The sequence of the 841 amino acid residues in each subunit (molecular weight 97,412) of rabbit muscle glycogen phosphorylase b (1,4-a-D-glucan:orthophosphate a-glucosyltransferase; EC 2.4.1.1) has been determined. The general strategy was based on limited proteolysis of native phosphorylase b by subtilisin BPN', yielding two large segments (light and heavy)which were fragmented by cleavage at methionyl-, asparaginyl-glycine, and aspartyl-proline bonds. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule yielded the overlap between the light and heavy fragments and the remainder ofthe sequence. The residues involved in the covalent aind allosteric control of the enzyme, and in the binding of the cofactor pyridoxal 5'-phosphate, were identified as serine-14, tyrosine-155, and lysine-679, respectively.Muscle glycogen phosphorylase (1,4-a-D-glucan:orthophosphate a-glucosyltransferase; EC 2.4.1.1) is one of the key enzymes directly involved in the metabolism of glycogen. Upon activation through a cascade of successive enzymatic reactions, the enzyme catalyzes the conversion of glycogen and phosphate to glucose-i-phosphate. Since its isolation and crystallization (1), phosphorylase has attracted the interest of numerous investigators because it is the first enzyme in which activity was shown to be controlled by both covalent and allosteric modifications (2-4).A full understanding of the regulation and mechanism of action of the enzyme requires a detailed analysis of the structure of the subunits of the protein and of their molecular assembly. As an initial step, Saari and Fischer (5) isolated 18 fragments produced by cleavage of phosphorylase with cyanogen bromide. Titani et al. (6) showed that one of these fragments (CB14) is acetylated at its amino-terminus and that serine-14 becomes phosphorylated in the conversion of the enzyme from the b to the a form.The sequence determination described herein took advantage of a finding by Raibaud and Goldberg (7) that subtilisin cleaves the native enzyme into two reasonably homogeneous and complementary segments of molecular weight about 30,000 and 70,000. Further chemical cleavages of each of these segments at asparaginyl-glycine, aspartyl-proline, and methionyl bonds facilitated the completion of the sequence analysis. The general strategy followed for establishing the primary structure is presented herein; the experimental details will be published elsewhere. METHODS

The sequence of the amino-terminal 349 residues of rabbit muscle glycogen phosphorylase (EC 2.4.1.1) has been determined. Limited proteolysis of native phosphorylase b (841 residues, subunit molecular weight 97 412) by subtilisin BPN', Streptomyces alkaline protease, or elastase yielded two large segments (light and heavy). The light segment isolated from the subtilisin digest was cleaved at methionyl bonds with cyanogen bromide to yield eight major fragments and two minor overlapping fragments. The alignment of the major fragments was obtained by analysis of the two minor fragments, of five tryptic peptides containing methionine and of one large fragment generated by cleavage of an aspartylproline bond. Analysis of two cyanogen bromide fragments (CB14 and CB17) isolated from the intact molecule identified the sites susceptible to limited proteolysis and the overlap between the light and the heavy segments. Serine-14 and tyrosine-155 were identified as the residues involved in the covalent and allosteric controls of the enzyme, respectively. Residues 108 and 142 were identified as the cysteine residues reported to be involved in the aggregation of subunits.

To investigate the physiologic significance of antibody to human T cell leukemia virus type 1 (HTLV-1) tax gene product (p40tax), 147 male and 243 female HTLV-1 carriers were examined for anti-p40tax, and 104 carriers were checked for anti-p40tax an average of 5.4 times during an 8-year period. Prevalence of anti-p40tax was significantly higher in female (62.6%) than in male subjects (51.0%; P less than .05). Anti-p40tax status did not change in most during the observation period. There were significantly more HTLV-1 carriers among children of anti-p40tax-positive mothers (45.3%) than among those from anti-p40tax-negative mothers (20.0%; P less than .01). However, no significant difference was observed between wives of p40tax-positive and -negative men. The p40tax antibody may be a marker of relative infectivity of HTLV-1, albeit an imperfect one.

A new enzyme‐linked immunosorbent assay (ELISA) for detecting the antibody to a human T‐lymphotropic virus type I (HTLV‐I) tax gene product, p40tax, has been developed. By this ELISA method, we have investigated the relationship between the presence of p40tax antibody in HTLV‐I‐infected mothers and the virus transmission rate from mothers to their children. The rate of mother‐to‐child transmission of HTLV‐I was higher in P40tax antibody‐positive mothers than in antibody‐negative ones. Thus, the presence of P40tax antibody may indicate an increased risk of vertical transmission of HTLV‐I.

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