The biochemistry and in vitro activity of soluble factors of activated lymphocytes

Summary Activated lymphocytes release numerous products which are either synthesized de novo or in increased amounts; some of these products play a role in the regulation of the immune response and are designated as mediators of cellular immune reactions or lymphokines. The first lymphokine described was the macrophage migration inhibitory factor (MIF) which has been studied most extensively with regard to its chemical and biological properties. Using sensitive radiolabelling techniques and an antiserum against highly purified fractions of MIF we were able to identify several products of activated guinea pig lymphocytes with different molecular weights of 15.000, 30.000, 45.000, 60.000 which all had an isoelectric point of 5.2 and were all inhibitory to macrophage migration. It is suggested, that these molecules are oligomers of a common subunit of molecular weight 15.000. It was further shown, that molecules of the same physical-chemical and serological characteristics are produced by activated B-cells, L₂C leukemia cells and growing fibroblasts, thus further substantiating earlier reports on the production of MIF by lymphoid and non-lymphoid cells. The described molecules were also shown not to contain determinants of the major histocompatibility complex and to be distinct from lymphotoxin, another lymphocyte activation product. It is concluded, that MIF is not sa single molecule but rather a system of structurally related molecules. Thier interaction with macrophages and possible relationship to macrophage activating factor is discussed.     

Fractionation of murine macrophage inhibitory factor into two distinct species

Murine migration inhibitory factor (MIF) produced by concanavalin A-stimulated lymph node cells from C57BL/6 mice was fractionated by Sephadex G-100 gel filtration, density gradient electrophoresis, and isoelectrofocusing in a sucrose density gradient and assayed on in vitro-cultivated bone marrow macrophages from C57BL/6 mice. Two major MIF species, pH3-MIF with an isoelectric point of 3.0–4.3 and pH5-MIF with an isoelectric point of 4.6 to 5.2, were obtained. The similarity of murine MIF to guinea pig and human MIF is discussed.     

Two migration inhibitory factors with different chromatographic behavior and isoelectric points.

Migration inhibitory factor (MIF), produced by stimulation of guinea pig lymph node cells with concanavalin A, was fractionated by Sephadex G-100 gel filtration, sucrose density gradient electrophoresis, and isoelectrofocussing. Two distinct species were identified and separated. One, pH 3-MIF, has an isoelectric point of 3.0 to 4.5 and elutes from Sephadex G-75 columns with molecules having an apparent m.w. of 65,000 (Kd of 0.05 to 0.12). The other, pH 5-MIF, has an isoelectric point of 5.0 to 5.5 and elutes with molecules having an apparent m.w. between 25,000 and 43,000 (Kd of 0.15 to 0.23).     

Antibodies to guinea pig lymphokines. II. Suppression of delayed hypersensitivity reactions by a "second generation" goat antibody against guinea pig lymphokines.

A "second generation" antibody to a highly purified lymphocyte product was raised in a goat against material eluted from a rabbit anti-guinea pig lymphokine immunoadsorbent column. This anti-lymphokine serum, in constrast to anti-lymphocyte serum (ALS) did not appear to contain cytotoxic antibodies directed against membrane antigens on guinea pig lymph node lymphocytes. Furthermore, the anti-lymphokine serum did not inhibit the formation of spontaneous T rosettes nor significantly depress lymphocyte response to mitogens. The anti-lymphokine serum totally suppressed the delayed skin reactivity to PPD and contact sensitivity to DNCB when injected intradermally around the site of antigen challenge. By contrast, intradermally injected ALS did not appear to suppress the PPD response in sensitized guinea pigs. Intravenously and i.p. administered anti-lymphokine serum was somewhat less effective in suppressing the delayed skin response to PPD. The intradermal injection of the antiserum had no effect on nonspecific inflammation evoked by turpentine-olive oil or on the extravasation of circulating Evans blue evoked by intradermally injected histamine. Histologic examination of 24-hr DNCB-induced skin lesions from sensitized guinea pigs treated with intradermally injected anti-lymphokine serum showed marked reduction of mononuclear infiltration of the dermis and of epidermal lesions, as compared with skin sites taken from sensitized animals pretreated with normal goat serum. The anti-lymphokine serum injected i.v. also markedly reduced the perivascular infiltration of the dermis and subcutis in skin reaction sites from sensitized animals challenged with PPD. Intravenous treatment with ALS for 3 consecutive days caused extensive depletion of the paracortical areas of peripheral lymph nodes whereas treatment with normal serum and anti-lymphokine serum caused no such depletion. It is proposed that the anti-lymphokine serum is directed against activated lymphocyte products, one of them being MIF. These products are involved in the mediation of delayed hypersensitivity reactions. This is in marked contrast to ALS, the suppressive action of which appears to be central rather than peripheral.     

Immortalization of BGDF (BCGF II)- and BCDF-producing T cells by human T cell leukemia virus (HTLV) and characterization of human BGDF (BCGF II)

Human peripheral T cells were transformed by human T cell leukemia virus (HTLV), and T cell lines producing BGDF (BCGF II) and BCDF were established. Among these cell lines, a cell line, TCL-Na1, secreted the highest level of both BGDF and BCDF, and the amount of BCDF secreted by TCL-Na1 cells was 900-fold more than that produced by PHA-stimulated T cells. Within the limits of our examination, none of the HTLV-transformed T cell lines produced IL 2 or BSF-p1 (BCGF I). BCDF produced by TCL-Na1 cells had a m.w. of 35,000 and a pI value of 5.5, being separated from BGDF, which was eluted in the fractions corresponding to m.w. of more than 60,000 and pI values of 5 to 6. BGDF induced both proliferation and IgM secretion in a mouse leukemic B cell line, BCL1, and these activities were not separated by either isoelectric focusing or gel filtration in the presence or absence of 0.1% Triton X-100, suggesting that the molecule designated BGDF exerted both growth and differentiation activities. BGDF acted on normal mouse B cells to induce proliferation as well as IgM secretion. The target cells of BGDF were in vivo activated B blast cells. BGDF acted on DXS-activated murine B cells to induce both proliferation and IgM secretion but not anti-Ig-activated B cells, indicating that BGDF and BSF-p1 were different molecules.     

Stimulated rat T cell-derived inhibitory factor for cellular DNA synthesis (STIF). I. Isolation and characterization

A lymphokine inhibitory for cellular DNA synthesis (termed STIF) was isolated from the culture supernatants of concanavalin A (Con A)-stimulated SD rat spleen cells. STIF inhibited the DNA synthesis of mouse bone marrow cells as well as mouse leukemia cells. STIF has an apparent m.w. of 45,000 to 50,000 and is separable from IL 2, m.w. 20,000 to 25,000, by Sephacryl S-200 gel filtration, but not from immune interferon (IFN) having the same m.w. as STIF. Con A-Sepharose chromatography of the fraction containing STIF and IFN could separate these lymphokines into Con A-unbound and Con A-bound fractions, respectively. Further fractionation of the STIF fraction by DEAE-Sephadex A-50 or Mono Q-FPLC anion exchange chromatography indicated that the STIF fraction contained two components of STIF activity, both showing the same pI value (5.1 to 5.6) on flat-bed isoelectric focusing. STIF was characterized as a sugar-free lymphokine of trypsin-sensitive protein nature.     

Production and in vivo effect of antibodies against guinea pig lymphokines

Supernatants from guinea pig lymph node lymphocytes stimulated with insoluble Concanavalin A in serum-free medium were fractionated by Sephadex chromatography and preparative electrophoresis. The isolated fraction possessed migration inhibition, mitogenic, and skin reactive activities. Associated with these were apparently two newly synthesized haemoproteins of unknown function. Antibodies were prepared against this partially purified lymphokine fraction. MIF produced by sensitized lymphocytes activated with an antigen (PPD tuberculin) could be completely absorbed from whole supernatants by immunoadsorbent columns prepared with that antibody whereas mitogenic factor and skin reactive factor were not retained. The anti-lymphokine antiserum totally inhibited the delayed skin response of sensitized guinea pigs challenged with PPD.     

Role of the macrophage-derived hybridoma growth factor in the in vitro and in vivo proliferation of newly formed B cell hybridomas

It has been shown recently that monocyte-macrophage cells produce a growth factor (HGF) active on newly formed B cell hybridomas. We have studied the effect of HGF on the proliferation of an HGF-sensitive clone of B cell hybridoma. Results obtained showed that the murine P388D1 cell-derived HGF has a m.w. of 29,000 and an isoelectric point (pI) of 6.2 whereas the human monocyte-derived HGF has a m.w. of 34,000 and a pI of 4.9. The HGF activity was not mediated by interleukin 1 because the two activities could be completely separated by gel filtration. Results obtained in in vivo experiments showed that HGF-sensitive cells are tumorigenic in mice. The finding that the HGF biochemical parameters (m.w. and pI) are similar to the ones of a recently described plasmacytoma growth factor suggests that HGF and the plasmacytoma growth factor are similar and that the HGF sensitivity of SP 2/O myeloma cells is reactivated after fusion with normal B lymphocytes.     

Sphingomyelinase activities in cultured skin fibroblasts from patients with Niemann-Pick disease

Summary Sphingomyelinase activity in cultured skin fibroblasts from a fetus affected with infantile-type Niemann-Pick disease was 0.5% of control activity; the activities in cells from two patients with adult-type disease (Cases 2 and 3) were 5.0% and 59.0%.Sphingomyelinase activity was separated into three peaks (I–III) by isoelectric focusing. The isoelectric points were 4.5, 4.9, and 5.2 for peaks I, II, and III, respectively. The three peaks in the Case 2 cells were drastically reduced; only a very small peak could be distinguished (pI of 4.7). On the other hand, three peaks were observed in the Case 3 cells. Peak I had a pI of 4.4, peak II a pI of 4.7, and peak III a pI of 5.2. Peak I was found at near normal level, but both peaks II and III were markedly reduced.Sphingomyelinase in the peak I fraction obtained from isoelectric focusing in Case 3 cells was found to have the same Km value as that in control cells.     

Human low molecular weight B cell growth factor induces surface IgM+/A- B cells to express and secrete IgA.

The regulation of Ig class expression has been a controversial area of research. It is well established that T cells, and/or their products, influence which Ig isotype is produced during an immune response. In this study the regulation of Ig secretion of activated human IgM+/A- B cells was examined. Human T cell supernatants induced PWM-activated IgM+/A- B cells to switch to IgA secretion. Purification of the lymphokine mediating this effect involved hydroxylapatite, ion exchange, and gel filtration chromatography. The purified lymphokine could induce switch of IgM+/A- B cells, and it was also capable of inducing proliferation of Staphylococcus aureus Cowan 1 strain (SAC)-activated IgM+/A- B cells. SDS-PAGE and isoelectric focusing indicated the protein mediating this activity had a molecular mass of approximately 14 kDa and a pI of 6.8. These results suggested that the observed activity might be due to low m.w. B cell growth factor (LMW-BCGF), a lymphokine which is capable of inducing proliferation of SAC-activated B cells and has a molecular weight and pI value in the range of the purified protein. Indeed, rLMW-BCGF was able to switch IgM+/A- B-cells to IgA expression and secretion as well as induce the proliferation of SAC-activated IgM+/A- B cells. These results demonstrate that LMW-BCGF is capable of inducing PWM-activated IgM+/A- B-cells to switch to IgA possibly by providing a proliferation signal which induces clonal expansion of IgM+/A- B cells, the progeny of which express a range of isotypes including IgA. This study also demonstrates that lymphokine induced isotype switching involves an intermediate stage of B cell development where human B cells coexpress IgM and a downstream isotype on their surface.     

The role of subcellular factors in pulmonary immune function: physicochemical characterization of two distinct species of lymphocyte-activating factor produced by rabbit alveolar macrophages

T cell stimulatory factors produced by rabbit alveolar macrophages were investigated. Physicochemical characterization revealed that alveolar macrophages (harvested by bronchopulmonary lavage and stimulated in tissue culture with bacterial lipopolysaccharide) released 2 predominant species of lymphocyte-activating factor (LAF) with isoelectric points of 4.6 and 7.4, and m.w. of 14,400 and 11,600 daltons, respectively, as calculated by the Svedberg equation. Using C3H/HeJ mouse thymocytes (and in some instances nylon wool-purified nonadherent rabbit spleen or lymph node cells) as target cells, rabbit LAF was found to induce proliferative responses directly, as well as enhance proliferative responses to phytomitogens. Both LAF species were inactivated by heating, treatment with trypsin, or at low (2.3) pH. The pI 7.4 LAF was also unstable at high pH (9.0). The thymocyte stimulatory activity of both LAF species was not inhibited by the anti-proteases alpha-1-anti-trypsin, Traysylol (aprotinin), leupeptin, or pepstatin.     

Comparative analysis of p21 proteins from various cell types by two-dimensional gel electrophoresis

The products of the ras gene family are related proteins at a molecular weight of 21 kDa, designated p21. In the present study we used two-dimensional gel electrophoresis to compare p21 proteins from five different normal and malignant cell lines. Using a known protein (3H-labeled translation initiation factor [eIF-4D]) as a standard internal marker for isoelectric point (pI), we show that p21 proteins from various cells differ only slightly in molecular weight (21-24 kDa) but express a wide variety in charge (pI 4.8 to 7) that could only be detected by the use of two-dimensional gel electrophoresis. p21 in NIH/3T3 cells was expressed as a single protein, which migrated at 21 kDa and pI 5.1. This peptide, which is probably the product of the normal cellular ras gene, was also detected in normal human lymphocytes. The synthesis of this peptide was not elevated in the transformed cells. However, transformation of NIH/3T3 fibroblasts and of human leukocytes was found to be associated with expression of qualitatively different forms of p21 peptides. Four additional p21-associated peptides of identical molecular weight (23 kDa), but multiple charge forms, were detected selectively in Kirsten murine sarcoma virus-transformed NIH/3T3 cells. Transformation of cells with Harvey murine sarcoma virus was found to be associated with prominent expression of two major pairs of p21-associated proteins, one at 21 kDa (pI, 5.2 and 5.3) and the other at 23 kDa (pI, 5.1 and 5.2). In HL-60 leukemic cells there was an additional, more acidic form (pI 5.0) of p21, which appeared to be absent or reduced in normal human lymphocytes. These results indicate that p21 from viral origin or cellular origin might be expressed in the cells in multiple charge forms. The capability to distinguish multiple forms of p21 and slight charge modifications associated with malignancy should call for the use of 2-D gel electrophoresis as an important tool in future studies involving p21 proteins.     

Integration and gene replacement in the Lactococcus lactis lac operon: induction of a cryptic phospho-beta-glucosidase in LacG-deficient strains.

Insertions, replacement mutations, and deletions were introduced via single or double crossover recombination into the lacE (enzyme IIlac) and lacG (phospho-beta-galactosidase) genes of the Lactococcus lactis chromosomal lacABCDFEGX operon. LacG production was abolished in strains missing the lacG gene or carrying multicopy insertions in the lacE gene that affected expression of the lacG gene. However, these LacG-deficient strains could still ferment lactose slowly and were found to contain an enzymatic activity that hydrolyzed the chromogenic substrate o-nitrophenyl-beta-D-galactopyranoside phosphate. Induction of this phospho-beta-glycohydrolase activity coincided with the appearance of a new 55-kDa protein cross-reacting with anti-LacG antibodies that had a size similar to that of LacG but a higher isoelectric point (pI 5.2) and was not found in wild-type cells during growth on lactose. Since the phospho-beta-glycohydrolase activity and this protein with a pI of 5.2 were highly induced in both mutant and wild-type cells during growth on cellobiose that is likely to be transported via a phosphoenolpyruvate-dependent phosphotransferase system, we propose that this induced activity is a phospho-beta-glucosidase that also hydrolyzes lactose-6-phosphate.     

Initial characterization of sheep T-cell growth factor and its species-restricted activity on human, rat, and mouse cells

Sheep T-cell growth factor (TCGF) was prepared from concanavalin A-activated sheep peripheral blood cells and subsequently characterized by ammonium sulfate precipitation, gel exclusion chromatography, and isoelectric focussing. The TCGF was found in the 60-80% ammonium sulfate fraction and was shown to have an apparent molecular weight of 32,500 and an isoelectric point in the range pI 5.2-5.5. The ability of the sheep TCGF to promote proliferation of activated human, sheep, mouse, and rat cells was compared with that of human TCGF prepared by phytohemagglutinin stimulation of lymphocytes from multiple donors and TCGF prepared from concanavalin A-stimulated rat and mouse spleen cells. Human TCGF was found to act across all species barriers, rat TCGF supported the growth of cells of all species except human, and mouse only promoted the growth of activated mouse and rat cells. Sheep TCGF was unique in being unable to support the growth of any cells except autologous cells.     

Identification and characterization of a B cell growth inhibitory factor (BIF) on BCGF-dependent B cell proliferation

The culture supernatants of Con A-activated human peripheral blood mononuclear cells (PBM) contained at least two regulatory factors upon B cell proliferation. One was B cell growth factor (BCGF), which activated antigen-stimulated B cells to proliferation and clonal expansion, and the other was its inhibitory factor, arbitrarily named B cell growth inhibitory factor (BIF). This BIF inhibited the effect of BCGF on anti-mu-stimulated B cells or the monoclonal mature B cell line (CLL-T.H.) obtained from the peripheral blood lymphocytes of B cell-type chronic lymphocytic leukemia patients, which were activated only with BCGF and without adding other proliferating stimuli (e.g., anti-mu). BIF activity was detected in the 24 hr culture supernatants of Con A-activated human PBM in FCS containing medium and also in serum-free RPMI 1640 medium. This substance with BIF activity could not be derived from FCS. Con A-induced BIF (m.w. of 80,000 and an isoelectric point of pH 5.4) was analyzed by Sephadex G-200 gel filtration and chromatofocusing. BIF was stable at pH 2.0 and at 56 degrees C for 30 min. Partially purified BIF had no effect on cell viability and almost no interferon activity (less than 1 IU/ml). BIF with high titer had a slight but significant inhibition on TCGF-dependent T cell growth and on PHA or Con A responses, but the extent of these inhibitions was far less than that of BCGF-dependent B cell growth. Absorption of BIF with Con A blasts made its inhibition on T cell growth even less. On the other hand, BIF activity could not be absorbed with Con A blasts but was almost absorbed with large numbers of CLL-T.H. cells. BIF had almost no inhibitory effect on the proliferation of a mouse fibroblast cell line (NIH 3T3), a mouse myeloma cell line (NS-1), human lymphoid cell lines (MOLT-4, HSB-2, and Daudi), or a human myeloid cell line (K-562). BIF-producing cells were estimated to be T cells and were identified as T8+ T cells. On the other hand, Con A-induced BCGF was demonstrated to be produced predominantly by T4+ T cells. These results show that human B cell proliferation is regulated by interaction between T4+ and T8+ cells via soluble factors, namely BCGF and BIF, respectively.     

Thymoma production of T cell growth factor (Interleukin 2)

Phorbol-12-myristate-13-acetate stimulates a subline of mouse EL-4 thymoma cells to produce, in vitro, in very high titer, T cell growth factor (Interleukin 2, IL 2). The EL-4-derived IL 2 has the same m.w. (30,000) and isoelectric point heterogeneity (pI 3.8-4.4) as the IL 2 produced by Con A-stimulated spleen cells. In addition, the thymoma-derived IL 2 exhibits the same spectrum of biologic activities as has been reported for spleen cell-derived IL 2.     

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.     

Evidence for shared antigenic determinants on rabbit and human interleukin 1 (IL 1)

An antiserum to human interleukin 1 (IL 1) was prepared by immunizing a goat with the isoelectric point (pI) 6.9 type of IL 1 in Freund's complete adjuvant. Serum-mediated inhibition of the biological activity of IL 1 appeared within 4 wk after the first immunization, and showed a progressive rise in titer over a 9-mo period. The inhibitory moiety was purified by sequential ammonium sulfate fractionation and DEAE-Sephacel ion exchange chromatography, and the activity was found to co-purify with the IgG fraction of the serum. The antibody neutralized the biological activity of the pI 6.9 type of human IL 1 derived from either human placental tissue or human peripheral blood adherent cells, but did not neutralize the pI 5.2 type of IL 1 derived from either source. When used as an affinity reagent, the antibody selectively absorbed the pI 6.9 human IL 1, but not the pI 5.2 human IL 1. Furthermore, the antibody neutralized the pI 7.4 type of IL 1 derived from rabbit alveolar macrophages, but had no activity against the pI 4.6 IL 1 derived from the same source. No inhibitory activity against rat spleen cell-derived IL 1 or murine P388D1 cell line-derived IL 1 was detected. These experiments support the concept that the differing pI types of IL 1 derived from the same species are both biochemically and antigenically distinct molecules, and IL 1 of similar pI type derived from different species may share antigenic determinants.     

Role of carbohydrate moiety of IL-5. Effect of tunicamycin on the glycosylation of IL-5 and the biologic activity of deglycosylated IL-5

IL-5 is a T cell-derived lymphokine that induces B cell growth and differentiation in murine systems. In this study, we examined the role of carbohydrate moiety of IL-5 in the expression of biological function. IL-5 polypeptides translated in Xenopus oocytes were heterogeneous in terms of isoelectric point (pI 4.7 to 8.0) and m.w. (45,000 to 60,000 under nonreducing conditions) and yielded m.w. of 25,000 to 30,000 under reducing conditions. Treatment of rIL-5 with N-glycanase under reducing conditions yielded an IL-5 monomer of m.w. 12,000 to 14,000. Furthermore, deglycosylated rIL-5 that had been translated in the presence of tunicamycin showed very limited heterogeneity by two-dimensional gel electrophoresis (first dimension, nonequilibrium pH gradient electrophoresis; second dimension, SDS-PAGE). The m.w. was 27,000 to 28,000 under non-reducing conditions and migrated to m.w. 13,000 to 14,000 under reducing conditions. These results indicate that IL-5 is a glycoprotein carrying the N-glycosidically-linked carbohydrates. Treatment of IL-5 with sialidase caused the decrease in the heterogeneity in isoelectric point of IL-5. Deglycosylated rIL-5 that had been obtained from tunicamycin-treated oocytes could bind to IL-5-responding cells (T88-M), which express both high- and low-affinity IL-5 receptors, as efficient as intact rIL-5 under high-affinity conditions. Scatchard plot analysis of equilibrium binding of 35S-labeled rIL-5 to T88-M cells revealed that the dissociation constants (Kd) of glycosylated rIL-5 and deglycosylated rIL-5 were 127 pM and 110 pM, respectively. IL-5 activities determined by both B cell growth and differentiation assays were not affected by deglycosylation. These results indicate that N-linked glycoside moiety of IL-5 molecules may not play an essential role in the expression of its activity.     

Evidence that polygalacturonic acid may not be a major source of carbon and energy for some colonic Bacteroides species.

Five Bacteroides species that are found in the human colon can utilize polygalacturonic acid (PGA) when they are grown in laboratory media: Bacteroides thetaiotaomicron, Bacteroides vulgatus, Bacteroides ovatus, Bacteroides fragilis subsp. a, and Bacteroides sp. strain 3452A (an unnamed DNA-DNA homology group). PGA-degrading enzymes from B. thetaiotaomicron have been isolated and characterized previously. To determine whether a PGA lyase activity in human feces could be attributed to any of these species, we first determined the properties of PGA lyases from the other four Bacteroides species. PGA lyases from all the Bacteroides species were soluble, cell associated, and inducible by PGA. All had similar pH optima (8.4 to 8.8) and similar molecular weights (50,000). All activities were enhanced by calcium. The PGA lyases from the five species differed with respect to isoelectric point: B. thetaiotaomicron (pI 7.5), B. vulgatus (pI 7.7), B. ovatus (pI 5.8, 7.2), B. fragilis subsp. a (pI 6.1), and Bacteroides sp. strain 3452A (pI 7.7). The PGA lyase activity in human feces resembled those of the Bacteroides PGA lyases in that it had a pH optimum of 8.4 to 8.8 and was enhanced by calcium. However, it differed from the Bacteroides PGA lyases both with respect to isoelectric point (pI 4.2 to 4.4) and molecular weight (100,000). On the basis of these findings, it appears that the PGA lyase activity in human feces is not produced by any of the Bacteroides species surveyed in this survey. Moreover, there was no detectable PGA lyase activity in feces that had the same properties as the Bacteroides enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)