From the
Muscle and brain glycogen phosphorylases differ in their
responses to activation by phosphorylation and AMP. The muscle isozyme
is potently activated by either phosphorylation or AMP. In contrast,
the brain isozyme is poorly activated by phosphorylation and its
phosphorylated a form is more sensitive to AMP activation when
enzyme activity is measured in substrate concentrations and
temperatures encountered in the brain. The nonphosphorylated b form of the brain isozyme also differs from the muscle isozyme
b form in its stronger affinity and lack of cooperativity for
AMP. To identify the structural determinants involved, six enzyme
forms, including four chimeric enzymes containing exchanges in amino
acid residues 1-88, 89-499, and 500-842 (C terminus),
were constructed from rabbit muscle and human brain phosphorylase
cDNAs, expressed in Escherichia coli, and purified. Kinetic
analysis of the b forms indicated that the brain isozyme amino
acid 1-88 and 89-499 regions each contribute in an additive
fashion to the formation of an AMP site with higher intrinsic affinity
but weakened cooperativity, while the same regions of the muscle
isozyme each contribute to greater allosteric coupling but weaker AMP
affinity. Kinetic analysis of the a forms indicated that the
amino acid 89-499 region correlated with the reduced response of
the brain isozyme to activation by phosphorylation and the resultant
increased sensitivity of the a form to activation by
saturating levels of AMP. This isozyme-specific response also
correlated with the glycogen affinity of the a forms. Enzymes
containing the brain isozyme amino acid 89-499 region exhibited
markedly reduced glycogen affinities in the absence of AMP compared to
enzymes containing the corresponding muscle isozyme region.
Additionally, AMP led to greater increases in glycogen affinity of the
former set of enzymes. In contrast, phosphate affinities of all a forms were similar in the absence of AMP and increased
approximately the same extent in AMP. The potential importance of a
number of isozyme-specific substitutions in these sequence regions is
discussed.
For key enzymes of many metabolic pathways in eukaryotes,
divergent evolution has spawned isozymic forms that tailor enzyme
function to the specific environment and requirements of differentiated
cells and tissues. In the case of glycogen phosphorylase, an enzyme
documented by detailed structural analyses, isozyme forms with
differential regulatory responses may provide a rich resource of
insight into the details of the allosteric regulatory mechanisms.
Glycogen phosphorylase (EC 2.4.1.1) is a complex allosteric enzyme
that catalyzes the regulated phosphorylysis of glycogen into glucose
1-phosphate (reviewed in Refs. 1-3). In mammals, phosphorylase
exists as a family of three isozymes, muscle (M),(
Detailed kinetic analysis, carried out primarily with the rabbit M
isozyme, has shown that phosphorylase activity is intricately regulated
(1-3). The enzyme is activated by intracellular ligands, such as
AMP and glycogen, which upon binding promote active conformers. In
contrast, glucose, glucose 6-phosphate, and purine nucleosides inhibit
enzyme activity by stabilizing inactive conformers. X-ray
crystallographic analysis of the rabbit M isozyme bound to a variety of
ligands
(11, 12, 13, 14, 15, 16, 17, 18) indicates that AMP, glycogen, glucose, and purine
nucleosides bind to distinct sites on the enzyme, while the glucose
6-phosphate site overlaps that used for AMP
binding
(11, 12, 18) . Specific covalent
phosphorylation at Ser-14 also activates the enzyme and is controlled
by extracellular hormonal factors regulating phosphorylase kinase and
phosphorylase phosphatase activity
(1, 2, 3) .
Less is known about allosteric control of the B isozyme. However,
interesting differences in the activation properties of the rabbit B
and M isozymes have been observed. While the M isozyme responds
strongly to activation by both phosphorylation and AMP, the B isozyme
responds less well to activation by phosphorylation when assayed at
physiological temperatures and substrate concentrations encountered in
the brain
(19) . AMP is required for full activation of the
phosphorylated a form and increases activity to a level more
comparable to that of the M isozyme a form. The AMP binding
affinity of the nonphosphorylated b form of the B isozyme is
stronger than that of the M isozyme
(19, 20) and while
the M isozyme binds AMP with high cooperativity, AMP binding in the B
isozyme is noncooperative
(20) . Finally, the glycogen affinity
of the B isozyme is reduced compared to that of the M isozyme, but is
markedly increased in the presence of AMP
(19) .
The
three-dimensional structure of the rabbit M isozyme reveals the AMP
binding site and Ser-14-P in close proximity to each other. Both AMP
and phosphorylation are thought to activate the enzyme by a similar
overall mechanism
(11, 12, 16, 17, 21) involving complex tertiary and quaternary changes within
the ``activation subdomain'' comprising amino acid residues
(aa) 1-120
(17, 22, 23) . It is therefore
of interest to consider how differential responses to activation by
phosphorylation and AMP arise in the B and M isozymes. In order to
assess the role of structural differences between these isozymes, we
have used rabbit M and human B phosphorylase cDNAs to construct a set
of chimeric enzymes containing exchanges in the regions aa 1-88,
89-499, and 500-842 (C terminus). The enzymes encoded by
these cDNAs were expressed in high amounts in Escherichia coli and purified for kinetic analysis.
M and B phosphorylase cDNAs containing
exchanges in amino acid coding regions aa 1-88, 89-499, and
500-C terminus were constructed as shown in Fig. 1. To
simplify nomenclature, enzymes are referred to by a three-letter
designation corresponding to the M or B isozyme origin of the above
three regions.
Chimeric phosphorylase cDNAs were generated
by restriction endonuclease digestions, followed by ligation to
exchange specific cDNA regions. Prior to transformation, unwanted
ligation products were reduced by linearization with selective
restriction endonuclease digestions (Fig. 1). All cDNA constructs
were identified and verified following transformation by diagnostic
restriction endonuclease digestions.
For
kinetic analysis of phosphorylase a forms, activity was
assessed in the direction of glycogenolysis by a
The location of these regions in the three-dimensional structure of
MMM is presented in Fig. 2(A and B). The
region aa 1-88 (see Fig. 2A) contains the majority
of residues in the activation subdomain (aa 1-120), including the
N-terminal tail (aa 1-23), which rotates to create a number of
intersubunit contacts following
phosphorylation
(12, 16, 17) , residues Arg 43`
and Arg 69 which are involved in Ser-14-P binding
(12) , and
residues in the CAP loop (aa 41`-47`) and helix-2 (aa
48-78), which comprise part of the AMP binding
site
(11, 12, 16) . (The prime symbol denotes
residues in the diad-related subunit.) The second exchanged region, aa
89-499, makes up the rest of the N-terminal domain and contains
residues in helix-8 (aa 290-312) and the aa 313-325 loop
that comprise the remainder of the AMP binding
site
(11, 12, 16, 17) , as well as
residues of the tower helix aa 262-277 that form intersubunit
contacts at the catalytic face of the dimer (see
Fig. 2B). This region also contains residues within
regions aa 205-215 and 354-362 and residues within aa
387-438 which comprise the minor and major glycogen storage
sites, respectively
(13, 14) . The third exchanged
region, aa 500-C terminus, comprises the C-terminal
domain
(1, 2) which contains the majority of residues
which make up the active site and pyridoxal phosphate cofactor binding
site. The active site itself is situated in a cleft between the N- and
C-terminal domains.
The initial immobilized
metal affinity column fractionation led to higher purification of MMM
and the chimeric phosphorylase, BMM, compared to BBB and the other
chimeric enzymes. This was due, in part, to differences in elution
behavior of these enzymes. As described previously
(28) , MMM
eluted at a higher concentration of imidazole (39 mM) than
that required for elution of BBB (34 mM). BMM eluted similarly
to MMM at 43 mM imidazole, and MBB eluted similarly to BBB at
35 mM imidazole, while MBM and BBM eluted at lower imidazole
concentrations of 24 mM and 20 mM, respectively.
Elution at higher imidazole concentrations led to greater removal of
impurities in the E. coli cell sonicates and, hence, resulted
in greater purification of MMM and BMM. Another factor contributing to
their higher purity was that both MMM and BMM were present in higher
amounts (3-10-fold) in E. coli sonicates compared to BBB
and the other chimeric enzymes. The recovered amount of all
phosphorylases, however, could be improved (approximately 3-fold) by
disrupting E. coli cells with a French press.
On
DEAE-Sepharose the b form enzymes eluted at different NaCl
concentrations (45-110 mM) that correlated with
estimated isoelectric points derived from their primary sequences. MMM
(pI = 6.9) and BMM (pI = 6.6) eluted at the lowest NaCl
concentrations, and this resulted in better removal of impurities than
in the case of BBB (pI = 6.3) and the other chimeric enzymes
(pI
M and B isozymes can be distinguished by their mobilities on native
polyacrylamide gels containing 100 µM glycogen (5, 9). On
native gels prepared as described previously
(36) , the b form of BBB migrated faster than that of MMM, while all of the
chimeric enzymes exhibited intermediate mobilities (data not shown).
The mobility patterns of the chimeric enzymes correlated roughly with
their estimated isoelectric points. Interestingly, all chimeric enzymes
containing the aa 89-499 region from the B isozyme exhibited
migration rates very similar to that of BBB, while BMM containing the
corresponding region from the M isozyme exhibited a mobility similar to
that of MMM. As the aa 89-499 region contains the glycogen
storage site, the mobility patterns of these enzymes may be determined
in part by differential enzyme affinity for glycogen in the gel.
Based on the S
Chimeric phosphorylases revealed interesting
correlations between sequence regions and the cooperativity of AMP
responses. When the C-terminal domain (aa 500-842) of the B
isozyme was replaced by that of the M isozyme, the resulting enzyme,
BBM, displayed the same noncooperative AMP response as BBB. Converting
MBB into MBM would involve a similar exchange of C domains, and these
enzymes, within experimental error, also did not differ significantly
in AMP cooperativity. This suggests that the property of AMP
cooperativity is invariant with respect to the origin of the C domain.
Comparing enzymes that differ only in the origin of the aa
89-499 region indicated that replacing the muscle isozyme aa
89-499 with the brain isozyme aa 89-499 region (MMM
While exchanging the aa 1-88 region in MMM
The apparent intrinsic AMP affinity of BBB was about 10-fold
stronger than that of MMM in this analysis. Values of
K`
The difference in the
apparent free energy of binding between the brain and muscle AMP
binding sites is about 1.4 kcal/mol, as determined from the expression
shown in Equation 1.
On-line formulae not verified for accuracy Similar determinations using the apparent intrinsic dissociation
constants for the other enzymes indicate that the brain isozyme aa
1-88 and 89-499 regions contribute equally (0.6-0.8
kcal/mol) and in an additive manner to the overall free energy change.
Glycogen is both a substrate and an
activator of phosphorylase
(1) . Increasing the glycogen
concentration to 50 mM in the assay led to a much reduced AMP
effect (approximately 2-fold) on all enzyme activities. For enzymes
containing the B isozyme aa 89-499 region, this reduced
activation by AMP was due primarily to an increase in their specific
activities in the absence of AMP. In contrast to enzyme activation by
AMP, changes in glycogen concentration in the physiological range (2
and 5 mM) did not lead to dramatic changes in enzyme activity
either in the absence or presence of AMP.
In order to distinguish
between the separate effects of phosphorylation and AMP on enzyme
activation, nonphosphorylated b forms were assayed under
similar conditions (data not shown). In 5 mM glycogen, all
phosphorylase b forms exhibited specific activities in the
range of 0.5-1.5 units/mg in the presence of 100 µM
AMP and 200-fold less activity in the absence of AMP. Phosphorylated
MMM and BMM exhibited 5-fold higher specific activities than their
corresponding AMP-activated b forms, while BBB and chimeric
enzymes with the B isozyme aa 89-499 region were activated to
similar extents whether by phosphorylation or by 100 µM
AMP. These findings again demonstrate the relative insensitivity of BBB
to activation by phosphorylation and indicate its correlation with the
aa 89-499 region.
Michaelis-Menten constants
for glycogen, K
K
Homotropic cooperativity
was not observed in the M, B, and chimeric enzymes when
K
In the absence of inhibitors, the activity reached by
phosphorylases in vivo depends on the AMP level, the degree of
phosphorylation and the concentration of substrates. In this report, we
have used chimeric enzymes to identify regions in the protein that
govern the differential responses of the M and B isozymes to activation
by phosphorylation and AMP.
These studies indicate that differential
cooperativity of AMP activation and apparent intrinsic AMP affinity of
the b forms are governed in an additive fashion by both the aa
1-88 and 89-499 regions of the protein. The brain aa
1-88 and 89-499 regions each contribute to forming an
improved (i.e. high affinity) AMP site and weakened
cooperativity, while the same regions of the M isozyme each contribute
to greater allosteric coupling but a weaker AMP site.
In contrast,
the differential response of the M and B isozymes to activation by
phosphorylation and the resultant differential sensitivities of the
a forms to saturating levels of AMP correlate solely with the
aa 89-499 region of the protein. Based on analysis of substrate
affinities, this is probably due to significant differences in the
glycogen affinities of the a forms which also correlate with
the aa 89-499 region. Verifying a previous report (19), this
differential response is most pronounced when activities are determined
in physiological temperatures and substrate concentrations encountered
in the brain. Under these conditions in the absence of AMP, the
glycogen affinity of the B isozyme was markedly lower than that of the
M isozyme and K
As a
basis for identifying structural elements that govern differential
allosteric responses of phosphorylase isozymes to activation, primary
sequence comparisons of M, B, and L isozymes have been carried out in
relation to the three-dimensional structure and function of the rabbit
M isozyme
(40, 41) . These studies indicate that there is
very high conservation of residues in the active site and pyridoxal
phosphate cofactor binding site, as well as high conservation of the
secondary structural elements containing these residues. High
conservation of these regions has also been noted for seven
nonmammalian phosphorylases (42). Hence, differences between the M and
B isozymes would not be expected to be related directly to differences
in active site structure or affinity for substrates at the active site.
In the aa 1-88 region, 13 amino acid substitutions occur
between the rabbit M and human B isozymes. Eleven of these are
clustered in the first 30 residues, and eight, located throughout the
region, have been designated as B isozyme-specific as they occur in B,
but not M and L isozymes regardless of species
(41) . While all
of the residues implicated in intersubunit contacts in this region are
exactly or highly conserved, it is reasonable to expect conformational
rearrangement in the N-terminal tail as a summative consequence of many
substitutions. The N-terminal tail upon activation may rotate and fold
into a crevice between the two subunits, creating new intersubunit
contacts that transmit conformational changes to residues of the other
subunit
(12, 16, 17) . Three consecutive, B
isozyme-specific substitutions, V(M isozyme)21L(B isozyme), E22G, and
N23D, clustered at the hinge of the N-tail rotation, could alter the
tail movement, thereby affecting intersubunit interactions and the
coupling of AMP sites. Other candidates for affecting AMP affinity and
cooperativity are B isozyme-specific Y52F and K77R substitutions in
helix-2 (aa 48-78), which form part of the dimer interface at the
AMP locus.
In the aa 89-499 region, there are 54 amino acid
differences between the rabbit M and human B isozymes, 29 of which are
B isozyme-specific (41). The major structural components in this region
that participate in AMP binding and allosteric activation are the tower
helix (aa 262-277) and adjacent loops, both involved in
intersubunit interactions, and a region at the end of helix-8 (aa
309-325) involved in AMP binding. Three B isozyme-specific
substitutions, F196Y, N253K, and Q264E, may alter AMP cooperativity
since they occur at or next to residues that are involved in dimer
contacts. An additional substitution, G261D, may also be important
since Asp-261 is present in both the B and L isozymes regardless of
species and the L isozyme is known to exhibit noncooperative AMP
activation
(1) . In addition, G261D and Q264E may alter
helix-helix stability and interactions of the tower helices. M isozyme
mutations that alter tower helix interactions have been shown to
significantly alter AMP cooperativity and binding
(43) . Two
other B isozyme substitutions, C171V and G172D, may also be important
since they occur in a structural element, the Dali loop, that is
thought to be involved in allosteric activation of the rabbit M
isozyme
(2) . The aa 265-324 stretch of residues is exactly
conserved between B and M isozymes and includes the aa 313-325
loop, which is an important AMP binding determinant governing AMP/IMP
specificity
(17) . The nearest B isozyme-specific substitutions
are an adjacent clustered set, N325C, D327E, and A328T, which may
produce a slight conformational rearrangement of this loop.
With
regard to the reduced affinity of the B isozyme a form for
glycogen, 12 B isozyme-specific substitutions in the aa 89-499
region lie within the minor and major glycogen storage
sites
(41) . The majority of these do not occur at sugar binding
residues, and of those that do, the amino acid change is generally
conservative. However, one substitution at aa 405 has been noted as
being potentially significant (41) since it is highly nonconservative
(Glu to Ala) and occurs at an amino acid residue in the rabbit M
isozyme, Glu-405, that interacts with multiple sugar residues
(S6-S9) in the major glycogen storage
site
(13, 14) . A second nonconservative, B
isozyme-specific substitution (Ala to Asp) also occurs in the major
glycogen storage site at aa 435
(41) . Although Ala-435 in the
rabbit M isozyme does not interact directly with sugar residues, the B
isozyme substitution may be significant since it occurs in a flexible
loop between two sugar binding residues, Glu-433 and Lys-437, which
moves upon oligosaccharide binding to maximize sugar
contacts
(13, 14) .
In contrast to substitutions in
the glycogen storage site that might directly affect glycogen affinity,
differences in glycogen affinity might also arise from isozyme
differences in the ability of the enzyme to respond properly to
activation by phosphorylation. In this case, weaker glycogen affinity
obtained from phosphorylation at Ser-14 might have been expected to be
due to changes in the N-terminal, aa 1-88 region since the
phosphoserine and its binding site are located in this
sequence
(1) . However, the transmission of energy from binding
of the phosphopeptide N-terminal tail also involves the N-terminal
domain up to aa 482
(12) . As mentioned above, substitutions at
or near dimer contact residues or within the Dali loop may play a role
in this regard.
Interestingly, the differential response of the M
and B isozymes to activation by phosphorylation does not correlate with
the N-terminal (aa 1-88) and C-terminal (aa 500-C terminus)
regions of the protein. The aa 500-C-terminal domain of the
protein, containing the majority of active site and pyridoxal phosphate
binding site residues, is mobile during activation
(23) . Hence,
residues in this region could be involved in the enzyme's
response to allosteric activation. The studies presented here, however,
demonstrate that the aa 500-C-terminal region is not used to
tailor the differential response. The N-terminal, aa 1-88 region
forms about 40% of the intersubunit contacts and although substitutions
in this region affect AMP coupling and affinity, they are not a
determining factor for the differential response of these enzymes to
activation by phosphorylation.
The difference in AMP and glycogen
affinities of the phosphorylated M and B isozymes is relevant to their
physiological roles. The B isozyme is prevalent in brain and fetal
tissues
(5, 6, 7, 8, 9, 10) and is thought to be responsible for providing an emergency
energy source during brief periods of anoxia or
hypoglycemia
(1) . Hence, this isozyme would be expected to be
more sensitive to intracellular control through changes in AMP
concentration than extracellular control via phosphorylation. The low
glycogen affinity of the B isozyme a form in the absence of
AMP implies poor enzyme activity in vivo when AMP
concentration is low. This would reduce glycogenolysis and therefore
increase glycogen retention under levels of stress when the
extracellular phosphorylation cascade system is active but internal
energy charge is high. This would be especially important for fetal
tissues and adult tissues such as the brain, which must maintain
optimal glycogen levels for internal use. Poor glycogen affinity in the
absence of AMP would also allow the a form to be more
sensitive to AMP. The higher AMP affinity of the a and b forms of the B isozyme
(19, 20) would also
contribute to increased sensitivity to lower than saturating AMP
concentrations. By comparison, in the 10-fold higher glycogen
concentrations that occur in muscle, the a form of the M
isozyme would be expected to be highly active and relatively
insensitive to AMP. This would be in accord with the enzyme's
physiological role of providing an internal energy source for muscle
contraction
(1) , since the enzyme must respond to extracellular
neural and hormonal control via phosphorylation regardless of the
internal energy state of the cell.
Our analysis of differential
responses of the phosphorylated M and B forms underscores the
importance of glycogen in the activation process of phosphorylase. In
the B isozyme, the reduced glycogen affinity of the a form has
tuned the enzyme so that the phosphorylated form in the absence of AMP
is only weakly active at physiological glycogen concentrations, which
are significantly below the K
n
We are grateful to Dr. John Hudson for assistance in
the construction of plasmids and to Arnold Chin for technical
assistance in the purification of enzymes.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
)
brain (B), and liver (L), named in association with the tissues
where they predominate. All three isozymes exist as homodimers
containing identical subunits of approximately 100 kDa and are encoded
by distinct but structurally related genes situated on separate
chromosomes in both the mouse
(4) and human
(1) . During
development, the B isozyme predominates in fetal tissues, but is
replaced partially or completely around birth by a distribution of the
different isozymes
(5, 6) . In the adult, each isozyme
exhibits its own distinct pattern of widespread, but regulated
expression across
tissues
(5, 7, 8, 9, 10) .
Construction of Domain Exchanges between M and B
Phosphorylase Isozymes
Rabbit M phosphorylase cDNA containing the entire protein
coding region was inserted previously
(24) into the pTACTAC
expression vector (25) at the unique NdeI and HindIII
sites downstream of the TACTAC promoter. cDNA containing the entire
protein coding region of human B phosphorylase was obtained by ligating
two overlapping partial cDNAs
(7) at a common NcoI site
at aa 792. To subclone the full-length B phosphorylase cDNA into the
pTACTAC vector, NdeI and HindIII restriction sites
were engineered into the 5`- and 3`-untranslated regions of the cDNA,
respectively, by oligonucleotide-directed mutagenesis using standard
protocols
(26) .
Figure 1:
Construction of phosphorylase cDNAs for
BMM, MBB, MBM, and BBM containing exchanges in amino acid regions aa
1-88, 89-499, and 500-842 (C
terminus).
For construction purposes, an EcoRI site was
introduced at aa 88 in MMM cDNA and an NheI site was
introduced into both BBB and BMM cDNAs at aa 499 without changing the
primary amino acid sequence. This was accomplished by site-directed
mutagenesis
(24, 27) using the following synthetic
oligonucleotide primers, where the asterisk indicates a mismatch.Single-stranded cDNA templates were obtained using the Bluescript vector
KSm (Stratagene), which had been modified to contain a NdeI
site in the polylinker.
Expression of Phosphorylase cDNAs in E. coli
Phosphorylase cDNAs were expressed in E. coli as
described previously
(24) . E. coli 25A6 cells (W3110;
tonA,lonD,galE,htpP)
containing phosphorylase cDNAs in the pTACTAC vector were grown
overnight in LB/amp (Luria broth plus 50 µg/ml ampicillin). Cells
were then pelleted, resuspended in the same volume of fresh LB/amp and
inoculated into a 40-fold greater volume of expression culture medium
containing LB/amp, 250 µM
isopropyl-1-thio-
-D-galactopyranoside (Sigma), 3
mM MnCl
, and 0.5 mM pyridoxine (Sigma).
The culture was grown for 48-50 h at 22 °C to allow high
phosphorylase expression. Cells were then pelleted, quick frozen in
liquid nitrogen, and stored at -70 °C.
Purification of Phosphorylase b Forms
Preparation of Cell Extracts
E. coli 25A6 cell pellets were resuspended in 1/12.5 of the original
expression culture volume in homogenization buffer containing 25
mM -glycerophosphate, pH 7.0, 250 mM NaCl, 0.3
mM
-mercaptoethanol, 0.3 mM EDTA, 0.2
mM phenylmethylsulfonyl fluoride (Sigma), 0.7 µg/ml
pepstatin A (Boehringer Mannheim), 0.5 µg/ml leupeptin (Boehringer
Mannheim), and 0.01% (w/v) benzamidine HCl (Sigma). Cell extracts were
generated either by sonication or with a French press and clarified by
centrifugation at 16,000
g for 45 min, followed by
filtration through a 0.45-µm filter (Nalgene).
Immobilized Metal Affinity Column
Chromatography
Cell extracts were fractionated by copper
affinity column chromatography as described previously
(28) .
Briefly, cell extracts were loaded onto columns of chelating Sepharose
(Pharmacia Biotech Inc.) equilibrated first with 50 mM
CuCl and then with 1 mM imidazole (Sigma) in 25
mM
-glycerophosphate, pH 7.0, and 250 mM NaCl.
Phosphorylases were then eluted with a linear gradient of 1-100
mM imidazole and collected into tubes containing EDTA and
-mercaptoethanol to make a final concentration of 0.5 mM
each. Fractions containing phosphorylase activity were pooled, dialyzed
against 25 mM
-glycerophosphate, pH 7.0, 0.3 mM
EDTA, 0.3 mM
-mercaptoethanol, 0.2 mM
phenylmethylsulfonyl fluoride, 0.01% (w/v) benzamidine HCl, and 10%
(v/v) glycerol.
DEAE-Sepharose Column Chromatography
For further
purification, phosphorylases were dialyzed against 25 mM
-glycerophosphate, pH 6.8, 1 mM EDTA, 1 mM
-mercaptoethanol and loaded onto DEAE-Sepharose Fast Flow
(Pharmacia) columns equilibrated with the same solution. Fractionation
by DEAE-cellulose chromatography has been used previously to separate
and purify rabbit M and B isozymes
(20) . The phosphorylase b forms in this analysis were eluted from DEAE-Sepharose with a
linear gradient of 0 M NaCl/25 mM
-glycerophosphate, pH 6.8 to 250 mM NaCl/25 mM
-glycerophosphate, pH 7.2. Fractions containing phosphorylase
activity were pooled, concentrated with a Centriprep 10 concentrator
(Amicon), and stored at -20 °C in 50% glycerol.
In Vitro Phosphorylation of
Phosphorylases
Conversion of phosphorylases from the b to a forms was carried out enzymatically with rabbit
muscle phosphorylase kinase, a gift from Dr. Donal Walsh, University of
California, Davis
(29) . Phosphorylation was carried out in 0.2
mM ATP, 5 mM MgCl, 1 mM
CaCl
, 100 mM Tris-HCl, pH 8.5, at 30 °C.
Preliminary test conversions were performed with
[
-
P]ATP (1100 cpm/pmol) to determine the
optimal incubation time for phosphorylation of each phosphorylase
construct. Incorporation of
P into phosphorylase was
determined by a filter paper assay (30).
DEAE-Sepharose Purification of Phosphorylase a
Forms
Following conversion, a forms of phosphorylases
were further purified by DEAE-Sepharose column chromatography. Prior to
chromatography, phosphorylases were diluted 20-fold in DEAE-Sepharose
loading buffer consisting of 25 mM -glycerophosphate, pH
6.8, 1 mM EDTA, and 1 mM
-mercaptoethanol.
Following loading in the above buffer, phosphorylases were eluted with
a linear gradient of 50 mM NaCl/25 mM
-glycerophosphate, pH 6.8, to 250 mM Nacl/25 mM
-glycerophosphate, pH 7.2. Conditions for concentration and
storage of the a forms were identical to those described for
the b forms.
Slot-blot Assay for Phosphorylase
Activity
Phosphorylase activity in column fractions was
determined in the direction of glycogen formation by a rapid slot-blot
assay. Approximately 100 µl of column fractions were slot-blotted
onto a nitrocellulose filter (Bio-Rad) presoaked for 10 min in 50
mM -glycerophosphate, pH 7.0, and 0.1% glycogen (bovine
liver type IX, Sigma). Following sample loading, the filter was
incubated for 0.5-1 h at room temperature in 25 mM
-glycerophosphate, pH 7.0, 1% (w/v) glycogen, 75 mM
glucose 1-phosphate, and 1 mM AMP. The extent of glycogen
formation on the filter was determined with iodine stain (0.4% KI plus
0.2% I
).
Kinetic Analysis
In order to determine kinetic parameters of AMP activation of
the phosphorylase b forms, a modification of the procedures of
Carney et al.(31) was used to assay phosphorylase in
the direction of glycogen synthesis. Assays at each condition were done
in triplicate. The enzymes were preincubated for 5 min at 30 °C in
10 mM BES, pH 6.8, 1 mM EDTA, 1 mM
dithiothreitol in the presence of 1% glycogen (equivalent to 62
mM glucose residue units) and varying concentrations of AMP.
The reaction was initiated by addition of glucose 1-phosphate to a
concentration of 30 mM. At the end of the reaction, 50%
trichloroacetic acid was added to a final concentration of 5% and the
reaction mixtures were placed on ice. The colorimetric detection of
phosphate was used to measure the amount of phosphate generated by the
conversion of glucose 1-phosphate into glycogen
(31) . Reaction
aliquots were added to the color reagent and incubated exactly 5 min at
room temperature before measurement of 316 nm absorbance.
P assay
originally developed by Gevers and Stalmans
(32) and modified
for small volumes as described previously
(33) . Phosphorylase
was incubated for 10 min at 37 °C in a 125-µl final volume of
reaction mixture containing varying concentrations of potassium
phosphate, pH 7.4, 1 µCi of [
P]dipotassium
monohydrogen phosphate (1.0 Ci/mmol, DuPont NEN) and varying
concentrations of bovine liver glycogen (type IX, Sigma) and AMP. Prior
to assay, the [
P]dipotassium monohydrogen
phosphate was incubated for 2 h at 100 °C with 4.5 volumes of 1
N HCl to hydrolyze polyphosphates and then neutralized with
NaOH. The phosphorylase reaction was terminated by adding 100 µl of
the reaction mixture to 25 µl of 4.0 M perchloric acid
plus 2 µl of 1.0 M nonradioactive potassium phosphate, pH
7.4, at 4 °C. A 475-µl solution of 31.7 mM ammonium
molybdate and 52.6 mM triethylamine, pH 5.0, was then added,
and the mixture was incubated at 4 °C for a minimum of 30 min to
precipitate unincorporated phosphate. Following centrifugation at
12,000
g for 30 min, 400 µl of the supernatant was
transferred to 6 ml of Biofluor (DuPont) for liquid scintillation
analysis. With this assay, one unit of phosphorylase activity is
defined as the amount of enzyme required to convert 1 µmol of
inorganic phosphate into ammonium molybdate-triethylamine-soluble
material per min. For specific activity determinations, protein
concentration was measured by the Bradford procedure
(34) .
Values obtained were used in nonlinear least squares fitting (JMP
version 2 software, SAS Institute) to determine refined estimates of
kinetic parameters.
Phosphorylase Chimeras
Four chimeric
phosphorylases, BMM, MBB, MBM, and BBM, were constructed by exchanging
rabbit M (MMM) and human B (BBB) phosphorylase cDNA regions encoding aa
1-88, 89-499, and 500-C terminus. The three-letter
designation of these enzymes corresponds to the M or B isozyme origin
of these regions in the N- to C-terminal direction. It should be noted
that DNA constructs for chimeras BMB and MMB were also prepared, but we
were unable to express enzymes in sufficient quantity for analysis.
Figure 2:
The location of exchanged regions between
the M and B isozymes in relation to the three-dimensional structure of
the rabbit M isozyme a form dimer complexed with glucose (Ref.
12; S. R. Sprang, E. J. Goldsmith and R. J. Fletterick, unpublished
results). Exchanged regions are colored as mediumgray for aa 1-88, lightgray for aa
89-499, and darkgray for aa 500-C
terminus. A, side view of the enzyme highlighting the aa
1-88 exchanged region within the activation subdomain. The
locations of the phosphorylation sites at Ser-14 and the AMP binding
sites in both subunits and the glycogen storage site with maltoheptaose
bound in the lower subunit are shown. B, front view of the
catalytic face of the dimer showing the two active site clefts
(containing glucose, phosphate, and the cofactor pyridoxal phosphate in
side view) located between the exchanged regions, aa 89-499 and
500-C terminus. The glycogen storage sites with malto- heptaose
bound are shown in the aa 89-499 exchanged region in both
subunits. In this orientation, the activation subdomain is in the back
of the molecule.
Phosphorylase Purification
The following general
scheme was developed for the expression and purification of all
phosphorylases. Phosphorylase b forms were expressed in E.
coli from cDNAs inserted in the pTACTAC vector under the control
of the double trp-lac promoter. Following sonication of E.
coli cells, phosphorylases were purified by chromatography on
immobilized metal affinity and DEAE-Sepharose columns. An additional
DEAE-Sepharose column fractionation was used to further purify in
vitro phosphorylated a forms.
= 6.4, pI
= 6.3, and
pI
= 6.2). In most cases, the a forms
eluted at higher NaCl concentrations (100-140 mM) than
the b forms. Hence, passage of the phosphorylase a forms over DEAE-Sepharose resulted in further purification. Lack
of detectable phosphorylase b forms in the elution profiles
confirmed the completeness of in vitro phosphorylation. All
enzymes were greater than 90% pure based on SDS-polyacrylamide gel
electrophoresis
(35) , and maximum yields of all phosphorylases
ranged from 10 to 40 mg/liter of starting E. coli culture.
AMP Affinity and Cooperativity of Phosphorylase b
Forms
Enzymatic activities of the phosphorylase b forms
were measured at varying concentrations of AMP in order to compare
their activation responses to AMP binding. Refined estimates of kinetic
parameters of AMP activation are summarized in .
V, S
(AMP), and Hill
coefficient (n
) were derived for each enzyme by
nonlinear least squares fitting to the Hill equation
(37) .
value, BBB was found to bind
AMP with about 2-fold stronger apparent affinity than MMM. BBB also
exhibited noncooperative AMP activation (n
= 1.14), while MMM exhibited high cooperativity
(n
= 1.75). The S
values of the other enzymes fell within the range of the MMM and
BBB values, indicating that the general activating response to AMP was
retained in all enzymes and not compromised by the replacement of
sequence regions.
MBM, BMM
BBM) leads to lower cooperativity, suggesting that
sequence substitutions in the aa 89-499 region play a role in
determining AMP cooperativity. This is further supported by the finding
that BMM was as highly cooperative as MMM and even exhibited stronger
apparent AMP affinity. In fact, the BMM data plus the observation that
AMP response is not significantly affected by swapping C domain regions
(aa 500-842) would sufficiently indicate that the aa 89-499
region strongly governs differential AMP cooperativity in the M and B
isozymes.
BMM conversion had no effect on AMP cooperativity, the same exchange in
the cases of BBB
MBB and BBM
MBM did produce a change in
cooperativity. When the M aa 1-88 region replaced the B aa
1-88 region in either of the noncooperative enzymes BBB or BBM,
the resulting enzyme exhibited increased cooperativity. Thus, it would
appear that the M isozyme aa 1-88 region also confers properties
of AMP cooperativity, although it is not sufficient to effect the full
cooperativity of the native muscle isozyme.
Comparison of Intrinsic AMP Affinity in the Phosphorylase
b Forms
While differences in S provide a
general comparison of AMP affinity, S
values are
an unreliable indicator of the nature of the AMP binding site in the
unbound phosphorylase dimer, particularly when strong homotropic
interactions exist between the two sites. Therefore, the activity data
was also analyzed to determine the apparent intrinsic dissociation
constant for AMP, K`
, as this provides a better
structural measure of how the sequence exchanges affect the formation
of a native unbound AMP site. presents estimates of the
apparent intrinsic AMP affinity and interaction coefficient,
,
obtained by fitting a velocity equation derived previously
(38) .
for the muscle isozyme from previous studies
would suggest an even larger difference between MMM and BBB (19, 20).
Comparisons between BBB and BBM or between MBB and MBM indicate that
exchanging the C-terminal aa 500-842 region has no effect upon
K`
. On the other hand, replacing an M aa
1-88 region with a B aa 1-88 region leads to a significant
change of K`
. In all three cases represented in
our test set (MBB
BBB, MBM
BBM, MMM
BMM),
K`
decreased, indicating that resulting enzymes
formed an AMP site with stronger binding affinity for the first
molecule of the activating ligand. Thus, substitutions in the aa
1-88 region between brain and muscle phosphorylases play a
determining role in the affinity of the site. From similar comparisons
for the effect of exchanging the aa 89-499 regions, the data
suggest that substitutions in the aa 89-499 region also affect
the affinity of the AMP site. Again, the effect observed is that
changing a muscle aa 89-499 region into a brain aa 89-499
results in an AMP site with improved affinity.
The Effect of Phosphorylation on Enzyme
Activity
The effect of phosphorylation in the absence or
presence of saturating levels (100 µM) of AMP on the
activities of these enzymes was determined at physiological temperature
(37 °C) and substrate concentrations (2 and 5 mM glycogen,
10 mM phosphate) that approximate conditions in the
brain
(19) . Enzyme activity was also assessed at high glycogen
concentration (50 mM). Specific activity values are presented
in . In agreement with previous analysis of the rabbit B
isozyme
(19) , the specific activity of the a form of
BBB was low when determined in the absence of AMP and in 2 and 5
mM glycogen. Activity, however, increased dramatically
(7-9-fold) in 100 µM AMP. In contrast, the specific
activity of the a form of MMM minus AMP was much higher
(6-8-fold) than that of BBB minus AMP and the degree of enzyme
activation by AMP was lower (3-4-fold). This led to specific
activity values for MMM that were 2-3-fold higher in AMP than
those for BBB. Replacing aa 1-88 in MMM with the corresponding B
isozyme region (i.e. BMM) did not appreciably change the
specific activity of the enzyme minus AMP or the extent of enzyme
activation by AMP. However, all chimeric enzymes containing the aa
89-499 region of BBB (MBB, MBM, and BBM) behaved more like BBB.
These enzymes exhibited the same low levels of activity as BBB in the
absence of AMP, and the degree of enzyme activation by AMP was
intermediate (4-7-fold). In AMP, the activity of these
``xBx'' chimeras was similar to or slightly
lower than that of BBB in AMP. Hence, isozymic differences in
activation by phosphorylation and in the degree of enzyme activation of
the a forms by saturating levels of AMP correlate with the
isozymic origin of the aa 89-499 region. Exchanges in the
C-terminal domain (aa 500-C terminus) do not correlate with these
isozymic differences since BBB/BBM and MBB/MBM pairs all exhibited
similar responses to activation by phosphorylation and AMP. Isozymic
differences also do not correlate with the aa 1-88 region since
the response of BMM was similar to that of MMM, while MBB responded
like BBB and MBM like BBM.
Glycogen Affinity of a Forms in the Absence and Presence
of AMP
Since the differential effect of phosphorylation and 100
µM AMP on the activation of the b and a forms, respectively, of the M and B isozymes correlated with the
aa 89-499 region and occurred only in moderate, physiological
glycogen concentrations, we considered whether isozyme-specific
differences in glycogen affinity determine the responsiveness of these
enzymes to phosphorylation and AMP. K values for glycogen were therefore assessed in all phosphorylase
a forms in the absence and presence of AMP. It should be noted
that glycogen binds to three distinct sites on a phosphorylase monomer.
Two of these together form the so-called glycogen storage
site
(13, 14) , while the third locus is the active site
itself. It has been shown previously that the affinity of MMM for
glycogen is at least 20-fold higher at the glycogen storage site than
at the active site
(39) . Hence, the K
values determined here probably reflect affinity for glycogen at
the glycogen storage site. The function of the glycogen storage site is
presumed to be 2-fold, to target the enzyme to its substrate for
localization and to promote substrate binding at the active
site
(1, 13, 14) .
(glyc), determined for the
a forms in the absence of AMP and at different, fixed
phosphate concentrations are presented in I. All
K
(glyc) values decreased with increasing
phosphate concentration. Interestingly,
K
(glyc) values for BBB were much higher
(5-6-fold) than those for MMM when determined in concentrations
of phosphate, 5 and 10 mM, that are in the physiological range
(1-10 mM). BMM exhibited
K
(glyc) values similar to those of MMM,
while the enzymes, MBB, MBM, and BBM, containing the B isozyme aa
89-499 region, exhibited K
(glyc)
values intermediate to those of MMM and BBB.
(glyc) values for the a forms were also determined in the presence of 100 µM
AMP and are shown in . In physiological (2-10
mM) phosphate concentrations, the
K
(glyc) values for BBB decreased
dramatically (12-20-fold) in AMP and were now more similar to
MMM, being only 2-3-fold higher.
K
(glyc) values for MMM were less
responsive to AMP, decreasing 3-7-fold in 5-10 mM
phosphate. BMM exhibited K
(glyc) values
similar to those of MMM, while all enzymes containing the B isozyme aa
89-499 region, MBB, MBM, and BBM, exhibited
K
(glyc) values similar to those of BBB.
Hence, isozyme-specific differences in the glycogen affinities of the
a forms of MMM and BBB determined in the absence and presence
of 100 µM AMP show significant correlation with the aa
89-499 region of the protein, but not with the regions containing
aa 1-88 or 500-C terminus.
Phosphate Affinity of a Forms in the Absence and Presence
of AMP
The affinities of the a forms for the second
substrate, phosphate, K (P
),
were also determined at 37 °C in the absence and presence of 100
µM AMP. Values are presented in Tables V and VI,
respectively. In the absence of AMP and in 10 mM glycogen,
K
(P
) values for all
phosphorylases were approximately the same although enzymes with the B
isozyme aa 89-499 region exhibited a significant degree of
homotropic cooperativity making exact comparisons difficult. For the
latter enzymes, data points were fit to the Hill equation
(37) to obtain estimates of the degree of cooperativity and
K
(P
) values. Homotropic
cooperativity of MMM and BMM for phosphate was observed at lower
concentrations of glycogen (2-5 mM). In higher glycogen
concentrations (25-100 mM),
K
(P
) values for enzymes with
the B isozyme aa 89-499 region decreased approximately 2-fold and
cooperativity was reduced (data not shown).
(P
) values were determined
in AMP. In 100 µM AMP and 10 mM glycogen,
K
(P
) values decreased
5-10-fold compared to the values determined minus AMP. In these
conditions, K
(P
) values were
approximately 2-fold higher for BBB and enzymes containing the B
isozyme aa 89-499 region compared to MMM and BMM, which exhibited
similar K
(P
) values. In lower
glycogen concentrations (0.5-5 mM),
K
(P
) values for all enzymes
increased slightly, up to 2-3-fold.
(glyc) values were
approximately 5-10-fold higher than physiological glycogen
concentrations (2-5 mM). Saturating levels of AMP,
however cause glycogen affinity of the B isozyme to increase by an
order of magnitude, bringing K
(glyc) into
the physiological range and closer to the
K
(glyc) of the M isozyme in AMP. Chimeras
containing the B-isozyme aa 89-499 region exhibited glycogen
affinities more similar to those of the B isozyme, while BMM,
containing the M isozyme aa 89-499 region, exhibited glycogen
affinities similar to those of the M isozyme. In contrast to their
marked differences in glycogen affinity, M, B, and chimeric enzymes
exhibited similar affinities to phosphate in the absence of AMP. AMP
increased phosphate affinities 5-10-fold. For all enzymes,
K
values were slightly higher or within
the physiological range of phosphate concentration
(19) .
value. The
greater responsiveness of the B isozyme a form to AMP arises
because AMP improves glycogen affinity to the point where
K
and physiological glycogen
concentration are similar. AMP also increases glycogen affinity of the
phosphorylated M isozyme, but the effect has less physiological
consequence because of the higher glycogen concentrations in muscle and
the enzyme's higher affinity for glycogen in the absence of AMP.
Table:
Comparison of phosphorylase b AMP cooperativity
and affinity
, Hill coefficient;
S
, concentration of AMP required for
half-maximal activation; V
, maximal enzyme
velocity;
, interaction coefficient; K`
,
apparent intrinsic AMP affinity (dissociation constant for first AMP
ligand bound to enzyme dimer).
Table:
Comparison of phosphorylase a specific
activities in 10 mM phosphate and varying concentrations of
glycogen, plus and minus AMP
Table: 1952671790p4in
K (mM) ± standard
error.(119)
Table:
Estimates of
K (glycogen) of phosphorylase a forms assayed in
100 µM AMP and varying phosphate concentrations
Table: 1953439843p4in
n
, Hill
coefficient.(119)
Table:
Estimates
of K (P
) for phosphorylase a
forms assayed in 100 µM AMP and varying glycogen
concentrations
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
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Copyright © 1995 by the American Society for Biochemistry and Molecular Biology.
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Molecular and Cellular Proteomics
Journal of Lipid Research
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