Human macrophage colony-stimulating factor heterogeneity results from alternative mRNA splicing, differential glycosylation, and proteolytic processing

The single gene for human macrophage colony-stimulating factor (M-CSF, or CSF-1) generates multiple mRNA species that diverge within the coding region. We have characterized translation products of these mRNA species from native and recombinant sources. Immunoblots of reduced native M-CSF indicate that multiple glycosylated species ranging from 25 kd to 200 kd are secreted by human monocytes and cell lines. In contrast, CV-1 cells expressing a short M-CSF clone secrete only 24 kd recombinant M-CSF. Synthetic peptide antibodies were developed to distinguish between secreted recombinant M-CSF from long and short mRNA splicing variants. Immunoblot analysis indicates that alternative mRNA splicing generates some M-CSF protein heterogeneity. Most secreted MIA PaCa-2 M-CSF reacts with long-clone-specific antibody. Lectin affinity chromatography shows that variable glycosylation contributes significantly to MIA PaCa-2 M-CSF size heterogeneity. In addition, cell lysates also contain larger M-CSF species that apparently undergo proteolytic processing before secretion. The data indicate that M-CSF protein heterogeneity results from both pre- and post-translational processing.     

Improvement of production,assay and purification of streptavidin

Summary The production of streptavidin byStreptomyces avidinii in several different media was examined at 24, 48 and 72 hours. Flask studies indicated that fermentation media containing either complex or multiple carbon sources resulted in higher yields of streptavidin than media with a single carbon source. Streptavidin could be detected in crude fermentation broths by use of a tritiated biotin binding assay. This assay appears to give useful estimates of streptavidin production. Depending upon the medium employed, streptavidin yields ranged from 0.5 mg/l to 53 mg/l. Production was successfully scaled up to ten liter fermentors. Streptavidin was purified in a one step process from centrifuged, concentrated fermentation broths by binding the protein to an iminobiotin column at pH 11 followed by elution at pH 4.0. Recovery percentages varied depending upon the solubility of the fermentation media ingredients.     

Phenylalanine biosynthesis in Arabidopsis thaliana. Identification and characterization of arogenate dehydratases

There is much uncertainty as to whether plants use arogenate, phenylpyruvate, or both as obligatory intermediates in Phe biosynthesis, an essential dietary amino acid for humans. This is because both prephenate and arogenate have been reported to undergo decarboxylative dehydration in plants via the action of either arogenate (ADT) or prephenate (PDT) dehydratases; however, neither enzyme(s) nor encoding gene(s) have been isolated and/or functionally characterized. An in silico data mining approach was thus undertaken to attempt to identify the dehydratase(s) involved in Phe formation in Arabidopsis, based on sequence similarity of PDT-like and ACT-like domains in bacteria. This data mining approach suggested that there are six PDT-like homologues in Arabidopsis, whose phylogenetic analyses separated them into three distinct subgroups. All six genes were cloned and subsequently established to be expressed in all tissues examined. Each was then expressed as a Nus fusion recombinant protein in Escherichia coli, with their substrate specificities measured in vitro. Three of the resulting recombinant proteins, encoded by ADT1 (At1g11790), ADT2 (At3g07630), and ADT6 (At1g08250), more efficiently utilized arogenate than prephenate, whereas the remaining three, ADT3 (At2g27820), ADT4 (At3g44720), and ADT5 (At5g22630) essentially only employed arogenate. ADT1, ADT2, and ADT6 had k(cat)/Km values of 1050, 7650, and 1560 M(-1) S(-1) for arogenate versus 38, 240, and 16 M(-1) S(-1) for prephenate, respectively. By contrast, the remaining three, ADT3, ADT4, and ADT5, had k(cat)/Km values of 1140, 490, and 620 M(-1) S(-1), with prephenate not serving as a substrate unless excess recombinant protein (>150 microg/assay) was used. All six genes, and their corresponding proteins, are thus provisionally classified as arogenate dehydratases and designated ADT1-ADT6.     

Photo-oxidation-induced inactivation of the selenium-containing protective enzymes thioredoxin reductase and glutathione peroxidase

Singlet oxygen ((1)O(2)) is a reactive oxygen species generated during photo-oxidation, inflammation, and via peroxidase-catalyzed reactions (e.g., myeloperoxidase and eosinophil peroxidase). (1)O(2) oxidizes the free amino acids Trp, Tyr, His, Cys, and Met, and those species present on peptides/proteins, with this resulting in modulation of protein structure and function. Impairment of the activity of antioxidant enzymes may be of relevance to the oxidative stress observed in a number of pathologies involving either light exposure or inflammation. In this study, the effects of (1)O(2) on glutathione peroxidase (GPx) and thioredoxin reductase (TrxR) activity, including the mechanisms of their inactivation, were investigated. Exposure of GPx or TrxR, either as purified proteins or in cell lysates, to Rose Bengal and visible light (an established source of (1)O(2)) resulted in significant, photolysis time-dependent reductions in enzyme activity (10-40%, P<0.05). More extensive inhibition (ca. 2-fold) was detected when the reactions were carried out in D(2)O, consistent with the intermediacy of (1)O(2). No additional inhibition was detected after the cessation of photolysis, eliminating a role for photo-products. Methionine, which reacts rapidly with (1)O(2) (k～10(7)M(-1) s(-1))(,) significantly reduced photo-inactivation at large molar excesses, presumably by acting as an alternative target. Reductants (NaBH(4), DTT, GSH, or NADPH) added after the cessation of (1)O(2) formation were unable to reverse enzyme inactivation, consistent with irreversible enzyme oxidation. Formation of nonreducible protein aggregates and/or fragments was detected for both photo-oxidized GPx and TrxR by SDS-PAGE. An oxidant concentration-dependent increase in protein carbonyls was detected with TrxR but not GPx. These studies thus demonstrate that the antioxidant enzymes GPx and TrxR can be irreversibly inactivated by (1)O(2).     

Physcomitrella patens has lipoxygenases for both eicosanoid and octadecanoid pathways

Mosses have substantial amounts of long chain C20 polyunsaturated fatty acids, such as arachidonic and eicosapentaenoic acid, in addition to the shorter chain C18 α-linolenic and linoleic acids, which are typical substrates of lipoxygenases in flowering plants. To identify the fatty acid substrates used by moss lipoxygenases, eight lipoxygenase genes from Physcomitrella patens were heterologously expressed in Escherichia coli, and then analyzed for lipoxygenase activity using linoleic, α-linolenic and arachidonic acids as substrates. Among the eight moss lipoxygenases, only seven were found to be enzymatically active in vitro, two of which selectively used arachidonic acid as the substrate, while the other five preferred α-linolenic acid. Based on enzyme assays using a Clark-type oxygen electrode, all of the active lipoxygenases had an optimum pH at 7.0, except for one with highest activity at pH 5.0. HPLC analyses indicated that the two arachidonic acid lipoxygenases form (12S)-hydroperoxy eicosatetraenoic acid as the main product, while the other five lipoxygenases produce mainly (13S)-hydroperoxy octadecatrienoic acid from α-linolenic acid. These results suggest that mosses may have both C20 and C18 based oxylipin pathways.     

The use of light-directed combinatorial peptide synthesis in epitope mapping

The application of light-directed combinatorial peptide synthesis to epitope mapping is described. Photolithography and solid phase peptide synthesis were combined in an automated fashion to assemble arrays containing 1024 peptide sequences on a glass support in ten steps with the precise location of each peptide known. The simultaneous synthesis of two slides containing three arrays of peptidtes each allowed for the independent screening of both a monoclonal antibody (mAb) and its Fab fragment at two different concentrations. A binary synthesis strategy was used to assemble the arrays, resulting in all deletions and truncations possible within the FLRRQFKVVT sequence being present and available for screening. The relative binding interactions of each peptide was determined by incubating the arrays with either mAb D32.39 and goat antimouse immunoglobulin G–FITC or mAb D32.39 Fab-FITC conjugate, followed by scanning the surface for fluorescence with an epifluorescence microscope. The fragment RQFKVVT was found to bind lightly to both the mAb and Fab fragment while tethered to the surface, and was measured to have 0.49 n M affinity in solution. The frame-shifted RRQFKVV sequence was found to have lower affinity both in solution (1.3 m M) and on the surface. The fragment RQFKVV was determined to be responsible for antibody recognition and was found to bind tightly when tethered to the surface, yet exhibited no binding in solution as the free acid, suggesting the requirement of an amidated C-terminus or an additional flunking residue. A deletion analysis revealed that the novel RQFKVT sequence exhibited higher affinity than the RQFKVV sequence while tethered to the surface. © 1994 John Wiley & Sons, Inc.     

Gibberellin biosynthesis in bacteria: Separate ent-copalyl diphosphate and ent-kaurene synthases in Bradyrhizobium japonicum

Gibberellins are ent-kaurene-derived diterpenoid phytohormones produced by plants, fungi, and bacteria. The distinct gibberellin biosynthetic pathways in plants and fungi are known, but not that in bacteria. Plants typically use two diterpene synthases to form ent-kaurene, while fungi use only a single bifunctional diterpene synthase. We demonstrate here that Bradyrhizobium japonicum encodes separate ent-copalyl diphosphate and ent-kaurene synthases. These are found in an operon whose enzymatic composition indicates that gibberellin biosynthesis in bacteria represents a third independently assembled pathway relative to plants and fungi. Nevertheless, sequence comparisons also suggest potential homology between diterpene synthases from bacteria, plants, and fungi.     

Induced phenylpropanoid metabolism during suberization and lignification: a comparative analysis

Induction of the biosynthesis of phenylpropanoids was monitored at the enzyme level through measurement of the temporal change in the activity of two marker enzymes of phenylpropanoid metabolism, phenylalanine ammonia-lyase, (PAL, E.C. 4.1.3.5) and 4-coumaryl-CoA ligase (4-CL, E.C. 6.2.1.12) and two marker enzymes for hydroxycinnamyl alcohol biosynthesis, cinnamoyl-CoA:NADP+ oxidoreductase (CCR, E.C. 1.2.1.44) and cinnamyl alcohol dehydrogenase (CAD, E.C. 1.1.1.195) in both suberizing potato (Solanum tuberosum) tubers and lignifying loblolly pine (Pinus taeda) cell cultures. While measurable activities of PAL, 4-CL and CAD increased upon initiation of suberization in potato tubers, that of CCR did not. By contrast, all four enzymes were induced upon initiation of lignification in pine cell cultures. The lack of CCR induction in potato by wound treatment is consistent with the channelling of hydroxycinnamoyl-CoA derivatives away from monolignol formation and toward other hydroxycinnamoyl derivatives such as those that accumulate during suberization.