The role of histidine-proline-rich glycoprotein as zinc chaperone for skeletal muscle AMP deaminase.

Metallochaperones function as intracellular shuttles for metal ions. At present, no evidence for the existence of any eukaryotic zinc-chaperone has been provided although metallochaperones could be critical for the physiological functions of Zn2+ metalloenzymes. We propose that the complex formed in skeletal muscle by the Zn2+ metalloenzyme AMP deaminase (AMPD) and the metal binding protein histidine-proline-rich glycoprotein (HPRG) acts in this manner.

Evidence that muscle cells do not express the histidine-rich glycoprotein associated with AMP deaminase but can internalise the plasma protein.

Histidine-rich glycoprotein (HRG) is synthesized by liver and is present at relatively high concentration in the plasma of vertebrates. We have previously described the association of a HRG-like molecule to purified rabbit skeletal muscle AMP deaminase (AMPD). We also provided the first evidence for the presence of a HRG-like protein in human skeletal muscle where a positive correlation between HRG content and total determined AMPD activity has been shown.

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. A new model for substrate interactions at a dinuclear cocatalytic Zn site.

We have previously provided evidence for a dinuclear zinc site in rabbit skeletal muscle AMPD compatible with a (micro-aqua)(micro-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site. XAS of the zinc binding site of the enzyme in the presence of PRN favors a model where PRN is added to the coordination sphere of one of the two zinc ions increasing its coordination number to five. The uncompetitive nature of the inhibition of AMPD by fluoride reveals that the anion probably displaces the nucleophile water molecule terminally coordinated to the catalytic Zn(1) ion at the enzyme C-terminus, following the binding of AMP at the Zn(2) ion located at N-terminus of the enzyme.

Characterization of the metallocenter of rabbit skeletal muscle AMP deaminase. Evidence for a dinuclear zinc site.

XAS of Zn-peptide binary and ternary complexes prepared using peptides mimicking the potential metal binding sites of rabbit skeletal muscle AMP deaminase (AMPD) strongly suggest that the region 48-61 of the enzyme contains a zinc binding site, whilst the region 360-372 of the enzyme is not able to form 1:1 complexes with zinc, in contrast with what has been suggested for the corresponding region of yeast AMPD. XAS performed on fresh preparations of rabbit skeletal muscle AMPD provides evidence for a dinuclear zinc site in the enzyme compatible with a (mu-aqua)(mu-carboxylato)dizinc(II) core with an average of two histidine residues at each metal site and a Zn-Zn distance of about 3.3 Angstrom.

Immunohistochemical analysis of human skeletal muscle AMP deaminase deficiency. Evidence of a correlation between the muscle HPRG content and the level of the residual AMP deaminase activity.

We have previously described that, in healthy human skeletal muscle, an anti-histidine-proline-rich-glycoprotein (HPRG) antibody selectively binds to type IIB fibers that are well known to contain the highest level of AMP deaminase (AMPD) activity, suggesting an association of the HPRG-like protein to the enzyme isoform M. The present paper reports an immunohistochemical study performed on human skeletal muscle biopsies from patients with AMPD deficiency and carried out utilizing both the anti-HPRG antibody and an anti-AMPD antibody specific for the isoform M. A correlation between the muscle content of the HPRG-like protein and the level of AMPD activity was demonstrated.

A calpain-like proteolytic activity produces the limited cleavage at the N-terminal regulatory domain of rabbit skeletal muscle AMP deaminase: evidence of a protective molecular mechanism.

On storage at 4 degrees C, rabbit skeletal muscle AMP deaminase undergoes limited proteolysis with the conversion of the native 85-kDa enzyme subunit to a 75-kDa core that is resistant to further proteolysis. Further studies have shown that limited proteolysis of AMP deaminase with trypsin, removing the 95-residue N-terminal fragment, converts the native enzyme to a species that exhibits hyperbolic kinetics even at low K+ concentration. The results of this report show that a 21-residue synthetic peptide, when incubated with the purified enzyme, is cleaved with a specificity identical to that reported for ubiquitous calpains.

Isolation by zinc-affinity chromatography of the histidine-proline-rich-glycoprotein molecule associated with rabbit skeletal muscle AMP deaminase. Evidence that the formation of a protein-protein complex between the catalytic subunit and the novel component is critical for the stability of the enzyme.

The histidine-proline-rich glycoprotein (HPRG) component of rabbit skeletal muscle AMP deaminase under denaturing and reducing conditions specifically binds to a Zn(2+)-charged affinity column and is only eluted with an EDTA-containing buffer that strips Zn(2+) from the gel. The isolated protein is homogeneous showing an apparent molecular weight (MW) of 95000 and the N-terminal sequence L-T-P-T-D-X-K-T-T-K-P-L-A-E-K-A-L-D-L-I, corresponding to that of rabbit plasma HPRG. The incubation with peptide-N-glycosidase F promotes the reduction of the apparent MW of isolated HPRG to 70000, characterizing it as a N-glycosylated protein.

XAS of dilute biological samples.

The experimental setup of beamline ID26 at ESRF (Grenoble) has been successfully exploited to obtain high-quality XAS (X-ray absorption spectroscopy) data from a biological sample where the metal concentration is about 100 micro M. The sample consists of the adenosine monophosphate deaminase (AMPD) histidine proline rich glycoprotein (HPRG) complex that contains 3-4 Zn(II) ions per dimer of approximately 320 kDa molecular weight. The experiment shows that third-generation X-ray sources equipped with insertion devices and appropriate optics and detectors allow the investigation of complex biological systems where the metal concentration is intrinsically low.

Characterization of the zinc-binding site of the histidine-proline-rich glycoprotein associated with rabbit skeletal muscle AMP deaminase.

The AMP deaminase-associated variant of histidine-proline-rich glycoprotein (HPRG) is isolated from rabbit skeletal muscle by a modification of the protocol previously used for the purification of AMP deaminase. This procedure yields highly pure HPRG suitable for investigation by x-ray absorption spectroscopy of the zinc-binding behavior of the protein. X-ray absorption spectroscopy analysis of a 2:1 zinc-HPRG complex shows that zinc is bound to the protein, most probably in a dinuclear cluster where each Zn(2+) ion is coordinated, on average, by three histidine ligands and one heavier ligand, likely a sulfur from a cysteine.

Regulation of skeletal muscle AMP deaminase: lysine residues are critical for the pH-dependent positive homotropic cooperativity behaviour of the rabbit enzyme.

Reaction of rabbit skeletal muscle AMP deaminase with a low molar excess of trinitrobenzene sulfonic acid (TNBS) results in conversion of the enzyme into a species with about six trinitrophenylated lysine residues per molecule which no longer manifests positive homotropic cooperativity at pH 7.1 or at the optimal pH value of 6.5 in the presence of low K+ concentrations.

Presence in human skeletal muscle of an AMP deaminase-associated protein that reacts with an antibody to human plasma histidine-proline-rich glycoprotein.

Histidine-proline-rich glycoprotein (HPRG) is a protein that is synthesized by parenchimal liver cells. The protein has been implicated in a number of plasma-specific processes, including blood coagulation and fibrinolysis. We have recently reported the association of an HPRG-like protein with rabbit skeletal muscle AMP deaminase (AMPD).

Association of purified skeletal-muscle AMP deaminase with a histidine-proline-rich-glycoprotein-like molecule.

Denaturation of rabbit skeletal-muscle AMP deaminase in acidic medium followed by chromatography on DEAE-cellulose in 8 M urea atpH 8.0 allows separation of two main peptide components of similar apparent molecular mass (75-80 kDa) that we tentatively assume correspond to two different enzyme subunits. Whereas the amino acid composition of one of the two peptides is in good agreement with that derived from the nucleotide sequence of the known rat and human AMPD1 cDNAs, the second component shows much higher contents of proline, glycine and histidine.

Regulation of skeletal-muscle AMP deaminase: involvement of histidine residues in the pH-dependent inhibition of the rabbit enzyme by ATP.

Reaction of rabbit skeletal-muscle AMP deaminase with a low molar excess of diethyl pyrocarbonate results in conversion of the enzyme into a species with one or two carbethoxylated histidine residues per subunit that retains sensitivity to ATP at pH 7.1 but, unlike the native enzyme, it is not sensitive to regulation by ATP at pH 6.5.

Evidence of a species-differentiated regulatory domain within the N-terminal region of skeletal muscle AMP deaminase.

Rabbit skeletal muscle AMP deaminase was submitted to limited proteolysis by trypsin that converts the native 80 kDa enzyme subunit to a stable product of approx. 70 kDa, which, in contrast to the native enzyme, is not sensitive to regulation by ATP at pH 6.5.

Regulation of skeletal-muscle AMP deaminase. Evidence for a highly pH-dependent inhibition by ATP of the homogeneous derivative of the rabbit enzyme yielded by limited proteolysis.

Limited proteolysis of rabbit skeletal-muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.

Regulatory properties of AMP deaminase isoenzymes from rabbit red muscle.

We examined the kinetic and regulatory properties of the two isoenzymes of red muscle AMP deaminase, forms A and B, corresponding respectively to the single isoenzymes present in the heart and white skeletal muscle. At the optimal pH value, 6.5, both enzymes show hyperbolic substrate-velocity curves and are inhibited by GTP, inducing sigmoid kinetics.

Interaction with troponin T from white skeletal muscle restores in white skeletal muscle AMP deaminase those allosteric properties removed by limited proteolysis.

Limited proteolysis of rabbit skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.

Effect of pH and KCl on aggregation state and sulphydryl groups reactivity of rat skeletal muscle AMP deaminase.

The effects of pH and KCl on sedimentation properties and SH groups reactivity of rat skeletal muscle AMP deaminase have been investigated. The values obtained for apparent molecular weight are consistent with an association of AMP deaminase subunits in response to increasing KCl concentration. Increasing pH value from 6.

pH-dependent cold lability of rabbit skeletal muscle AMP deaminase.

Rabbit skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.4.

Inactivation of rat muscle 5'-adenylate aminohydrolase by tyrosine nitration with tetranitromethane.

Reaction of rat muscle AMP deaminase with low molar excess of tetranitromethane results in a rapid loss of free thiol groups and a concomitant decrease in enzyme activity at high, but not at low, AMP concentration. This modification appears to be limited to the same non-essential thiol groups reactive towards specific reagents in non-denaturing conditions. On incubation with higher molar excess of tetranitromethane, a loss of enzyme activity is observed, which correlates with nitration of tyrosine residues.

Effects of storage on activity and subunit structure of rabbit skeletal-muscle AMP deaminase.

On storage, AMP deaminase is converted into a form exhibiting hyperbolic kinetics even at low KCl concentration. This effect results from cleavage of the enzyme subunit (mol.wt.

Effect of pH on the kinetic properties of rat skeletal muscle AMP deaminase.

1. The optimal pH for activity of rat skeletal muscle AMP deaminase depends on substrate and salt concentrations. 2.

Adenylate metabolism in the heart. Regulatory properties of rabbit cardiac adenylate deaminase.

The kinetic properties of a 300-fold purified cardiac AMP deaminase were studied and compared with those of the corresponding enzyme from skeletal muscle. The heart enzyme is activated by ATP and less efficiently by ADP, and is inhibited by Pi, phosphocreatine and GTP. ATP, even at micromolar concentrations, is able to abolish the effects of the inhibitors.

Negative homotropic cooperativity in rat muscle AMP deaminase. A kinetic study on the inhibition of the enzyme by ATP.

1. Rat skeletal muscle AMP deaminase (AMP aminohydrolase, EC 3.5.