Vol. 109, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS December 15, 1982 Pages 769-775 REGULATION OF MUSCLE PHOSPHORYLASE ACTIVITY BY CARNOSINE AND ANSERINE Peter Johnson*, Joanne S. Fedyna*,tAndrew Schindzielorz Cindylu Montz Smith* and tPeter J. Kasvinsky *Department of Chemistry and College of Osteopathic Medicine, Ohio University Athens, OH 45701, and tDepartment of Biochemistry, Marshall University Medical School, Huntington, WV 25704 Received October 22, 1982 SUMMARY. Carnosine (B-alanyl-L-histidine) activates rabbit muscle phosphorylase 5 inpresence and absence of AMP and phosphorylase b in the presence of AMP in a biphasic manner with a maximal activation at about 50mM carnosine and with phos- phorylase b showing a greater degree of activation than phosphorylase a. Anserine (B-alanyl-L-NT-methyl-histidine) activates phosphorylase a to a lesser extent than carnosine up to a concentration of 90mM, whereas with phosphorylase b a weak acti- vation below 30mM and a concentration-dependent inhibition above thiS concentration occurs. These effects are specific for the dipeptides and are not shown by their constituent amino acids. Carnosine and anserine activate phosphorylase 2 in the presence of the allosteric inhibitors ATP, D-glucose and caffeine, and the inhibi- tion of phosphorylase b by anserine is also observed in the presence of these inhibitors. Carnosine (B-alanyl-L-histidine) and anserine (8-alanyl-L-N*-methyl-histidine) are found in a wide variety of vertebrate muscles at concentrations approaching 40 millimolar in some cases (1). The dipeptides are generally found at higher con- centrations in anaerobic (fast twitch) muscles whereas highly aerobic muscles (such as cardiac muscle) have little or no detectable amounts of these compounds (2). The ratio of carnosine to anserine varies widely between different muscles and it has also been reported (3,4) that this ratio and the absolute levels of the dipeptides are not constant in a given muscle but vary with development, exercise and denervation. In addition to its presence in muscle, carnosine is also found in other excitable tissues (5,6), and considerable interest has centered on the high and variable amounts of the dipeptide in olfactory epithelium and bulb (7). Despite the relatively widespread distributions of these dipeptides, their precise physiological role(s) are not clearly established although several have 0006-291X/82/230769-07$01.00/0 769 Copyright 0 1982 by Academic Press, Inc. All rights of reproduction in any form reserved.
Transcript
Vol. 109, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS December 15, 1982 Pages 769-775
REGULATION OF MUSCLE PHOSPHORYLASE ACTIVITY BY CARNOSINE AND ANSERINE
Peter Johnson*, Joanne S. Fedyna*,tAndrew Schindzielorz Cindylu Montz Smith* and tPeter J. Kasvinsky
*Department of Chemistry and College of Osteopathic Medicine, Ohio University Athens, OH 45701, and tDepartment of Biochemistry, Marshall University Medical
School, Huntington, WV 25704
Received October 22, 1982
SUMMARY. Carnosine (B-alanyl-L-histidine) activates rabbit muscle phosphorylase 5 inpresence and absence of AMP and phosphorylase b in the presence of AMP in a biphasic manner with a maximal activation at about 50mM carnosine and with phos- phorylase b showing a greater degree of activation than phosphorylase a. Anserine (B-alanyl-L-NT-methyl-histidine) activates phosphorylase a to a lesser extent than carnosine up to a concentration of 90mM, whereas with phosphorylase b a weak acti- vation below 30mM and a concentration-dependent inhibition above thiS concentration occurs. These effects are specific for the dipeptides and are not shown by their constituent amino acids. Carnosine and anserine activate phosphorylase 2 in the presence of the allosteric inhibitors ATP, D-glucose and caffeine, and the inhibi- tion of phosphorylase b by anserine is also observed in the presence of these inhibitors.
Carnosine (B-alanyl-L-histidine) and anserine (8-alanyl-L-N*-methyl-histidine)
are found in a wide variety of vertebrate muscles at concentrations approaching 40
millimolar in some cases (1). The dipeptides are generally found at higher con-
centrations in anaerobic (fast twitch) muscles whereas highly aerobic muscles
(such as cardiac muscle) have little or no detectable amounts of these compounds
(2). The ratio of carnosine to anserine varies widely between different muscles
and it has also been reported (3,4) that this ratio and the absolute levels of
the dipeptides are not constant in a given muscle but vary with development,
exercise and denervation. In addition to its presence in muscle, carnosine is
also found in other excitable tissues (5,6), and considerable interest has centered
on the high and variable amounts of the dipeptide in olfactory epithelium and
bulb (7).
Despite the relatively widespread distributions of these dipeptides, their
precise physiological role(s) are not clearly established although several have
0006-291X/82/230769-07$01.00/0
769 Copyright 0 1982 by Academic Press, Inc.
All rights of reproduction in any form reserved.
Vol. 109, No. 3, 1982 BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
been suggested for carnosine in particular including buffering power (2), myosin
regulation (8,9), divalent ion chelation (lO,ll), neurotransmission (12), and
histamine biosynthesis (13). In this report, we present evidence that the
dipeptides might be involved in the in vivo regulation of muscle phosphorylase --
as both of the dipeptides activate both a and k forms of the enzyme at physio- -
logical dipeptide concentrations.
MATERIALS AND METHODS
Carnosine, anserine nitrate, NADP+ (N-0505), glycogen (Type III), phospho- glucomutase (P-3397), glucose 6-phosphate dehydrogenase (Type XV), and rabbit muscle phosphorylase a (P-1261) were purchased from Sigma Chemical Co., Missouri. Before use, the glycogen was further purified by chromatography on BioRad AG 1x8 (200-400 mesh) resin (14), Rabbit muscle phosphorylase b was prepared by the procedure of Fischer and Krebs (15) and after four recryFtallisations, the protein was chromatographed on a Sephadex G-25 column to remove contaminating AMP (16),
Assays of the phosphorolytic activity of phosphorylase were performed at 30°C according to the general procedure of Helmreich and Cori (17) as described in (18) using a Gilford model 250 spectrophotometer equipped with a Thermoset temperature control and a model 6051 recorder. A final volume of 0.5ml per assay was used in which the initial concentrations of glycogen and phosphate were 0.5% (w/v) and 1OmM respectively, and the amount of enzyme used was approximately 0.1 ug per assay. Assays of the glycogenic activity of phos- phorylase were performed according to the procedure of Engers et al (19) as described in (14) using 2mM glucose l-phosphate. Specific activities were calculated based on protein concentrations for phosphorylase determined spec- trophotometrically from El% at 280 nm = 13.2 (14). The respective specific activities for the phosphd#lytic activity of phosphorylase a and phosphorylase bwhen assayed under these conditions in the presence of 0.5iM AMP were found to be 22.6 umol/min/mg enzyme and 10.1 umol/min/mg enzyme.
RESULTS
When phosphorylase a and b activities were measured in the presence of -
0.5mM AMP and in the presence of varying amounts of carnosine, it was found
that the dipeptide had an activating effect which was maximal at about 20-40mM
carnosine (Fig. 1A) for both enzymes. In these experiments it was found that
phosphorylase b was considerably more sensitive to activation by carnosine
than was phosphorylase a.
Because of the inhibitory effect of nitrate ion on phosphorylase (20), the
effects of anserine (as the nitrate salt) were studied using a series of controls
containing appropriate concentrations of nitrate ion. These results (Fig. 1B)
showed that anserine was a more effective activator of phosphorylase awhen compared
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20 40 60 80
[CAI~N~~INE] .mM
20 40 60 80
[ANSERINE] ,mM
Fiq. 1. Effects of Carnosine and Anserine on Phosphorylase a and b Activities. Assays of phosphorolytic and glycogenic activities of the enzymes were made in the presence of 0.5mM AMP and variable amounts of carnosine or anserine as described in Methods. The results are expressed as a percentage of the control activity recorded for the enzyme in the absence of dipeptide. Studies with anserine were performed using controls containing appropriate nitrate concen- trations. A, variable carnosine; B, variable anserine. Phosphorolytic activity of a, M ; glycogenic activity of a-,&+ ; phosphorolytic activity of b M; glycogenic activity of b, M.
to phosphorylase b, and with the latter enzyme, the dipeptide was inhibitory at
higher concentrations.
Control experiments revealed that these results were not caused by effects
of the dipeptides on the auxiliary enzymes (phosphoglucomutase and glucose
6-phosphate dehydrogenase) used in the phosphorolysis assay, and the dipeptide
effects were also observed when the glycogenic activity of the enzymes was
assayed.
The effects of carnosine and anserine were shown to be different from
those of their constituent amino acids (Table I). These results showed that
B-alanine has little effect on phosphorylase activities, whereas L-histidine
is inhibitory and L-N'-methyl-histidine is activating. When equimolar concen-
trations of B-alanine and either of the other amino acids were used, the effect
on phosphorylase activity was different from that of the same concentration of
the corresponding dipeptide, indicating that peptide bond formation is a
prerequisite for the dipeptide effect.
As phosphorylase is known to contain at least three allosteric sites (21),
studies were also performed on the effect of the dipeptides in the presence of
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TABLE I
Effect of Free Amino Acids on Phosphorylase Activity
Amino acid Relative Activity (%)b in assaya
Phosphorylase a Phosphorylase b
@alanine 95 110
L-histidine 58 9
L-NT-methyl-histidine 120 129
B-alanine + L-histidine 55 21
B-alanine + L-NV-methyl-histidine 105 76
aPresent at 25mM concentration. b
Results expressed relative to the phosphorolytic activities of control samples of phosphorylases assayed in the presence of 0.5mM AMP and the absence of free amino acids.
known effecters in order to investigate if the dipeptides are bound at any of
the effecters sites (see Table II). In studies on the effect of the presence of
AMP on the activation effect of carnosine, it was found that carnosine activated
phosphorylase a regardless of the presence of AMP, whereas no phosphorylase b
activity was detectable in the presence of carnosine and the absence of AMP.
In the presence of AMP, carnosine was found to activate both forms of phosphory-
lase in the presence of the allosteric inhibitors D-glucose, ATP and caffeine,
and the activating effect of anserine on phosphorylase a in the presence of AMP
was also evident in the presence of these inhibitors. At an inhibitory anserine
concentration (50mM) and in the presence of the allosteric inhibitors (Table II),
the inhibitory effect of anserine was still evident, but because almost complete
inhibition of the enzyme occurred in these cases, it was not possible to make
conclusions about possible interactive effects of anserine and the other inhibitors.
DISCUSSION
These studies clearly demonstrate that carnosine is a non-essential
activator (22) of both phosphorylase 5 and b. As carnosine activates phosphory-
lase 5 in the presence or absence of the activator AMP but cannot activate
phosphorylase b in its absence, it appears that carnosine does not bind at the
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TABLE II
Effects of Carnosine and Anserine on Phosphorylase Activity
in the Presence of AMP, ATP, D-glucose and Caffeine
Effecters in Assaya
Relative Activity (%)b
Phosphorylase a Phosphorylase b
None 93 0
AMP 100 100
Carnosine 120 0
Anserine 115 0
AMP + ATP 72 5
AMP + D-Glucose 76 20
AMP + Caffeine 60 2
AMP + Carnosine + ATP 117 25
AMP t Carnosine + D-Glucose a4 24
AMP t Carnosine + Caffeine a2 a
AMP + Anserine + ATP 80 2
AMP + Anserine + D-Glucose a9 5
AMP + Anserine t Caffeine 73 2
aConcentrations used in the assays were: AMP, 0.5mM; carnosine, 25mM; ATP, 9mY; D-glucose, 25mM; caffeine, 2mM; anserine, 25mM in studies with phosphorylase 5 and 50mM with phosphorylase k.
b Results are expressed relative to the phosphorolytic activities of phosphorylase a and b in the presence of 0.5mM AMP and the absence of any of the other effecters.
nucleotide-binding site of the enzyme in competition with AMP (22). That the
activation curves for both enzymes show a biphasic character also suggests
that there may be at least two binding sites for carnosine on phosphorylase.
Anserine can also activate phosphorylase a in a biphasic concentration-
dependent manner although the extent of activation is less than that obtained
with carnosine. However, in contrast to carnosine, anserine significantly
inhibits phosphorylase b at higher dipeptide concentrations (depending on the -
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direction of the assay), and the biphasic response of phosphorylase b activity
to increasing anserine concentration suggests that there may be more than one
type of binding site for anserine on the enzyme. The difference in the responses
of phosphorylase a and b to anserine also suggests that the binding event which
causes inhibition in phosphorylase b either does not occur or is not inhibitory
in phosphorylase a.
Because of the similarities in dipeptide structures and effects on phos-
phorylase, it is tempting to speculate that carnosine and anserine may bind
at the same site(s) on phosphorylase, with differences in binding and effects
on activity being related to the presence of the NT-methyl group in anserine.
In these preliminary studies, carnosine appears to be a more effective activator
of phosphorylase bthan a, and methylation of carnosine to anserine appears to
favor inhibition of phosphorylase band activation of phosphorylase a,
The Possibility of physiological control of phosphorylase by the dipeptides
is suggested by the fact that activation effects are demonstrable at physiological
concentration of the dipeptides and that the response of phosphorylase a to each
dipeptide is quite different from that of phosphorylase b0 Furthermore, the
regulatory effects of the dipeptides on phosphorylase are uniquely related to
dipeptide structure as is evident from the studies on the effects of the consti-
tuent free amino acids on phosphorylases. The specificity of the effects of
carnosine and anserine on phosphorylase markedly contrasts with earlier
observations on the putative physiological effects of carnosine on fructose
1,Gbisphosphatase (10) and myofibrillar ATPase (23,24) which were not specific
to the dipeptide and could be elicited by other imidazole compounds.
In addition to activation of phosphorylase in the presence of the allosteric
activator AMP, the dipeptides also increased the activities of both phosphorylase
forms in the presence of the allosteric inhibitors ATP, D-glucose and caffeine,
and anserine could also exert its inhibitory effect in the presence of these
inhibitors. These initial studies do not indicate if carnosine and anserine
are binding to the previously identified nucleoside and glucose allosteric sites
on phosphorylase (21), or if separate binding sites exist on the enzyme for the
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dipeptides. More detailed kinetic investigations of the effects of carnosine
and anserine on phosphorylase are in progress to answer these questions and to
investigate the possible physiological significance of these findings.
ACKNOWLEDGEMENT
This research was supported in part by N.I.H. grant AM 27155 to P.J.K.
