Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes

Teizo Yoshimura, E. A. Robinson, S. Tanaka, E. Appella, E. J. Leonard

Research output: Contribution to journalArticle

219 Citations (Scopus)

Abstract

Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics-molecular mass, basic isoelectric point, and dye column binding - were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.

Original languageEnglish
Pages (from-to)1956-1962
Number of pages7
JournalJournal of Immunology
Volume142
Issue number6
Publication statusPublished - 1989
Externally publishedYes

Fingerprint

Mononuclear Leukocytes
Chemotactic Factors
Phytohemagglutinins
Monocytes
Glioma
High Pressure Liquid Chromatography
Amino Acids
Peptides
Sepharose
Polyacrylamide Gel Electrophoresis
Agarose Chromatography
Isoelectric Point
Gel Chromatography
Coloring Agents

ASJC Scopus subject areas

  • Immunology

Cite this

Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes. / Yoshimura, Teizo; Robinson, E. A.; Tanaka, S.; Appella, E.; Leonard, E. J.

In: Journal of Immunology, Vol. 142, No. 6, 1989, p. 1956-1962.

Research output: Contribution to journalArticle

@article{d02fe97a40eb4e86832ed55f56cb9a8d,
title = "Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes",
abstract = "Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40{\%} of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics-molecular mass, basic isoelectric point, and dye column binding - were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50{\%} of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.",
author = "Teizo Yoshimura and Robinson, {E. A.} and S. Tanaka and E. Appella and Leonard, {E. J.}",
year = "1989",
language = "English",
volume = "142",
pages = "1956--1962",
journal = "Journal of Immunology",
issn = "0022-1767",
publisher = "American Association of Immunologists",
number = "6",

}

TY - JOUR

T1 - Purification and amino acid analysis of two human monocyte chemoattractants produced by phytohemagglutinin-stimulated human blood mononuclear leukocytes

AU - Yoshimura, Teizo

AU - Robinson, E. A.

AU - Tanaka, S.

AU - Appella, E.

AU - Leonard, E. J.

PY - 1989

Y1 - 1989

N2 - Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics-molecular mass, basic isoelectric point, and dye column binding - were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.

AB - Physicochemical characteristics of monocyte chemotactic activity in the culture fluid of PHA-stimulated human mononuclear leukocytes (MNL) were investigated. Among several chemotactic activity peaks eluted from a TSK-2000 gel filtration column, one peak, corresponding to a molecular mass of 17 kDa, accounted for about 40% of total chemotactic activity. On a chromatofocusing column, most of the 17-kDa activity eluted in a pH range of 9.4 to 7.9. It could bind to Orange-A Sepharose. These three characteristics-molecular mass, basic isoelectric point, and dye column binding - were similar to those of human glioma-derived monocyte chemotactic factor (GDCF), recently purified in our laboratory. Therefore, the MNL-derived chemoattractant was purified by the same procedures used for purification of GDCF, namely Orange-A Sepharose chromatography, carboxymethyl (CM)-HPLC, and reverse phase (RP) HPLC. About 50% of the culture fluid chemotactic activity bound to Orange-A Sepharose and was eluted in a single peak by a NaCl gradient. The active pool from the Orange-A column was separated into two sharp peaks by CM-HPLC, each of which eluted at identical acetonitrile concentrations from a RP HPLC column. By SDS-PAGE, the peptides had apparent molecular masses of 15 and 13 kDa and appeared homogeneous. Amino acid analysis showed that the composition of the two peptides was almost identical; and the N terminus of each peptide was apparently blocked. Shared characteristics of these peptides and the GDCF peptides include identical elution patterns from CM- and RP HPLC columns, identical SDS-PAGE migration, almost identical amino acid composition, and blocked N terminus. This suggests that the monocyte attractants isolated from culture fluid of PHA-stimulated MNL are identical to those derived from human glioma cells.

UR - http://www.scopus.com/inward/record.url?scp=0024590507&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0024590507&partnerID=8YFLogxK

M3 - Article

VL - 142

SP - 1956

EP - 1962

JO - Journal of Immunology

JF - Journal of Immunology

SN - 0022-1767

IS - 6

ER -