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. N-Terminal sequence analysis of the fragments liberated by limited proteolysis with trypsin of the novel peptide reveals a striking similarity to the fragments produced by plasmin cleavage of the rabbit plasma protein called histidine-proline-rich glycoprotein (HPRG). However, some divergence is observed between the sequence of one of the fragments liberated from AMP deaminase by a more extensive trypsinization and rabbit plasma HPRG in the region containing residues 472-477. A fragment with a blocked N-terminus, which was found among those liberated by proteolysis with pepsin of either whole AMP deaminase or the novel component of the enzyme, shows an amino acid composition quite different from that of the N-terminus of the known subunit of AMP deaminase. By coupling this observation with the detection in freshly prepared AMP deaminase of a low yield of the sequence (LTPTDX) corresponding to that of HPRG N-terminus, it can be deduced that in comparison with HPRG, the putative HPRG-like component of AMP deaminase contains an additional fragment with a blocked N-terminus, which is liberated by a proteolytic process during purification of the enzyme. The implications of the association to rabbit skeletal-muscle AMP deaminase of a HPRG-like protein species are discussed.

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SUMMARY 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). The results of the immunological analysis reported here demonstrate that an antibody against human plasma HPRG reacts with an AMPD preparation from human skeletal muscle. To probe the localization of the putative HPRG-like protein in human skeletal muscle, serial sections from frozen biopsy specimens were processed for immunohistochemical and histoenzymatic stains. A selective binding of the anti-HPRG antibody to Type IIB muscle fibers was detected, suggesting a preferential association of the novel protein to the AMPD isoenzyme contained in the fast-twitch glycolytic fibers. Histidine-proline-rich glycoprotein (HPRG) is a protein present at a relatively high concentration in the plasma of vertebrates. Its specific function remains unclear, although it has been implicated in several phenomena, including blood coagulation and fibrinolysis (Peterson et al. 1987). In a recent article we reported the isolation from purified rabbit skeletal muscle AMP deaminase (AMPD) of an approximately 75 kD novel peptide with an amino acid composition significantly different from that derived from the available AMPD cDNAs (Ranieri-Raggi et al. 1997). N-terminal sequence analysis of the fragments liberated by limited proteolysis revealed a striking similarity of this protein to rabbit plasma HPRG (Borza et al. 1996) although, in comparison with mature HPRG, the AMP deaminase-associated variant probably contains a unique N-terminal extension. Among the 60 amino acids sequenced up to now in the novel HPRG isoform, four substitutions were found with respect to the published rabbit HPRG sequence, all of them localized in the 472-477 region that also differs in five amino acid residues compared with the homologous region of the human protein (residues 461-466) (Koide et al. 1986). This divergence enabled us to raise a rabbit antibody against a synthetic peptide equivalent to residues 462-471 of human plasma HPRG. The similarity between the 461-466 region of human plasma HPRG (S-F-P-L-P-H) and the corresponding sequence of the HPRG-like molecule isolated from rabbit skeletal muscle (S-F-S-L-R-H) prompted us to utilize the antibody to probe the immunohistochemical localization of the putative HPRG-like protein in human skeletal muscle. The experimental results show that the anti-HPRG antibody reacts with an AMPD preparation from human skeletal muscle. Moreover, a clear positive reaction was detected at the level of Type IIB fibers, giving evidence of the presence of an HPRGlike peptide in human skeletal muscle. These observations suggest a correlation of this protein as well as Correspondence to: Prof.

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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. In the specimens from patients with Acquired AMPD deficiency the HPRG-immunoreactivity was less intense than that shown by the control subjects and was related to the residual AMPD activity. The patients affected by Primary and Coincidental AMPD deficiency, which were characterized by an absence of enzyme activity and AMPD immunoreactivity, showed the lowest HPRG immunoreactivity that was clearly detectable by Western blot analysis, but not by immunohistochemistry. The interpretation of the significance of these observations suggests a physiological mutual dependence between skeletal muscle HPRG and AMPD polypeptides with regard to their stability