- Email: [email protected]
The amino acid sequence of bovine thymus prothymosin α
ARCHIVES
OF BIOCHEMISTRY
Vol. 265, No. 2, September,
AND
BIOPHYSICS
pp. 454-457,
1988
The Amino Acid Sequence of Bovine Thymus C. PANNEERSELVAM, Department
of Biochemistry,
Cornell Received
D. WELLNER,
University March
Medical
College,
11, 1988, and in revised
AND
Prothymosin
B. L. HORECKER’
1300 York Avenue, form
(Y
May
New
York, New
York
10021
23, 1988
Prothymosin LY has been purified from calf thymus and its amino acid sequence determined. It contains 109 amino acid residues and closely resembles human prothymosin 01,with only two substitutions, glutamic acid for aspartic acid at position 31 and alanine for serine at position 83. This is in contrast to six differences between rat and bovine prothymosins, including four substitutions and two deletions. The structural similarity of the bovine and human polypeptides makes the former a good candidate for studies on the evaluation of the biological activities of prothymosin LY in human systems. 0 1988 Academic Press, Inc.
Prothymosin a! is a highly acidic polypeptide first isolated from rat thymus by Haritos et al. (1) as the putative precursor of thymosin (Ye, one of the biologically active peptides present in bovine thymosin fraction 5 (2, 3). Thymosin fraction 5 had been reported to restore parameters of immune function evaluated in a variety of animal models and in vitro test systems, and thymosin a1 exhibited some, but not all, of these activities (for reviews see Refs. (3-5)). Thymosin (Y~ and several related peptides present in preparations of thymosin fraction 5 (6) all contain the NHz-terminal sequence of prothymosin cy and differ only in length at the COOH-terminus. They appear to be proteolytic fragments derived from prothymosin CYby the action of endogenous proteinases during the preparation of thymosin fraction 5. This view is supported by the fact that thymosin LYE could not be isolated from thymic extracts prepared by procedures that prevented proteolytic modification (1, 6-8). Regarding the biological function of prothymosin (Y only preliminary evidence is available. Rat prothymosin LYappears to protect susceptible strains of mice against ’ To whom 0003-9861/88 Copyright All rights
correspondence
should
$3.00
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
be addressed. 454
infection with Candida albicans (9), and in this test was more effective than thymosin (Y~(6,10). Rat prothymosin LYalso has been reported to restore both autologous and allogeneic mixed lymphocyte responses in lymphocytes from patients with clinically active multiple sclerosis or systemic lupus erythematosus (11, 12). The complete amino acid sequences of rat and human prothymosin LYhave been established, based on conventional amino acid sequencing (13, 14) and on the cloned cDNA sequences (15,16). The human polypeptide was found to differ from rat prothymosin (Y at six positions, including four substitutions and two deletions, containing 109 amino acid residues as compared to 111 for rat prothymosin (Y. Human prothymosin (Y was found to be less effective than rat prothymosin cy in protecting mice against challenge with C. a&cans (14), suggesting species specificity for the biological activities of these polypeptides. It was therefore of interest to determine the structure of prothymosin CY from other species of mammals, and particularly from a species from which material could be isolated for eventual clinical trials. We report here the isolation and sequence analysis of prothymosin Q from calf thymus. The bovine polypeptide was
AMINO
ACID
SEQUENCE
OF
found to closely resemble human prothymosin 01, difFering at only two positions. EXPE:RIMENTAL
PROCEDURES
Materials. B,ovine thymus glands collected at the slaughterhouse were frozen immediately in liquid Nz and stored al; -70°C. Trypsin [L-1-tosylamino-2phenylmethyl chloroketone treated] was purchased from Worthington, clostripain from Sigma, and hydroxylamine from Mallinckrodt. Other reagents and solvents were chromatography-grade commercial preparations; solvents were redistilled as required. Buffer A was 1 M formic acid containing 0.2 M pyridine, pH 2.8. Preparation and isolation of peptide fragments. Digestion with trypsin (13) and clostripain (17) was carried out as. described. Cleavage of Asn-Gly bonds was by a modification (17) of the method of Bornstein and Balian (18). For the separation of proteolytic or hydroxylamine peptide fragments, the reaction mixtures were lyophilized and the residues, dissolved in buffer A, were fractionated by reversedphase HPLC using ODS Cl8 columns and a gradient of 0 to 40% acetonitrile in buffer A, as described (6). Amino acid analyses. These were carried out with a Waters Pica-Tag amino acid analyzer using the
10 xSDAAVDTSSEITTXDLXEXXEVVEEAENGREAPANGNANE
BOVINE
PROTHYMOSIN
method described by Heinrikson and Meredith (19) with minor modifications. Sequence analysis. Automated Edman degradation was carried out with an Applied Biosystems Model 4’70A gas-phase sequencer with an on-line PTH analyzer (Applied Biosystems Model 120A) for identification and analysis of the phenylthiohydantoin derivatives, following the procedures specified by the manufacturer. Isolation of prothymosin o( from calf thymus. Bovine prothymosin (Y was isolated by the procedure described for rat thymus (17). In brief, pulverized frozen thymus tissue was added slowly to 10 vol of boiling water and, after it cooled, the boiled tissue suspension was homogenized with a Polytron homogenizer (Brinkman) at top speed for 2 min. Insoluble material was removed by centrifugation and the extract was diluted with an equal volume of buffer A. After centrifugation, the clear supernatant solution was concentrated by ultrafiltration in a Diaflo YM2 filter. The peptides were then separated by chromatography on Sephacryl S-200 followed by HPLC as described (1). RESULTS
AND
DISCUSSION
Automated sequence analysis out with a sample of purified
20
carried bovine 40
30 eT7
T5 50 ENGEQEADNEVDEEEEEGGEEEEEEEEGDG[Z3B]GDEDEE
455
01
60
70
80 ---_----_---_---__--
T7
90
100
109
Hl
AEAATGXRAAEDDEDDDVDTXXQXTDEDD
FIG. 1. The amino acid sequence of bovine prothymosin (Y. Peptides generated by treatment with clostripain, trypsin, and hydroxylamine are designated C, T, and H, respectively. The numbers refer to the order of their elution in HPLC (see Experimental Procedures). The location of peptides T5 and T, in the sequence is based on the known sequence of thymosin LYE, the 35-amino acid peptide derived from the NHz-terminus (6). The location of other peptides is based on overlapping sequences and expected homologies with human prothymosin (Y (15). Sequences established by automated sequence analysis are shown by the heavy lines. The amino acid content of the unsequenced segment (residues 71-74) was calculated from the amino acid composition of peptide T7 and the results obtained by automated sequencing of peptide H1. The sequence of the first 20 residues is that published for calf thymosins (Ye (2) and ~~~ (6).
456
PANNEERSELVAM,
WELLNER,
10 rat
humm calf
AND
HORECKER
20
30
40 *
l
xSDAAVDTSSEITTKDLKEKKEVVEEAENGNAQN xSDAAVDTSSEITTKDLKEKKEVVEEAENGRDAPANGNA-N xSDAAVDTSSEITTKDLKEKKEVVEEAENGRKAPANGNA-N
50
60
70
SO
EENGEQEADNEVDEEEEEGGEEEEEEEEGDGEEEDGDEDE EENGEQEADNEVDEEEEEGGEEEEEEEEGDGEEEDGDEDE EENGEQEADNEVDEEEEEGGEEEEEEEEGDG[Z3B]GDEDE
90 l
*
l
110
100 *
total
l
111 109 109
EAEAPTGKRVAEDDEDDDVETKKQKKTDEDD EAESATGKRAAEDDEDDDVDTKKQK-TDEDD EAEAATGKRAAEDDEDDDVDTKKQK-TDEDD
FIG. 2. Comparison of amino acid sequences of rat, human, and bovine prothymosin cy’s. Asterisks indicate residues found in only one of the three polypeptides. Acetylation of the NHa-termini has been positively identified for bovine and rat prothymosin LY(1,2). The blocking group X has not been identified for human prothymosin N.
prothymosin LYindicated that the NH,-terminus is blocked, as previously shown for bovine thymosins (Y~(2) and all (6), the 28and 35-amino acid fragments considered to be generated by endogenous proteinases during the preparation of thymosin fraction 5 (6). Digestion of bovine prothymosin 01 with trypsin and automated sequence analysis of tryptic peptides T5 and T7 isolated by HPLC (see Experimental Procedures) provided the sequence of residues 21 through 70 (Fig. 1). Sequence analysis of the largest tryptic peptide, TV, was successful for 40 residues. The remainder of the sequence, except for a short segment containing three Glu/Gln residues and one Asp/Asn residue, was established by analysis of peptides obtained by digestion with clostripain and chemical cleavage with hydroxylamine (see Fig. 1). Based on the known susceptibility of peptide bonds to hydroxylamine (18), we would expect an Asn residue to precede GUYED. However, cloning of the cDNAs for human (15) and rat (16) prothymosin LY’S has identified Asp at this position in both of these polypeptides (see Fig. 2). The sequence of the missing segment in bovine prothymosin 01 is probably Glu71-Glu-Glu-Asp74. The primary structure of bovine thymus prothymosin a! is almost identical to that
of prothymosin LYfrom human spleen (Fig. 2). It contains the same number of amino acid residues, 109, and differs in having two substitutions, Glu for Asp at position 31 and Ala for Ser at position 83. The identification of Glu at position 31 confirmed the result obtained by sequence analysis of bovine thymosin all. Additional evidence for Alasa in bovine prothymosin (Y is provided by amino acid analysis of peptides T7 and H1, neither of which contained serine. Bovine prothymosin IX differs from rat prothymosin cz at six positions; five of these differences, including the deletions corresponding to positions 39 and 106 of the rat polypeptide, are shared with human prothymosin LY.The similarities in structure of human and bovine prothymosin (Y suggest that the latter may be a good candidate for eventual clinical trials with prothymosin 01. ACKNOWLEDGMENTS This work was supported by grants from the National Institute on Aging (2 PO1 AG00541) and the National Institute of Allergy and Infectious Diseases (2 ROl AI22901). C. Panneerselvam was the recipient of International Fellowship 3 F05 TW03629-01Sl from the Fogarty International Center, National Institutes of Health.
AMINO
ACID
SEQUENCE
OF
BOVINE
REFERENCES 1. HARITOS, A. A., GOODALL, G. J., AND HORECKER, B. L. (1984) Proc. N&l. Acad. Sci. USA 81, 1008-1011. 2. GOLDSTEIK, A. L., Low, T. L. K., MCADOO, M., MCCLURE, J., THURMAN, G. B., ROSSIO, J., LAI, C.-Y., CHANG, D., WANG, S.-S., HARVEY, C., RAMEL, A. H., AND MEIENHOFER, J. (1977) Proc. NatL Acad. Sci USA 74,725-729. 3. HOOPER, .I A., MCDANIEL, M. C., THURMAN, G. B., COHEN, G. H., SCHULOF, R. S., AND GOLDSTEIN, A. L. (1975) in Thymus Factors in Immunity (Friedman, H., Ed.) Vol. 249, pp. 125-144, New York Acad. of Sciences, New York. 4. WHITE, A. (1980) in Biochemical Actions of Hormones I:Litwack, G., Ed.), Vol. VII, pp. l-46, Academic Press, New York. 5. GOLDSTEIPI, A. L., Low, T. L. K., THURMAN, G. B., ZATZ, II. M., HALL, N., CHEN, J., Hu, S.-K., NAYLOA., P. B., AND MCCLURE, J. E. (1981) Recent Prog. Harm. Res. 37, 369-415. 6. CALDARELLA, J., GOODALL, G. J., FELIX, A. M., HEIMEA., E. P., SALVIN, S. B., AND HORECKER, B. L. (1.983) Proc. Natl. Acad. Sci. USA 80, 7424-7427. 7. HANNAPPIIL, E., DAVOUST, S., AND HORECKER, B. L. (1982) Biochem. Biophys. Res. Commun. 104,266-271. 8. Low, T. L. K., MCCLURE, J. E., NAYLOR, P. H., SPANGE:LO, B. L., AND GOLDSTEIN, A. L. (1983) J. Chromatogr. 266,533-544. 9. HARITOS, A. A., SALVIN, S. B., BLACHER, R.,
10. 11.
12.
13.
14.
15.
16.
17.
18.
19.
PROTHYMOSIN
a
457
STEIN, S., AND HORECKER, B. L. (1985) Proc. NatL Acad. Sci. USA 82,1050-1053. SALVIN, S. B., AND NETA, R. (1983) Cell Zmmunol. 75,160-172. RECLOS, G. J., BAXEVANIS, C. N., SFAGOS, C., PAPAGEORGIOU, C., TSOKOS, G. C., AND PAPAMICHAIL, M. (1987) Clin. Exp. Zmmunol. 70, 336-344. BAXEVANIS, C. N., RECLOS, G. J., PAPAMICHAIL, M., AND TSOKOS, G. C. (1987) Zmmunophamnaco1 Zmmunotoxicol. 9, 429-440. HARITOS, A. A., BLACHER, R., STEIN, S., CALDARELLA, J., AND HORECKER, B. L. (1985) Proc. NatL Acad. Sci. USA 82,343-346. PAN, L.-X., HARITOS, A. A., WIDEMAN, J., KoMIYAMA, T., CHANG, M., STEIN, S., SALVIN, S. B., AND HORECKER, B. L. (1986) Arch. Bicthem. Biophys. 250,197-201. GOODALL, G. J., DOMINGUEZ, F., AND HORECKER, B. L. (1986) Proc. Natl. Acad. Sci. USA 83, 8926-8928. FRANGOU-LAZARIDIS, M., CLINTON, M., GOODALL, G. J., AND HORECKER, B. L. (1988) Arch. Bie them. Biophys. 263,305-310. KOMIYAMA, T., PAN, L.-X., HARITOS, A. A., WIDEMAN, J. W., PAN, Y.-C. E., CHANG, M., ROGERS, I., AND HORECKER, B. L. (1986) Proc. Natl. Acad Sci USA 83,1242-1245. BORNSTEIN, P., AND BALIAN, G. (1977) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., Eds.), Vol. 47, pp. 132-145, Academic Press, San Diego. HEINRIKSON, R. L., AND MEREDITH, S. C. (1984) Anal. Biochem. 136,65-74.
OF BIOCHEMISTRY
Vol. 265, No. 2, September,
AND
BIOPHYSICS
pp. 454-457,
1988
The Amino Acid Sequence of Bovine Thymus C. PANNEERSELVAM, Department
of Biochemistry,
Cornell Received
D. WELLNER,
University March
Medical
College,
11, 1988, and in revised
AND
Prothymosin
B. L. HORECKER’
1300 York Avenue, form
(Y
May
New
York, New
York
10021
23, 1988
Prothymosin LY has been purified from calf thymus and its amino acid sequence determined. It contains 109 amino acid residues and closely resembles human prothymosin 01,with only two substitutions, glutamic acid for aspartic acid at position 31 and alanine for serine at position 83. This is in contrast to six differences between rat and bovine prothymosins, including four substitutions and two deletions. The structural similarity of the bovine and human polypeptides makes the former a good candidate for studies on the evaluation of the biological activities of prothymosin LY in human systems. 0 1988 Academic Press, Inc.
Prothymosin a! is a highly acidic polypeptide first isolated from rat thymus by Haritos et al. (1) as the putative precursor of thymosin (Ye, one of the biologically active peptides present in bovine thymosin fraction 5 (2, 3). Thymosin fraction 5 had been reported to restore parameters of immune function evaluated in a variety of animal models and in vitro test systems, and thymosin a1 exhibited some, but not all, of these activities (for reviews see Refs. (3-5)). Thymosin (Y~ and several related peptides present in preparations of thymosin fraction 5 (6) all contain the NHz-terminal sequence of prothymosin cy and differ only in length at the COOH-terminus. They appear to be proteolytic fragments derived from prothymosin CYby the action of endogenous proteinases during the preparation of thymosin fraction 5. This view is supported by the fact that thymosin LYE could not be isolated from thymic extracts prepared by procedures that prevented proteolytic modification (1, 6-8). Regarding the biological function of prothymosin (Y only preliminary evidence is available. Rat prothymosin LYappears to protect susceptible strains of mice against ’ To whom 0003-9861/88 Copyright All rights
correspondence
should
$3.00
0 1988 by Academic Press, Inc. of reproduction in any form reserved.
be addressed. 454
infection with Candida albicans (9), and in this test was more effective than thymosin (Y~(6,10). Rat prothymosin LYalso has been reported to restore both autologous and allogeneic mixed lymphocyte responses in lymphocytes from patients with clinically active multiple sclerosis or systemic lupus erythematosus (11, 12). The complete amino acid sequences of rat and human prothymosin LYhave been established, based on conventional amino acid sequencing (13, 14) and on the cloned cDNA sequences (15,16). The human polypeptide was found to differ from rat prothymosin (Y at six positions, including four substitutions and two deletions, containing 109 amino acid residues as compared to 111 for rat prothymosin (Y. Human prothymosin (Y was found to be less effective than rat prothymosin cy in protecting mice against challenge with C. a&cans (14), suggesting species specificity for the biological activities of these polypeptides. It was therefore of interest to determine the structure of prothymosin CY from other species of mammals, and particularly from a species from which material could be isolated for eventual clinical trials. We report here the isolation and sequence analysis of prothymosin Q from calf thymus. The bovine polypeptide was
AMINO
ACID
SEQUENCE
OF
found to closely resemble human prothymosin 01, difFering at only two positions. EXPE:RIMENTAL
PROCEDURES
Materials. B,ovine thymus glands collected at the slaughterhouse were frozen immediately in liquid Nz and stored al; -70°C. Trypsin [L-1-tosylamino-2phenylmethyl chloroketone treated] was purchased from Worthington, clostripain from Sigma, and hydroxylamine from Mallinckrodt. Other reagents and solvents were chromatography-grade commercial preparations; solvents were redistilled as required. Buffer A was 1 M formic acid containing 0.2 M pyridine, pH 2.8. Preparation and isolation of peptide fragments. Digestion with trypsin (13) and clostripain (17) was carried out as. described. Cleavage of Asn-Gly bonds was by a modification (17) of the method of Bornstein and Balian (18). For the separation of proteolytic or hydroxylamine peptide fragments, the reaction mixtures were lyophilized and the residues, dissolved in buffer A, were fractionated by reversedphase HPLC using ODS Cl8 columns and a gradient of 0 to 40% acetonitrile in buffer A, as described (6). Amino acid analyses. These were carried out with a Waters Pica-Tag amino acid analyzer using the
10 xSDAAVDTSSEITTXDLXEXXEVVEEAENGREAPANGNANE
BOVINE
PROTHYMOSIN
method described by Heinrikson and Meredith (19) with minor modifications. Sequence analysis. Automated Edman degradation was carried out with an Applied Biosystems Model 4’70A gas-phase sequencer with an on-line PTH analyzer (Applied Biosystems Model 120A) for identification and analysis of the phenylthiohydantoin derivatives, following the procedures specified by the manufacturer. Isolation of prothymosin o( from calf thymus. Bovine prothymosin (Y was isolated by the procedure described for rat thymus (17). In brief, pulverized frozen thymus tissue was added slowly to 10 vol of boiling water and, after it cooled, the boiled tissue suspension was homogenized with a Polytron homogenizer (Brinkman) at top speed for 2 min. Insoluble material was removed by centrifugation and the extract was diluted with an equal volume of buffer A. After centrifugation, the clear supernatant solution was concentrated by ultrafiltration in a Diaflo YM2 filter. The peptides were then separated by chromatography on Sephacryl S-200 followed by HPLC as described (1). RESULTS
AND
DISCUSSION
Automated sequence analysis out with a sample of purified
20
carried bovine 40
30 eT7
T5 50 ENGEQEADNEVDEEEEEGGEEEEEEEEGDG[Z3B]GDEDEE
455
01
60
70
80 ---_----_---_---__--
T7
90
100
109
Hl
AEAATGXRAAEDDEDDDVDTXXQXTDEDD
FIG. 1. The amino acid sequence of bovine prothymosin (Y. Peptides generated by treatment with clostripain, trypsin, and hydroxylamine are designated C, T, and H, respectively. The numbers refer to the order of their elution in HPLC (see Experimental Procedures). The location of peptides T5 and T, in the sequence is based on the known sequence of thymosin LYE, the 35-amino acid peptide derived from the NHz-terminus (6). The location of other peptides is based on overlapping sequences and expected homologies with human prothymosin (Y (15). Sequences established by automated sequence analysis are shown by the heavy lines. The amino acid content of the unsequenced segment (residues 71-74) was calculated from the amino acid composition of peptide T7 and the results obtained by automated sequencing of peptide H1. The sequence of the first 20 residues is that published for calf thymosins (Ye (2) and ~~~ (6).
456
PANNEERSELVAM,
WELLNER,
10 rat
humm calf
AND
HORECKER
20
30
40 *
l
xSDAAVDTSSEITTKDLKEKKEVVEEAENGNAQN xSDAAVDTSSEITTKDLKEKKEVVEEAENGRDAPANGNA-N xSDAAVDTSSEITTKDLKEKKEVVEEAENGRKAPANGNA-N
50
60
70
SO
EENGEQEADNEVDEEEEEGGEEEEEEEEGDGEEEDGDEDE EENGEQEADNEVDEEEEEGGEEEEEEEEGDGEEEDGDEDE EENGEQEADNEVDEEEEEGGEEEEEEEEGDG[Z3B]GDEDE
90 l
*
l
110
100 *
total
l
111 109 109
EAEAPTGKRVAEDDEDDDVETKKQKKTDEDD EAESATGKRAAEDDEDDDVDTKKQK-TDEDD EAEAATGKRAAEDDEDDDVDTKKQK-TDEDD
FIG. 2. Comparison of amino acid sequences of rat, human, and bovine prothymosin cy’s. Asterisks indicate residues found in only one of the three polypeptides. Acetylation of the NHa-termini has been positively identified for bovine and rat prothymosin LY(1,2). The blocking group X has not been identified for human prothymosin N.
prothymosin LYindicated that the NH,-terminus is blocked, as previously shown for bovine thymosins (Y~(2) and all (6), the 28and 35-amino acid fragments considered to be generated by endogenous proteinases during the preparation of thymosin fraction 5 (6). Digestion of bovine prothymosin 01 with trypsin and automated sequence analysis of tryptic peptides T5 and T7 isolated by HPLC (see Experimental Procedures) provided the sequence of residues 21 through 70 (Fig. 1). Sequence analysis of the largest tryptic peptide, TV, was successful for 40 residues. The remainder of the sequence, except for a short segment containing three Glu/Gln residues and one Asp/Asn residue, was established by analysis of peptides obtained by digestion with clostripain and chemical cleavage with hydroxylamine (see Fig. 1). Based on the known susceptibility of peptide bonds to hydroxylamine (18), we would expect an Asn residue to precede GUYED. However, cloning of the cDNAs for human (15) and rat (16) prothymosin LY’S has identified Asp at this position in both of these polypeptides (see Fig. 2). The sequence of the missing segment in bovine prothymosin 01 is probably Glu71-Glu-Glu-Asp74. The primary structure of bovine thymus prothymosin a! is almost identical to that
of prothymosin LYfrom human spleen (Fig. 2). It contains the same number of amino acid residues, 109, and differs in having two substitutions, Glu for Asp at position 31 and Ala for Ser at position 83. The identification of Glu at position 31 confirmed the result obtained by sequence analysis of bovine thymosin all. Additional evidence for Alasa in bovine prothymosin (Y is provided by amino acid analysis of peptides T7 and H1, neither of which contained serine. Bovine prothymosin IX differs from rat prothymosin cz at six positions; five of these differences, including the deletions corresponding to positions 39 and 106 of the rat polypeptide, are shared with human prothymosin LY.The similarities in structure of human and bovine prothymosin (Y suggest that the latter may be a good candidate for eventual clinical trials with prothymosin 01. ACKNOWLEDGMENTS This work was supported by grants from the National Institute on Aging (2 PO1 AG00541) and the National Institute of Allergy and Infectious Diseases (2 ROl AI22901). C. Panneerselvam was the recipient of International Fellowship 3 F05 TW03629-01Sl from the Fogarty International Center, National Institutes of Health.
AMINO
ACID
SEQUENCE
OF
BOVINE
REFERENCES 1. HARITOS, A. A., GOODALL, G. J., AND HORECKER, B. L. (1984) Proc. N&l. Acad. Sci. USA 81, 1008-1011. 2. GOLDSTEIK, A. L., Low, T. L. K., MCADOO, M., MCCLURE, J., THURMAN, G. B., ROSSIO, J., LAI, C.-Y., CHANG, D., WANG, S.-S., HARVEY, C., RAMEL, A. H., AND MEIENHOFER, J. (1977) Proc. NatL Acad. Sci USA 74,725-729. 3. HOOPER, .I A., MCDANIEL, M. C., THURMAN, G. B., COHEN, G. H., SCHULOF, R. S., AND GOLDSTEIN, A. L. (1975) in Thymus Factors in Immunity (Friedman, H., Ed.) Vol. 249, pp. 125-144, New York Acad. of Sciences, New York. 4. WHITE, A. (1980) in Biochemical Actions of Hormones I:Litwack, G., Ed.), Vol. VII, pp. l-46, Academic Press, New York. 5. GOLDSTEIPI, A. L., Low, T. L. K., THURMAN, G. B., ZATZ, II. M., HALL, N., CHEN, J., Hu, S.-K., NAYLOA., P. B., AND MCCLURE, J. E. (1981) Recent Prog. Harm. Res. 37, 369-415. 6. CALDARELLA, J., GOODALL, G. J., FELIX, A. M., HEIMEA., E. P., SALVIN, S. B., AND HORECKER, B. L. (1.983) Proc. Natl. Acad. Sci. USA 80, 7424-7427. 7. HANNAPPIIL, E., DAVOUST, S., AND HORECKER, B. L. (1982) Biochem. Biophys. Res. Commun. 104,266-271. 8. Low, T. L. K., MCCLURE, J. E., NAYLOR, P. H., SPANGE:LO, B. L., AND GOLDSTEIN, A. L. (1983) J. Chromatogr. 266,533-544. 9. HARITOS, A. A., SALVIN, S. B., BLACHER, R.,
10. 11.
12.
13.
14.
15.
16.
17.
18.
19.
PROTHYMOSIN
a
457
STEIN, S., AND HORECKER, B. L. (1985) Proc. NatL Acad. Sci. USA 82,1050-1053. SALVIN, S. B., AND NETA, R. (1983) Cell Zmmunol. 75,160-172. RECLOS, G. J., BAXEVANIS, C. N., SFAGOS, C., PAPAGEORGIOU, C., TSOKOS, G. C., AND PAPAMICHAIL, M. (1987) Clin. Exp. Zmmunol. 70, 336-344. BAXEVANIS, C. N., RECLOS, G. J., PAPAMICHAIL, M., AND TSOKOS, G. C. (1987) Zmmunophamnaco1 Zmmunotoxicol. 9, 429-440. HARITOS, A. A., BLACHER, R., STEIN, S., CALDARELLA, J., AND HORECKER, B. L. (1985) Proc. NatL Acad. Sci. USA 82,343-346. PAN, L.-X., HARITOS, A. A., WIDEMAN, J., KoMIYAMA, T., CHANG, M., STEIN, S., SALVIN, S. B., AND HORECKER, B. L. (1986) Arch. Bicthem. Biophys. 250,197-201. GOODALL, G. J., DOMINGUEZ, F., AND HORECKER, B. L. (1986) Proc. Natl. Acad. Sci. USA 83, 8926-8928. FRANGOU-LAZARIDIS, M., CLINTON, M., GOODALL, G. J., AND HORECKER, B. L. (1988) Arch. Bie them. Biophys. 263,305-310. KOMIYAMA, T., PAN, L.-X., HARITOS, A. A., WIDEMAN, J. W., PAN, Y.-C. E., CHANG, M., ROGERS, I., AND HORECKER, B. L. (1986) Proc. Natl. Acad Sci USA 83,1242-1245. BORNSTEIN, P., AND BALIAN, G. (1977) in Methods in Enzymology (Hirs, C. H. W., and Timasheff, S. N., Eds.), Vol. 47, pp. 132-145, Academic Press, San Diego. HEINRIKSON, R. L., AND MEREDITH, S. C. (1984) Anal. Biochem. 136,65-74.