERRILLEES
The phosphorylase activities of carp, tench and trout ventricular muscle did not differ significantly from one another and, accordingly, results relating to these preparations have been pooled in Fig. 1. A similar relatively high level of phosphorylase activity, per g wet weight was displayed by the ventricular muscle of these fish when incubation was effected at other temperatures, including 20 and 30°C. These experiments were performed in random sequence throughout the T A B L E 1 - - T O T A L PHOSPHORYLASE ACTIVITY AND PERCENTAGE OF THE ENZYME PHOSPHORYLASE IN THE a FORM IN TROU% CARP~ TENCH, LAMPREY AND RABBIT HOMOGENIZED VENTRICULAR MUSCLE INCUBATED AT 2 5 ° C
Preparation
Total phosphorylase activity (/~molesPO4/g wet wt.)
Trout (4) Carp (4) Tench (4) Toad (4) Lamprey (4) Rabbit (4)
98'6 + 11-3 99"7 ± 7-9 100"9 + 2-1 26"5 + 2"2 22'9 _+4.06 42"15 ± 0.72
% Phosphorylase a 62'9 + 3-3 61 '7 _+2'9 61.6 ± 1'4 60.6 ± 3"8 61 '2 ± 5.9 575 +_2-3
Results expressed as means and standard deviations. Numbers in brackets refer to number of experiments. year so that the difference between the total phosphorylase activity displayed per g wet weight by the various fish ventricular preparations and that of the other ventricular preparations used cannot be attributed to seasonal changes in activity. Other observations indicate, however, that some of the scatter within individual groups probably is due to such seasonal variations in total activity. The percentage of the enzyme which was active in the absence of added adenylic acid, and which therefore was present in the a form, was approximately the same in all these preparations. Resuks listed in Table 1 show that, when studied under the present experimental conditions, more than half of the phosphorylase enzyme present in trout, carp, tench, toad, lamprey and rabbit ventricular muscle appears to be present in the a form.
Phosphorylase activity in microsomal fractions Phosphorylase activity was readily demonstrable in microsomal fractions isolated from the various non-frozen ventricular preparations used during these experiments. The total phosphorylase activity displayed by trout, rabbit and toad microsomal preparations per mg protein, when incubated at 30°C in the presence of substrate, with and without adenylic acid is listed in Table 2. Similar results were obtained when incubation was effected at 25°C and when the microsomal fractions have been isolated from ventricles which had been frozen prior to homogenization and differential centrifugation.
935
CARDIAC PHOSPHORYLASE ACTIVITY
These results show that the phosphorylase activity in trout microsomal fraction, per mg protein, is significantly (P > 0.01) less than that in rabbit, but significantly (P > 0.001) greater than that found in microsomal fractions similarly prepared from either toads or lampreys. The activity per mg protein present in carp and tench microsomal fractions closely resembled that for trout ventricular muscle, as is shown in this same table (Table 2). TABLE
2 - - P H O S P H O R Y L A S E ACTIVITY IN MICROSOMAL FRACTIONS ISOLATED FROM TROUT, CARP, TENCH~ TOAD, LAMPREY AND RABBIT VENTRICULAR MUSCLE
Preparation Trout (7) Carp (4) Tench (4) Toad (6) Lamprey (4) Rabbit (4)
Microsomal phosphorylase activity ~/o Phosphory(/zmolesPOJmg microsomal protein) lase a 2"55 _+0"335 2"62 + 0"284 2"46 + 0"216 0"98 + 0"14 0-88 _+0"12 3"63 +_0"52
1"35 + 0"15 1"29 + 0"21 1"38 + 0"16 38"5 + 3-6 7"22 + 0"98 6'55 + 1"09
Results expressed as means and standard deviations. Numbers in brackets refer to number of experiments. Microsomal fractions separated from toad ventricular muscle differed markedly from those separated from tench, trout, carp or rabbit ventricles in that approximately 38 per cent of the activity was present in the a form, compared with only 1.4 per cent in trout and 6.5 per cent in rabbit microsomal preparations. These results are summarized in Table 2. DISCUSSION • These results indicate that the total activity of the enzyme phosphorylase, isolated from homogenized ventricles of certain fish including carp, tench and trout, is significantly higher than that found in ventricular muscle similarly isolated from a variety of other animals, including lampreys, tortoises, toads and rabbits, provided that the total activity is expressed in terms of the wet weight of the muscle. Despite this difference in total activity, the percentage of the enzyme which appeared to be in the a form, and hence active in the absence of added adenylic acid, was approximately the same for all species studied, again provided that estimations were made on homogenized non-fractionated muscle. Estimations of phosphorylase activity in the microsomal subcellular fractions prepared from ventricles isolated from these same species gave results which contrasted sharply with those obtained when whole muscle was used. Thus the phosphorylase activity present in the microsomal fraction of the various fish ventricles was found to be less per mg microsomal protein than that displayed by microsomal preparations similarly prepared from rabbit ventricular muscle, but
36
WINIFRED G. NAYLER AND N. C. R. MERRILLEES
was greater than that found in these same fractions isolated f r o m toad or lamprey ventricular muscle. Moreover, the percentage of the phosphorylase enzyme which appeared to be in the a f o r m in these microsomal preparations was m u c h higher for toad than for any of the other preparations used, a finding which m a y suggest that this fraction, when isolated f r o m toad ventricular muscle, contains another subcellular c o m p o n e n t capable of transforming phosphorylase b to the a form. I f this is so then it m u s t follow that this subcellular c o m p o n e n t is either present in lesser concentrations or absent from the other microsomal preparations used, and that its effectiveness in transforming phosphorylase b to a is enhanced by its isolation f r o m other subcellular components. REFERENCES BELFORD J. & FEINLEIBM. R. (1959) Phosphorylase activity of heart muscle under various conditions affecting force of contraction. J. Pharm. exp. Ther. 127~ 257-264. CORI C. F., Corn G. T. & GREEN A. A. (1943) Crystalline muscle phosphorylase--III. Kinetics. J. biol. Chem. 151~ 39-55. CoRI G. T. & ILL:V:CWORTHB. (1956) The effect of epinephrine and other glycogenolytic agents on the phosphorylase a content of muscle. Biochim. biophys. Acta 21~ 106-110. KREBS E. G., GRAVESD. J., & FISCHERE. H. (1959) Factors affecting the activity, of muscle phosphorylase b kinase. J. biol. Chem. 234~ 2867-2873. LowRY O. H. & LOPEZ J. A. (1946) The determination of inorganic phosphate in the presence of labile phosphate esters, ft. biol. Chem. 162~ 421-428. LOWRy D. H., ROSEBROUGHN. J., LEwis FARRA. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193~ 265-275. MAYER E. & MORAN N. C. (1960) Relation between pharmacologic augmentation of cardiac contractile force and the activation of myocardial glycogen phosphorylase, ft. Pharmacol. exp. Ther. 129~ 271-281. NAYLER W. G. & WRIGHT J. E. (1963) The effect of adrenaline on the mechanical and phosphorylase a activity of normo- and hypothermic hearts. Circular. Res. 13~ 199-206. RALL T. W. & SUTHERLANDE. W. (1958) Formation of a cyclic adenine ribonucleotide by tissue particles, o% (]~ol. Chem. 232~ 1065-1076. RALLT. W., WOSILAITW. D. & SUTHERLANDE. W. (1956) The interconversion of phosphorylase a and phosphorylase b from dog heart muscle. Biochim. biol)hys. Acta 20~ 69-76. SUTH~RLAND E. W. & RALL T. W. (1960) The relation of adenosine-3'5-phosphate and phosphorylase to the actions of catecholamines and other hormones. Pharmacol. Rev. 12~ 265-299.
SPECIES-DETERMINED DIFFERENCES IN CARDIAC PHOSPHORYLASE ACTIVITY* W I N I F R E D G. N A Y L E R t and N. C. R. M E R R I L L E E S ~ Baker Medical Research Institute, Melbourne, Victoria, Australia, and Department of Anatomy, University of Melbourne, Victoria, Australia (Received 28 February 1966)
Abstract--1. The activity of the enzyme phosphorylase in muscle excised from a variety of animals was determined. 2. Total phosphorylase activity in ventricular muscle excised from trout, carp and tench fish was significantly greater than that in ventricular muscle excised from other animals, including rabbits, lampreys, toads and tortoises, provided that activity was expressed on a wet weight basis. 3. The percentage of the enzyme phosphorylase in the a form in toad microsomal preparation was significantly greater than that in microsomal preparations similarly separated from ventricular muscle derived from a variety of other animals. INTRODUCTION THE enzyme phosphorylase is present in a wide variety of tissues (Sutherland & Rall, 1960). This enzyme, which catalyses the reaction glycogen + inorganic phosphate -- glucose-l-phosphate + glycogen: glucose residue, occurs in both cardiac and skeletal muscle in two forms, the a form which is active in the absence of adenosine monophosphate and the b form which requires the presence of adenylic acid as a co-factor to elicit activity (Cori et al., 1943 ; Cori & Illingworth, 1956; Rail et al., 1956; Belford & Feinleib, 1959; Mayer & Moran, 1960). The addition of minute amounts of adrenaline to either skeletal or cardiac muscle promotes transformation of phosphorylase b to the a form, this transformation being an indirect effect involving increased synthesis of cyclic adenosine monophosphate (Rail & Sutherland, 1958; Sutherland &Rall, 1960). During a series of experiments planned to investigate the effect of inotropically active drugs on hearts isolated from a wide variety of animals the occurrence of a marked species-dependent difference in cardiac phosphorylase activity was noted. These findings are described in this paper. * This work was done during the tenure of Grants-in-Aid from the National Heart Foundation of Australia. f Postal address: Commercial Road, Prahran S.1, Victoria, Australia. ++Postal address: Department of Anatomy, University of Melbourne, Parkville, Victoria, Australia. 931
932
WINIFRED G. NAYLER AND N . C. R. MERRILLEES
MATERIALS AND METHODS Hearts were removed from lampreys (Mordacia), rainbow trout (Salmo gairdneri), Asiatic carp (Carassius auratus), European tench ( Tinca tinca), tortoises (Chelodina longicollis) and rabbits (Orictologus cuniculus), all of which had been stunned by a blow on the head, and from toads (Bufo marinus) which had been pithed. Atria and superficially located fat deposits were rapidly discarded and the ventricles either immediately covered with an icecold aqueous solution containing 0"25 M sucrose + 0"005 M histidine, pH 7"4, or frozen by means of forceps which previously had been chilled in a dry-ice isopentane mixture. T h e frozen ventricles were weighed, pulverized and assayed for phosphorylase activity as described below. Ventricles which had been immersed in 0'25 M sucrose+0.005 M histidine solution were weighed, homogenized and then used for the isolation of particular subcellular fractions as required.
Determination of phosphorylase activity in ventricular muscle Each 100 mg of frozen pulverized ventricle was extracted with 5 ml of ice-cold 0-02 M sodium fluoride containing 0'001 M disodium ethylenediaminetetraacetic acid, pH 7'0. After centrifugation at 1000 g for 30 min at 0°C the tissue extract was added to 0"5 ml of substrate mixture with and without adenosine-5-phosphate, to provide duplicate tissueextract concentrations of 1/150 as described previously (Nayler & Wright, 1963). T h e substrate mixture contained 1% glycogen (as rabbit liver glycogen, Nutritional Biochem. Lab. purified as described by Krebs et al., 1959), 0-016 glucose-l-phosphate and, for the determination of total phosphorylase activity, 0"001 M adenosine-5-phosphate. T h e pH was adjusted to 6.1. Inorganic phosphate was determined by the method of Lowry & Lopez (1946), before and after 5 rain incubation at either 25 or 30°C as required. T h e reaction was terminated by the addition of trichloroacetic acid. Phosphorylase activity was calculated as described by Belford & Feinleib (1959). Thus the ratio PO4 formed without AMP x 100 -PO4 formed with AMP is designated in the present paper as the percentage of active phosphorylase a. All determinations were made in duplicate.
Preparation of microsomal subcellular fractions Ventricles which had been immersed in ice-cold 0'25 M sucrose + 0"005 M histidine solution were homogenized, using a Potter-Elvejehm type homogenizer operating at 1425 rev/min. The internal diameter of the smooth glass tube of the particular homogenizer used was 2"545 cm and the outside diameter of the pestle 2"53 era. During homogenization a cooling jacket (2°C) was used to prevent excessive heating of the homogenate, the final volume of which was adjusted to provide a 10% suspension (w/v referred to wet weight of tissue) in 0"25 M sucrose+0"005 M histidine solution. T h e resultant suspension was centrifuged at 600 g for 30 rain at 2°C in a Serval refrigerated centrifuge. The sediment was discarded and the supernatant centrifuged at 10,000 g for 30 min at 2°C after which the resultant sediment was discarded and the supernatant centrifuged at 104,000 g for 60 rain in a No. 30 head of a Spinco preparative ultracentrifuge. T h e resultant pale pink sediment, the microsomalfraction, was resuspended in an aqueous solution containing 0-25 M sucrose, 0"005 M histidine, 0"02 M sodium fluoride and 0.001 M disodium ethylenediaminetetraacetic acid so that the suspension contained approximately 0"4 mg protein/ml. Protein estimations were made according to the method of Lowry et al. (1951) using bovine albumin as the standard. T h e presence of small membrane-limited vesicles in this fraction was confirmed by electron microscopy.
933
CARDIAC P H O S P H O R Y L A S E A C T I V I T Y
In some additional experiments ventricular muscle isolated from toads, trout and rabbits was frozen and pulverized before being homogenized in ice-cold sucrose--histidine solution as described above. Mierosomal fractions were then isolated as described above and used for the determination of phosphorylase activity.
Determination of phosphorylase activity in microsomal subcellular fraction A microsomal suspension containing approximately 0"2 mg protein in 0-5 ml of the suspending medium was added to the substrate solution containing glycogen and glucose-l-phosphate with and without adenosine-5-phosphate as described above. Inorganic phosphate liberated during 5 rain incubation at either 25 or 30°C was determined as previously described. RESULTS
Ventricular phosphorylase activity T h e total phosphorylase activity displayed by extracts prepared from h o m o genized ventricles of toads, tortoises, lampreys and rabbits was found to be
120
g
÷ 4÷÷÷ ÷~ ÷÷ ÷÷ ÷ ÷ ÷
ioo
> ~_ 80
o
÷
.++ +++
~ 4o
~
20
÷ 4. ÷ 4 4..F4-4. 4-
TOAD
÷ 4÷÷4.÷4 ÷ 4. ÷ 4-4.÷
LAMPREY
÷
4. ÷+ ÷ ÷ 44..
TORTOISE
RABBIt
FISH
FIG. 1. Total phosphorylase activity, as pmoles PO4 liberated per g wet weight of ventricular muscle, isolated from toads, lampreys, tortoises, rabbits and fish. Incubation was effected at 25°C in the presence of added adenylic acid. significantly* ( P > 0.001) lower, per g wet weight, than that of similar extracts prepared under identical conditions from h o m o g e n i z e d ventricles of the various fish hearts used in this study. T h e results of a series of experiments, in which phosphorylase activity was established by incubating the various tissue extracts with the substrate mixture at 25°C, are summarized in Fig. 1 and Table 1. * Tests of significance calculated according to the Student's t test.
934
W I N I F R E D G . N A Y L E R AND N . C . R . MERRILLEES
The phosphorylase activities of carp, tench and trout ventricular muscle did not differ significantly from one another and, accordingly, results relating to these preparations have been pooled in Fig. 1. A similar relatively high level of phosphorylase activity, per g wet weight was displayed by the ventricular muscle of these fish when incubation was effected at other temperatures, including 20 and 30°C. These experiments were performed in random sequence throughout the T A B L E 1 - - T O T A L PHOSPHORYLASE ACTIVITY AND PERCENTAGE OF THE ENZYME PHOSPHORYLASE IN THE a FORM IN TROU% CARP~ TENCH, LAMPREY AND RABBIT HOMOGENIZED VENTRICULAR MUSCLE INCUBATED AT 2 5 ° C
Preparation
Total phosphorylase activity (/~molesPO4/g wet wt.)
Trout (4) Carp (4) Tench (4) Toad (4) Lamprey (4) Rabbit (4)
98'6 + 11-3 99"7 ± 7-9 100"9 + 2-1 26"5 + 2"2 22'9 _+4.06 42"15 ± 0.72
% Phosphorylase a 62'9 + 3-3 61 '7 _+2'9 61.6 ± 1'4 60.6 ± 3"8 61 '2 ± 5.9 575 +_2-3
Results expressed as means and standard deviations. Numbers in brackets refer to number of experiments. year so that the difference between the total phosphorylase activity displayed per g wet weight by the various fish ventricular preparations and that of the other ventricular preparations used cannot be attributed to seasonal changes in activity. Other observations indicate, however, that some of the scatter within individual groups probably is due to such seasonal variations in total activity. The percentage of the enzyme which was active in the absence of added adenylic acid, and which therefore was present in the a form, was approximately the same in all these preparations. Resuks listed in Table 1 show that, when studied under the present experimental conditions, more than half of the phosphorylase enzyme present in trout, carp, tench, toad, lamprey and rabbit ventricular muscle appears to be present in the a form.
Phosphorylase activity in microsomal fractions Phosphorylase activity was readily demonstrable in microsomal fractions isolated from the various non-frozen ventricular preparations used during these experiments. The total phosphorylase activity displayed by trout, rabbit and toad microsomal preparations per mg protein, when incubated at 30°C in the presence of substrate, with and without adenylic acid is listed in Table 2. Similar results were obtained when incubation was effected at 25°C and when the microsomal fractions have been isolated from ventricles which had been frozen prior to homogenization and differential centrifugation.
935
CARDIAC PHOSPHORYLASE ACTIVITY
These results show that the phosphorylase activity in trout microsomal fraction, per mg protein, is significantly (P > 0.01) less than that in rabbit, but significantly (P > 0.001) greater than that found in microsomal fractions similarly prepared from either toads or lampreys. The activity per mg protein present in carp and tench microsomal fractions closely resembled that for trout ventricular muscle, as is shown in this same table (Table 2). TABLE
2 - - P H O S P H O R Y L A S E ACTIVITY IN MICROSOMAL FRACTIONS ISOLATED FROM TROUT, CARP, TENCH~ TOAD, LAMPREY AND RABBIT VENTRICULAR MUSCLE
Preparation Trout (7) Carp (4) Tench (4) Toad (6) Lamprey (4) Rabbit (4)
Microsomal phosphorylase activity ~/o Phosphory(/zmolesPOJmg microsomal protein) lase a 2"55 _+0"335 2"62 + 0"284 2"46 + 0"216 0"98 + 0"14 0-88 _+0"12 3"63 +_0"52
1"35 + 0"15 1"29 + 0"21 1"38 + 0"16 38"5 + 3-6 7"22 + 0"98 6'55 + 1"09
Results expressed as means and standard deviations. Numbers in brackets refer to number of experiments. Microsomal fractions separated from toad ventricular muscle differed markedly from those separated from tench, trout, carp or rabbit ventricles in that approximately 38 per cent of the activity was present in the a form, compared with only 1.4 per cent in trout and 6.5 per cent in rabbit microsomal preparations. These results are summarized in Table 2. DISCUSSION • These results indicate that the total activity of the enzyme phosphorylase, isolated from homogenized ventricles of certain fish including carp, tench and trout, is significantly higher than that found in ventricular muscle similarly isolated from a variety of other animals, including lampreys, tortoises, toads and rabbits, provided that the total activity is expressed in terms of the wet weight of the muscle. Despite this difference in total activity, the percentage of the enzyme which appeared to be in the a form, and hence active in the absence of added adenylic acid, was approximately the same for all species studied, again provided that estimations were made on homogenized non-fractionated muscle. Estimations of phosphorylase activity in the microsomal subcellular fractions prepared from ventricles isolated from these same species gave results which contrasted sharply with those obtained when whole muscle was used. Thus the phosphorylase activity present in the microsomal fraction of the various fish ventricles was found to be less per mg microsomal protein than that displayed by microsomal preparations similarly prepared from rabbit ventricular muscle, but
36
WINIFRED G. NAYLER AND N. C. R. MERRILLEES
was greater than that found in these same fractions isolated f r o m toad or lamprey ventricular muscle. Moreover, the percentage of the phosphorylase enzyme which appeared to be in the a f o r m in these microsomal preparations was m u c h higher for toad than for any of the other preparations used, a finding which m a y suggest that this fraction, when isolated f r o m toad ventricular muscle, contains another subcellular c o m p o n e n t capable of transforming phosphorylase b to the a form. I f this is so then it m u s t follow that this subcellular c o m p o n e n t is either present in lesser concentrations or absent from the other microsomal preparations used, and that its effectiveness in transforming phosphorylase b to a is enhanced by its isolation f r o m other subcellular components. REFERENCES BELFORD J. & FEINLEIBM. R. (1959) Phosphorylase activity of heart muscle under various conditions affecting force of contraction. J. Pharm. exp. Ther. 127~ 257-264. CORI C. F., Corn G. T. & GREEN A. A. (1943) Crystalline muscle phosphorylase--III. Kinetics. J. biol. Chem. 151~ 39-55. CoRI G. T. & ILL:V:CWORTHB. (1956) The effect of epinephrine and other glycogenolytic agents on the phosphorylase a content of muscle. Biochim. biophys. Acta 21~ 106-110. KREBS E. G., GRAVESD. J., & FISCHERE. H. (1959) Factors affecting the activity, of muscle phosphorylase b kinase. J. biol. Chem. 234~ 2867-2873. LowRY O. H. & LOPEZ J. A. (1946) The determination of inorganic phosphate in the presence of labile phosphate esters, ft. biol. Chem. 162~ 421-428. LOWRy D. H., ROSEBROUGHN. J., LEwis FARRA. & RANDALLR. J. (1951) Protein measurement with the Folin phenol reagent. J. biol. Chem. 193~ 265-275. MAYER E. & MORAN N. C. (1960) Relation between pharmacologic augmentation of cardiac contractile force and the activation of myocardial glycogen phosphorylase, ft. Pharmacol. exp. Ther. 129~ 271-281. NAYLER W. G. & WRIGHT J. E. (1963) The effect of adrenaline on the mechanical and phosphorylase a activity of normo- and hypothermic hearts. Circular. Res. 13~ 199-206. RALL T. W. & SUTHERLANDE. W. (1958) Formation of a cyclic adenine ribonucleotide by tissue particles, o% (]~ol. Chem. 232~ 1065-1076. RALLT. W., WOSILAITW. D. & SUTHERLANDE. W. (1956) The interconversion of phosphorylase a and phosphorylase b from dog heart muscle. Biochim. biol)hys. Acta 20~ 69-76. SUTH~RLAND E. W. & RALL T. W. (1960) The relation of adenosine-3'5-phosphate and phosphorylase to the actions of catecholamines and other hormones. Pharmacol. Rev. 12~ 265-299.