Characterization of monoclonal antibodies generated against bovine and porcine prostacyclin synthase and quantitation of bovine prostacyclin synthase.

Monoclonal antibodies were raised against prostacyclin synthases purified from bovine and porcine aortae, respectively. Two monoclonal antibodies, RS1 and RS2, were purified and characterized. As shown by enzyme activity precipitation and Western blot analysis, in solubilized bovine and porcine aortae microsomes the monoclonal antibodies reacted only with prostacyclin synthase. The monoclonal antibody RS1 cross-reacts with partially purified prostacyclin synthase from human umbilical veins in an ELISA-based assay. None of the antibodies inhibited the enzyme activity. By combination of the monoclonal antibody RS2 with a polyclonal antibody we established an enzyme-linked immunosorbent assay (ELISA) for quantitation of bovine prostacyclin synthase. ELISA data were confirmed by Western blot analysis. Among different bovine tissues, aortae with 1665 +/- 200 ng/mg microsomal protein showed the highest content of PGIS. Significant lower concentrations were observed in tongue, lung, kidney and thymus ranging from 49 +/- 13.4 to 2.7 +/- 0.9 ng/mg protein. The monoclonal antibody RS1 binds to endothelial cells and vascular smooth muscle cells in human liver tissue.

Modulation of accessory cell function of immortalized bone marrow-derived macrophages by granulocyte/macrophage colony-stimulating factor.

To generate cloned macrophage populations with sensitivity towards granulocyte/macrophage colony-stimulating factor (GM-CSF), bone marrow-derived macrophages (BMM phi) were immortalized by transformation with SV40. A panel of transformed clones was established. The majority of clones represented independently derived transformants, as evidenced by restriction fragment length polymorphism using genomic DNA digested with EcoRI and TaqI and the 5.2 kb SV40 DNA for hybridization analysis. The cells belong to the macrophage lineage according to several criteria, e.g. the presence of nonspecific esterase, their phagocytic capacity and their morphology. Many clones were potent antigen-presenting cells (APC), without exogenous stimulation. Two clones, which did not act efficiently as APC when used untreated, were positively responsive to GM-CSF. GM-CSF stimulation of both clones resulted in potent APC capacity. I-A alpha, I-A beta and gamma chain-specific transcripts were observed upon stimulation with GM-CSF, corresponding to detectable levels of class II surface display as revealed by cytofluorometric analysis. Thus the macrophage clones established will allow dissection of the differential effects of GM-CSF on the parameters of antigen presentation.

Dendritic cells from mouse bone marrow: in vitro differentiation using low doses of recombinant granulocyte-macrophage colony-stimulating factor.

Dendritic cells (DC) are potent stimulator cells that are crucially involved in primary T cell responses. Since DC comprise a minor population in lymphoid cell suspensions tedious and time consuming procedures are required for their preparation. This work outlines an in vitro culture system that promotes the differentiation of DC from unfractionated mouse bone marrow (BM) cells in the presence of low doses of recombinant granulocyte-macrophage colony-stimulating factor (GM-CSF). Unlike co-induced BM-macrophages the outgrowing BM-DC express high levels of MHC class II molecules; they are negative for specific and nonspecific esterase and are nonphagocytic. A rapid one step purification procedure employing immunomagnetic bead selection yielded up to 95% BM-DC enriched cell fractions. The bead-selected BM-DC were powerful inducers of the allogeneic mixed leukocyte reaction. Thus, our findings demonstrate that low dose rGM-CSF-driven in vitro culture of BM cells provides convenient access to substantial numbers of DC and will greatly facilitate their further exploration.

The invariant chains of mouse class II antigens: biochemical properties and molecular relationship.

The proteins p40 (Mr = 40 000), p32 (Mr = 32 000), p28 (Mr = 28 000), p20 (Mr = 20 000) and p10 (Mr = 10 000) are described which occur in noncovalent association with the polymorphic alpha, beta heterodimer of class II antigens. They were investigated with respect to their molecular characteristics and their mutual structural relationship. p32, the predominant species of this group corresponds to the invariant chain gamma (Ii). In contrast to the polymorphic subunits alpha and beta, proteins p40, p28, p20 and p10 migrated like gamma in electrophoretically constant positions, when class II molecules of different subregions and different alleles were assessed by two-dimensional gel electrophoresis [1st dimension, isoelectric focusing; 2nd dimension, sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis]. Analogous to gamma, they are therefore designated invariant chains. The low Mr species of this group do not arise from higher Mr forms as preparation artefacts. Short-term pulse-chase analysis and cell-free translation of sucrose gradient-fractionated mRNA in conjunction with specific immunoprecipitation rendered the possibility unlikely that individual components of this set of proteins existed in a precursor-product relationship within the cell. Comparative enzymatic fragmentation on SDS polyacrylamide gels as well as tryptic peptide map comparisons by high performance liquid chromatography revealed a high structural relatedness among all members of this group of invariant proteins.

Mouse invariant chain gamma exhibits structural homology to both polymorphic subunits of the alpha, beta-core complex of I-Ak antigens.

The 3 major constituents of the I-Ak subregion-associated complex alpha, beta and gamma were obtained from splenocytes in homogeneous form by differential isolation methods. alpha, beta and gamma were compared on the primary structural level by enzymatic fragmentation procedures and tryptic peptide map analysis of radiolabeled proteins. The data indicate that the invariant chain gamma exhibits extensive structural homology to the polymorphic beta-light and the alpha-heavy chain. Thus, although not being encoded within the MHC gamma appears to belong structurally to the MHC-encoded class II proteins.

Isolation of the I-Ak core complex and its associated gamma-polypeptide by affinity chromatography on monoclonal immunoadsorbent 10-2.16 sepharose CL-4B.

Spleen cell-derived I-Ak antigens have previously been shown by us to be comprised of four noncovalently associated polypeptide chains (alpha, gamma, beta 1, beta 2). Here we report that pretreatment of the detergent solubilized four-chain structure with chaotropic ions yielded an alloantigenically reactive core complex (alpha/beta 1, beta 2) and a dissociated polypeptide gamma-chain which was not recognized by monoclonal antibody 10-2.16 (anti-I-Ak). The lack of alloantigenic determinants on gamma and the observation that gamma did not reassociate with alpha/beta 1, beta 2 upon removal of the chaotropic reagent suggested a rapid purification procedure for both moieties. A pure preparation of the serologically intact core complex was achieved by repeated affinity chromatography on hybridoma immunoadsorbent 10-2.16 Sepharose CL-4B. Concurrently its pertinent gamma-polypeptide was obtained in a highly enriched form and was purified to homogeneity by an additional gel electrophoretic step.

Phosphotransferase properties of human erythrocyte phosphoglycolate phosphatase.

1. Human erythrocyte phosphoglycolate phosphatase (PGP) (EC 3.1.3.18) shows transferase properties. Using p-nitrophenylphosphate (p-NPP) as substrate, methanol, at a concentration of 4.9 M, was the most efficient phosphate acceptor tested (60% phosphate transfer). 2. The branched alcohols i-propanol and i-butanol accept the phosphate better than the unbranched compounds. The acceptor potency is methanol greater than ethanol greater than i-propanol greater than n-propanol greater than i-butanol greater than n-butanol. 3. The relative transferase activity could be demonstrated to be independent of substrate concentration, pH, and the inhibitory effect of NaF at 2 and 4 mM. 4. PGP shows no transferase activity towards glucose and fructose, and is even inhibited by 250 mM of lactose and lactic acid to 67 and 55%, respectively. 5. Using p-nitrophenyl-[32P]-phosphate (p-NP[32P]P), the direct transfer of phosphate from donor to acceptor could be demonstrated.

Purification, isolation and characterization of a phosphoglycolate phosphatase isoenzyme from human erythrocytes.

1. Preparation, purification and characterization of a phosphoglycolate phosphatase (PGP) isoenzyme from human erythrocytes was achieved by DEAE-Sepharose CL-6B chromatography and isoelectric focusing using carrier ampholytes, pH 4-6. 2. The isoenzyme has an isoelectric point of 5.00 +/- 0.05 and could be purified 33,000 fold to a specific activity of 32.7 U/mg of protein. It represents the PGP phenotype 1 consisting of a single isoenzyme. 3. The enzyme is composed of two subunits (mol. wt 35,000) which are identical and not connected by SS-bridges. 4. At 4 degrees C the isoenzyme is more stable in the pH range of 7-9 than at acid pH values. 5. Incubation at 30 and 40 degrees C for 4 hr does not affect the activity of the isoenzyme. 6. It has a Km-value of 0.28 mM for phosphoglycolate (PG) as substrate.

Partial purification and characterization of human erythrocyte phosphoglycollate phosphatase.

Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5'-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.