Human T-lymphocyte chemotactic activity: nature and production in response to antigen

Previous studies have shown that supernatants from concanavalin A-stimulated human peripheral blood mononuclear cells and isolated Leu-2 suppressor/cytotoxic T cells are chemotactic for Leu-3 helper/inducer T cells. The current study shows that lymphocyte chemotactic factor (LCF) is also produced following antigen (tetanus toxoid) challenge of mononuclear cells obtained from recently immunized human donors. LCF was detected in 24-hr supernatants from mononuclear cells challenged with tetanus and was produced maximally at 48 hr. Tetanus toxoid challenge of mononuclear cells obtained from individuals whom had not received a tetanus immunization for 7 to 10 years prior to testing showed little or no production of LCF. Serial studies of these individuals following a tetanus booster immunization showed that LCF was produced by antigen-challenged mononuclear cells obtained 1-5 days postimmunization, was produced optimally 5-15 days postimmunization, and was still produced by antigen-challenged mononuclear cells obtained 6 weeks later. Fractionation of mononuclear cells from immunized donors into glass wool nonadherent lymphocytes, T lymphocytes, and non-T lymphocytes showed that tetanus-toxoid-induced LCF was produced by nonadherent lymphocytes and T cells but not non-T cells. Further fractionation of T lymphocytes into Leu-2 and Leu-3 T-cell subpopulations showed that LCF production by antigen-challenged isolated subpopulations was limited to the Leu-2 suppressor/cytotoxic T-cell subset. Characterization of both Con A and tetanus toxoid-induced LCF by gel filtration on Sephadex G-100 demonstrated the presence of two peaks of LCF corresponding to molecular weights of approximately 14,000-17,000 and 40,000-50,000.     

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

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.     

Purification and immunohistochemical study of actin in mitochondrial matrix

Actin was purified to apparent homogeneity from the matrix of ultra-pure mitochondria of rat livers by DNase-I affinity chromatography and HPLC gel filtration. The mitochondrial actin was immunologically identified by an anti-actin antibody, and its apparent molecular weight was 43 KDa, as determined by SDS-polyacrylamide gel electrophoresis. The immunohistochemical study revealed the localization of the mitochondrial actin in the matrix space and on the internal surface of inner membrane. The actin fraction eluted from a DNase-I column by KCl-EGTA solution underwent polymerization and bundling in vitro.     

Characterization and partial purification of a non-interferon macrophage activating factor produced by human leukemic T cell line

Culture supernatants from several human leukemic T cell lines were found to contain a macrophage activating factor which enhanced hydrogen peroxide release from human peripheral blood monocyte-derived macrophages. The macrophage activating factor from a T cell line, CCRF-CEM, was characterized biochemically and compared with interferon-gamma, which is also an immunological product of T cells and has a potent macrophage activating activity. In contrast to interferon-gamma, the macrophage activating factor in the culture supernatants bound to an anion exchanger and did not adsorb onto concanavalin A gel. Culture supernatants and active fractions from chromatographies were essentially devoid of anti-viral activity. Anti-human interferon-gamma monoclonal antibody also failed to neutralize the macrophage activating factor from CCRF-CEM. MAF was eluted in the fractions with molecular weight of 40,000 to 60,000 on gel filtration in the presence of a detergent and a salt. MAF was partially purified to about 1,300-fold by the methods described above: chromatography with anion exchangers and gel filtration. It was concluded that MAF from CCRF-CEM was biochemically and immunologically different from interferon-gamma.     

Purification and chemical characterization of human hexosaminidases A and B.

N-Acetyl-beta-hexosaminidases A and B were purified to homogeneity from human placenta. In the initial step of purification, the enzymes were adsorbed on concanavalin A-Sepharose 4B and eluted from the column with alpha-methyl D-mannosides. Subsequent purification steps included DEAE-cellulose column chromatography, QAE-Sephadex [diethyl-(2-hydroxypropyl)aminoethyl-Sephadex] column chromatography, Sephadex G-200 gel filtration and preparative disc polyacrylamide-gel electrophoresis, followed by another QAE-Sephadex chromatography for the hexosaminidase A preparation, and DEAE-cellulose column chromatography, calcium phosphate gel chromatography, Sephadex G-200 gel filtration, QAE-Sephadex chromatography and CM-cellulose chromatography for the hexosaminidase B preparation. The purified preparations, particularly hexosaminidase A, had significantly higher specific enzyme activities than previously reported. The preparations moved on polyacrylamide-gel electrophoresis as single protein bands, which also stained for enzyme activity. Sedimentation-equilibrium centrifugation indicated homogenous dispersion of the enzymes, and the molecular weight was estimated as about 110000 for both enzymes. Complete amino acid and carbohydrate compositions of the two isoenzymes were determined, and, in contrast with previous suggestions, no sialic acid was found in the enzymes.     

Suppression of human lymphocyte chemotaxis and transendothelial migration by anti-LFA-1 antibody

The role of lymphocyte function-associated antigen 1 (LFA-1) in human T cell chemotaxis was investigated by using mAb specific to the beta-chain (TS1/18) (CD18) and alpha-chain (TS1/22) (CD11a) of LFA-1. T cell chemotaxis in response to IL-2 and to lymphocyte chemotactic factor (LCF) was markedly suppressed by the addition of TS1/18. TS1/22 was a less effective inhibitor than TS1/18 with only LCF stimulated responses showing significant inhibition when compared in seven different T cell preparations. Neither TS1/18 nor TS1/22 antibody inhibited random T cell migration. Control mAb to CD4 T cells failed to inhibit T cell random migration or chemotaxis. Additional studies to evaluate the adherence and migration of T cells through IL-1-stimulated human umbilical vein endothelial cell (HUVEC) monolayers showed that both TS1/22 and TS1/18 suppressed T cell migration through HUVEC, but failed to inhibit adherence of T cells to these cells. These studies indicate that LFA-1 plays a role in the migration of T cells through HUVEC and in the in vitro chemotactic response of T lymphocytes to IL-2 and LCF.     

Purification and characterization of an endothelial cell growth factor from serum-free culture medium of human diploid fibroblast cells

Serum-free culture supernatants of human embryo fibroblast cells contain endothelial cell growth factor (f-ECGF) which supports the serial propagation of human umbilical vein endothelial cells in the serum-free culture medium (medium A). This growth-stimulating activity has been partially purified from serum-free culture-conditioned medium. The stability of the activity to acid (pH 4.0-4.5) was utilized for the first step in purification. f-ECGF had a high affinity to heparin-Sepharose CL-6B, and was isolated by the methods of heparin affinity, of ion-exchange and gel filtration chromatography from the serum-free culture-conditioned medium preparation. The purified f-ECGF had an isoelectric point in the pH range 4.5-6, and a molecular weight of approx. 30 kDa determined by either gel filtration or SDS-polyacrylamide gradient gel electrophoresis. The f-ECGF has high affinity for concanavalin A column, and the activity was partially eluted from the column with ethylene glycol and alpha-methylmannose. The results indicate that f-ECGF is an acidic-glyco-protein with heterogeneous sugar chain(s).     

Insulin-stimulating peptide from a tryptic digest of bovine serum albumin: purification and characterization

A procedure was established for isolation of a low molecular weight polypeptide with insulin-stimulating activity in apparent homogeneity from a tryptic digest of bovine serum albumin on a semipreparative scale. Purification of this insulin-stimulating peptide (ISP) was monitored by an adipose-explant assay in which stimulation of fatty acid synthesis from glucose by insulin was measured. The polypeptide was purified by a combination of DEAE-cellulose column chromatography, gel filtration on Bio-Gel P-10, hydrophobic chromatography on a semipreparative C18 reversed-phase HPLC column, and ion exchange chromatography on an SP-5PW HPLC column. The primary structure of ISP was deduced. ISP is a two-chain polypeptide consisting of 71 amino acid residues, and corresponds essentially to residues 115-143 and 144-184 (185) of bovine serum albumin connected to each other by a disulfide bridge. But comparison of the sequence of ISP with that of the relevant regions of bovine serum albumin determined by Brown indicated the presence of one tyrosine insertion between residues 155 and 156 of albumin. Therefore, the molecular weight of ISP was calculated to be 8,496.     

Certain characteristics of human lymphocyte migration inhibitory factors (LyMIF) and the effect of ampicillin on their appearance in unstimulated and PHA-stimulated cultures

Supernatants of human blood mononuclear cells stimulated with PHA, contained factors inhibitory for in vitro migration of human lymphocytes and granulocytes. After ultrafiltration of supernatants through Amicon PM-10 some stimulatory activity appeared in the bottom fraction. Sephadex G-100 gel filtration chromatography of supernatants showed three zones of lymphocyte migration inhibitory activity: In the range of molecules of m.w. 35 000-90 000, heat-stable; Factors of m.w. from several to from ten to twenty thousand daltons, heat unstable; Low molecular weight substances, resistant to heat. The possible relationship of these factors to lymphotoxins, soluble lymphocyte T receptors for SRBC, lymphocyte chemotactic factor and prostaglandins is discussed. Ampicillin in doses of 10, 20 and 50 micrograms/ml potentiated both the development of lymphocyte migration inhibitory factors and the production of factors with an opposing effect (stimulating lymphocyte migration).     

Suppression of T lymphocyte chemotactic factor production by the opioid peptides beta-endorphin and met-enkephalin

The opioid peptides beta-endorphin and met-enkephalin have been shown to modulate human lymphocyte proliferation, mononuclear cell locomotion, natural killer cell activity, and neutrophil locomotion. This study demonstrates that beta-endorphin and met-enkephalin inhibit the production of a T lymphocyte chemotactic factor (LCF) by concanavalin A (Con A)-stimulated peripheral blood mononuclear cells. Inhibition of LCF production was observed by using concentrations of 10(-11) to 10(-6) M beta-endorphin or met-enkephalin but not alpha-endorphin. A bimodal pattern of suppression of LCF production was observed with both met-enkephalin and beta-endorphin when titrated from 10(-12) to 10(-6) M concentrations, with the peaks of suppressive activity occurring at concentrations of 10(-11) M and 10(-6) M. Timed studies of the production of LCF over a 54-hr period showed that there was an appreciable lag in the onset of measurable LCF activity in mononuclear supernatants produced in the presence of beta-endorphin and met-enkephalin. The suppression of LCF production mediated by opioid peptides in mononuclear supernatants was abrogated by depletion of glass-adherent mononuclear cells before culturing with opioids and Con A. The inhibitory effect of opioid peptides on LCF production was prevented by the addition of indomethacin to cell cultures. Additional experiments showed that exogenous prostaglandin E2 (PGE2) suppressed Con A-stimulated LCF production when added at concentrations ranging from 10(-6) to 10(-8) M. Other studies suggested that the mechanism of opioid peptide-mediated suppression of LCF production was due to an enhanced sensitivity of mononuclear cells to the inhibitory action of PGE2. These data provide further evidence for modulation of the immune response in humans by the neuroendocrine hormones beta-endorphin and met-enkephalin and further suggest a link between this modulation and arachidonic acid metabolism.     

Purification of mouse interleukin 2 to apparent homogeneity

A procedure has been developed for the rapid purification of mouse interleukin 2 (IL2) to apparent homogeneity, using gel filtration, anion exchange, hydrophobic chromatography, and reverse phase high pressure liquid chromatography (RP-HPLC). IL2 eluted at a high concentration of acetonitrile on HPLC (approximately 40%), well removed from other proteins. This protocol did not resolve isoelectric variant forms of IL2. Both the biological activity and protein migrated as a band of apparent molecular weight 22,000-23,000 on SDS-polyacrylamide gel electrophoresis. It had a high potency, producing 30% of the maximal response in T cell growth at a concentration of 2-4 X 10(-12) M. Mouse Il2 synthesized in a wheat germ cell-free translation system behaved similarly on RP-HPLC as the form secreted by EL4 cells. Thus, the hydrophobicity of mouse IL2, which facilitates its purification, is an intrinsic property of the protein, determined primarily by its amino acid sequence.     

Purification and characterization of small molecular weight myeloperoxidase from human promyelocytic leukemia HL-60 cells

Human myeloperoxidase was purified to homogeneity from human promyelocytic leukemia HL-60 cells. A small molecular weight myeloperoxidase was found in these cells and was separated from three other forms of myeloperoxidase of large molecular weight by carboxymethyl-Sepharose CL-6B column chromatography and Sephacryl S-200 gel filtration. The S20,w values of the molecular weights of the small and large myeloperoxidases were found to be 5.2 and 8.07 S, respectively, by sucrose density gradient centrifugation. From these S20,w values, the molecular weights of the small and large myeloperoxidases were estimated to be 79 000 and 153 000, respectively. On electrophoresis in sodium dodecyl sulfate--polyacrylamide gel, the small and large myeloperoxidases each gave two bands of protein corresponding to molecular weights of 59 300 and 10 150. The small myeloperoxidase could not be distinguished from the large enzymes by the Ouchterlony double immunodiffusion test, but it could be distinguished from them by the microcomplement fixation text. One of the three large molecular weight myeloperoxidases was eluted at a lower concentration of methyl alpha-D-mannoside than the other two on concanavalin A--Sepharose chromatography. This suggested that the heterogeneity of the myeloperoxidases with large molecular weight may be partly due to differences in their sugar moieties.     

Purification and partial sequence of rabbit polymorphonuclear leukocyte-derived lymphocyte proliferation potentiating factor resembling IL-1 beta

A rabbit polymorphonuclear leukocytes (PMN)-derived lymphocyte proliferation-potentiating factor (PMN factor) was finally purified to homogeneity. PMN factor was released from early inflammatory peritoneal exudate cells (98% of PMN) stimulated with kaolin under roller bottle culture conditions. PMN factor was purified by large sequential scale steps, using membrane-type ion exchangers and gel filtration, followed in this order by HPLC steps with cationic ion exchangers and a hydroxylapatite column. Homogeneity was manifested based on the criteria of a single m.w. 18, 500 band on silver-stained polyacrylamide gel, a superimposable activity on a UV absorbance peak in analytic HPLC gel filtration, and detection of a single amino-terminal sequence. The homogeneous PMN factor had an isoelectric value of 7.2 and an activity of 1.9 x 10(7) U/mg in the thymocyte comitogenic assay. PMN factor stimulated one-half of the maximal response of thymocyte proliferation at 2.8 x 10(-12) M. Because of similarities in the physicochemical properties, specific activity, and amino-terminal sequence between rabbit PMN factor and human IL-1 beta, this PMN factor is therefore considered to be a rabbit IL-1 beta.     

Functional characteristics of histamine receptor-bearing mononuclear cells. II. Identification and characterization of two histamine-induced human lymphokines that inhibit lymphocyte migration

Although functional histamine receptors have generally been restricted to those human T lymphocytes expressing suppressor cell functions, more recent evidence suggests that histamine receptor-bearing human T lymphocytes are functionally heterogeneous and capable of other immunomodulatory activities. Lymphocyte chemoattractant factor (LCF) is a cationic sialoprotein with an apparent m.w. of 56,000, whose production is limited to histamine-type 2 receptor-bearing human T cells. LCF is selectively chemokinetic for T lymphocytes, and presumably contributes to the recruitment of unsensitized effector lymphocytes at inflammatory sites. In addition to LCF, Sephadex G-100 gel filtration of histamine-induced lymphocyte supernatants revealed two regions of migration inhibitory activity for human blood T and rat splenic lymphocytes. These regions corresponded to m.w. of 70,000 to 80,000 (LyMIF75K) and 30,000 to 40,000 (LyMIF35K). LyMIF75K had a single pI of 7.5 to 8.0, and its biologic activity was sensitive to trypsin but not to neuraminidase or heat (56 degrees C). LyMIF35K had a single pI of 8.5 to 8.8, and its biologic activity was sensitive to neuraminidase and heat but not to trypsin. These LyMIFs therefore appeared to be distinct from one another and physicochemically different from other migration inhibitory lymphokines. All three lymphokine activities appeared within 4 hr of incubation. The minimum concentration of histamine required to stimulate production of the LyMIF was 10(-6) M. Lymphocytes that did not adhere to a histamine affinity matrix were unable to produce either LyMIF upon subsequent stimulation with histamine or concanavalin A (Con A). Lymphocytes incubated with histamine and diphenhydramine produced LCF but neither LyMIF, whereas cells incubated with histamine in the presence of cimetidine produced both LyMIF but not LCF. These data suggest that a subset of lymphocytes defined by the presence of histamine-type 1 receptors are capable of producing two distinct species of lymphocyte migration inhibitory activity. These cells may contribute to the immobilization of effector T lymphocytes chemokinetically attracted to certain inflammatory sites.     

Isolation of a low molecular weight inhibitor of lymphocyte proliferation from tumorous ascites

Intraperitoneal growth of P-815 mastocytoma cells in syngeneic DBA/2 mice produces ascites fluid which strongly inhibits mitogen-stimulated lymphocyte proliferation. The less than 10,000 m.w. fraction from gel filtration chromatography of tumorous ascites on Sephadex G-150 showed no inhibition of proliferation when eluted under physiologic conditions but was inhibitory when eluted with a high ionic strength, acidic buffer. The organic phase of a chloroform/methanol extract of the low m.w. fraction contained all the inhibitory activity. Purification of the inhibitor to relative homogeneity was achieved by reverse phase, HPLC with a gradient of acetonitrile in dilute acetate buffer. Inhibitory activity eluted between 30 and 35% acetonitrile. The active fraction contained less than 30 pg/ml PGE by RIA which was insufficient to inhibit proliferation and may actually have been stimulatory. Inhibition comparable to that produced by the ascites fraction required greater than 300 pg/ml of PGE. This low m.w. (less than 10,000), lipid-like inhibitor of lymphocyte proliferation is acid stable, not sensitive to proteolytic enzymes, soluble in both aqueous and organic solvents and occurs normally bound to a higher m.w. carrier molecule.     

Purification and characterization of a growth factor from guinea pig harderian gland

A polypeptide growth factor, Harderian gland-derived growth factor (HGDGF), has been purified approximately 43,000-fold from guinea pig Harderian gland by column chromatography on TSK gel DEAE-5PW, blue-Sepharose CL-6B, and Superose 12. The yield was approximately 10%. The Superose 12 fraction was further purified by Aquapore BU-300 reversed-phase chromatography to apparent homogeneity. HGDGF was eluted from TSK gel DEAE-5PW at 0.20-0.35 M NaCl, with a linear gradient of 0.15-0.80 M NaCl and at 2.20 M NaCl from blue-Sepharose CL-6B. The activity of HGDGF toward human embryonic cells (TIG-3) was quantitated, [3H]thymidine incorporation for 48 h being stimulated in a linear and dose-dependent manner. Purified HGDGF has a molecular weight of approximately 13,000, as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and molecular sieve column chromatography. HGDGF is labile to treatment with SH reagents or acetic acid. Both trypsin digestion and boiling decrease the activity of HGDGF. Its pI is 5.1. HGDGF stimulates the multiplication of TIG-3 cells but has no effect on human endothelial cells K2T1 or A2T2 which require fibroblast growth factor for growth. HGDGF appears to differ from other growth factors, suggesting that it is a previously undescribed growth factor.     

Similar molecular properties of granulocyte-macrophage colony-stimulating factors produced by different mouse organs in vitro and in vivo.

Granulocyte-macrophage colony-stimulating factor (GM-CSF) was partially purified from post-endotoxin serum and conditioned media produced by organs from both normal and endotoxin-injected C57BL mice. The organs used to condition medium were heart, thigh muscle, salivary gland, thymus, spleen, kidney, brain, and femur shaft. The charge properties, molecular weights, and concanavalin A binding profiles of these GM-CSFs were analyzed and compared to purified mouse lung GM-CSF. All the GM-CSFs examined were shown to be gycoproteins since a proportion of the activity (80 to 100%) bound to concanavalin A-Sepharose. The organ-conditioned medium GM-CSFs were purified (3- to 13-fold) by absorption to calcium phosphate gel and chromatography on DEAE-Sepharose (further 2- to 10-fold). Analysis of the DEAE-Sepharose elution profiles indicated that there were two major charge species of GM-CSF eluting at conductivities of 10 and 14 mmho. These partially purified GM-CSFs showed considerable differences in their apparent molecular weights on Sephacryl S-200 (37,000 to 200,000). However, these differences could be eliminated by treating the GM-CSFs with neuraminidase and performing molecular sizing experiments under dissociating conditions (Sepharose CL-6B, 6 M guanidine hydrochloride). Although some of the GM-CSFs showed anomalously high molecular weights (40,000) on gel filtration columns, even under dissociating conditions, this appeared to be due to properties of the sialic acid residues. After neuraminidase treatment all of the conditioned medium GM-CSFs eluted from DEAE-Sepharose as a single peak of biological activity at a conductivity of 10 mmho and from gel filtration columns in the presence of 6 M guanidine hydrochloride as a single molecular weight species of approximately 23,000. GM-CSF from post-endotoxin serum (produced in vivo) eluted from the gel filtration column with an apparent molecular weight of 39,000, but analysis using polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that this GM-CSF also had an apparent molecular weight of 23,000.     

Purification of the phosphofructokinase regulatory factors

In this paper, the existence and purification of two species of phosphofructokinase regulatory factor activity are reported. The purification procedure included liver homogenization and ultracentrifugation, a 93 degrees C heat step on the supernate, precipitation with ammonium sulfate, DEAE-cellulose column chromatography, and Sephadex G-75 (fine) chromatography. Two discrete regions of factor activity were eluted from the DEAE-cellulose column with a 0 to 0.5 M linear NaCl gradient. The lesser anionic fraction was not significantly retarded by DEAE-cellulose at pH 7.6, and was referred to as factor A. The more anionic form, factor B, eluted at about 0.2 M NaCl. The presence of two active fractions was confirmed by separation of factor activity (prior to DEAE-cellulose chromatography) into two discrete species by preparative isoelectric focusing on granulated gel. The isoelectric points were approximately 7.0 for factor B and 8.5 for factor A. Factor A and factor B exhibited quite different elution volumes, i.e., apparent molecular weights, when applied to a Sephadex G-75 column. Rechromatography on a Sephadex G-75 column was used for further purification and estimation of native molecular weight. The gel filtration method yielded a molecular weight of 13,800 +/- 1,800 for factor A. Factor A activity eluted as a symmetrical protein peak of constant specific activity, suggesting a homogeneous preparation. For factor B, the absorption at 280 nm and activity profile did not directly overlap. When the peak absorbance at 280 nm was considered, a molecular weight range of 39,000 +/- 4,000 was found, and on the basis of activity the molecular weight range was 36,000 +/- 4,000. After the final Sephadex G-75 chromatographic step, sodium dodecyl sulfate (SDS)-polyacrylamide slab gel electrophoresis of each SDS-treated factor preparation indicated that factor A, after visualization by silver staining, was homogeneous, with a subunit molecular weight of approximately 12,000. The factor B preparation consisted of two major polypeptides (11,000 and 18,000). The data appeared to support the conclusions that factor B was a dimer of the 18,000-Da subunit, and that the major contaminant was a tetramer of the 11,000-Da subunit.     

Neutrophil chemotactic factor produced by lipopolysaccharide (LPS)-stimulated human blood mononuclear leukocytes: partial characterization and separation from interleukin 1 (IL 1)

LPS stimulated human blood mononuclear leukocytes to produce a chemotactic factor for human neutrophils. The effect of LPS was dose-dependent; 10 micrograms/ml was optimal for production of chemotactic factor. Chemotactic activity was detected 3 hr after LPS stimulation, and reached its peak at 12 hr. No activity was detected in culture supernatants of unstimulated cells, provided LPS-free media were selected. Isoelectric point of the factor, determined by chromatofocusing, was approximately 8 to 8.5. Molecular weight was approximately 10 kilodaltons by Sephacryl S-200 gel filtration or by HPLC gel filtration on TSK-2000 and -3000 columns in succession. The gel filtration fractions were also assayed for IL 1 activity. The elution position of IL 1 activity corresponded to a m.w. of 18. There was no chemotactic activity in the IL 1 activity peak. Furthermore, highly purified natural Il 1 alpha and -beta and recombinant Il 1 alpha and -beta did not exhibit chemotactic activity for neutrophils in our assay. Among mononuclear leukocytes, the monocyte was the principal producer of neutrophil chemotactic factor. These results suggest that a chemotactic factor for neutrophils, different from IL 1, is produced by LPS-stimulated blood monocytes.     

Purification and characterization of the trifunctional beta-subunit of anthranilate synthase from Neurospora crassa

The trifunctional beta-subunit of anthranilate synthase complex of Neurospora crassa has been purified from a mutant which produces no detectable alpha-subunit. The isolated beta-subunit appeared to be a highly asymmetric dimer with a s20,w of 7.35 and an apparent molecular weight of 200,000 as determined by gel filtration on Sephacryl S-300 compared with a monomer molecular weight of approximately 84,000 Da as determined by sodium dodecyl sulfate-gel electrophoresis. The purified subunit was cleaved by elastase, trypsin, or chymotrypsin into fragments which retained the three enzyme activities. After elastase digestion, two active fragments were separated by gel filtration and ion exchange chromatography. A 30,000-Da fragment, which behaved as a monomer on gel filtration, interacted with free alpha-subunit to produce glutamine-dependent anthranilate synthase activity. A second 56,000-Da fragment, which behaved as an asymmetric dimer (apparent molecular weight 140,000) on gel filtration, retained both N-(5'-phosphoribosyl)anthranilate isomerase and indole-3-glycerol phosphate synthase activity. The failure to detect an NH2-terminal amino acid residue on either the intact beta-subunit or the 30,000-Da complementing fragment, while the 56,000-Da fragment possessed an NH2-terminal histidine residue, indicated that the complementing fragment was derived from the NH2-terminal sequence of the beta-subunit.