The leaf peroxisomal form (MFP IV) of multifunctional protein functioning in fatty-acid β-oxidation

We have purified for the first time from green leaves a multifunctional protein (MFP) involved in fatty acid -oxidation. The protein, designated MFP IV, was extracted from green leaves of three-week-old cucumber (Cucumis sativus L.) plants. Chromatography on cation exchanger, separation on hydroxylapatite, and fast-protein liquid chromatography on Phenylsuperose led to a more than 7000-fold purification and to the isolation of an apparently homogeneous 80-kDa monomeric protein. This protein is immunologically related to the glyoxysomal MFP II, as evidenced by immunodecoration with antiserum raised against MFP II. Comparison of molecular masses of all MFPs presently known revealed that the MFP prepared from green leaves (MFP IV) is distinct from MFP II (76.5 kDa) and MFP I (74 kDa) from dark-grown cotyledons. By including other properties in this comparison, we demonstrated that MFP IV can also be distinguished from the glyoxysomal MFP III (81 kDa) and the bacterially expressed MFP-a (80 kDa). Moreover, MFP IV is a constituent of leaf peroxisomes and contains the activities of 2-enoyl-CoA hydratase (EC 4.2.1.17),l-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-hydroxyacyl-CoA epimerase.Abbreviation MFP multifunctional protein This work was supported by grants from the Deutsche Forschungsgemeinschaft and the Fonds der Chemischen Industrie.     

Pathways,pathway tubes,pathway docking,and propagators in electron transfer proteins

The simplest views of long-range electron transfer utilize flat one-dimensional barrier tunneling models, neglecting structural details of the protein medium. The pathway model of protein electron transfer reintroduces structure by distinguishing between covalent bonds, hydrogen bonds, and van der Waals contacts. These three kinds of interactions in a tunneling pathway each have distinctive decay factors associated with them. The distribution and arrangement of these bonded and nonbonded contacts in a folded protein varies tremendously between structures, adding a richness to the tunneling problem that is absent in simpler views. We review the pathway model and the predictions that it makes for protein electron transfer rates in small proteins, docked proteins, and the photosynthetic reactions center. We also review the formulation of the protein electron transfer problem as an effective two-level system. New multi-pathway approaches and improved electronic Hamiltonians are described briefly as well.     

Purification and partial characterization of an acidic ribosomal protein kinase from maize

Phosphorylation and dephosphorylation of ribosomal proteins have been suggested to participate in the regulation of protein synthesis in eukaryotic organisms. The present research focuses on the purification and partial characterization of a protein kinase from maize ribosomes that specifically phosphorylates acidic ribosomal proteins. Ribosomes purified from maize axes were used as the enzyme source. Purification of ribosomes was performed by centrifugation through a 0.5 M sucrose, 0.8 M KCl cushion. A protein kinase activity present in this fraction was released by extraction with 1.5 M KCl and further purified by diethylaminoethyl cellulose column chromatography. A peak containing protein kinase activity was eluted around 400 m M KCl. Analysis of this fraction by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed one band of 38 kDa molecular mass, which cross-reacted in a western blot with antibodies raised against proteins from the large ribosomal subunit. This enzyme specifically phosphorylates one of the acidic ribosomal proteins (P₂). Its activity is inhibited by Ca²⁺ and Zn²⁺ and is activated by Mg²⁺, polylysine and spermine. The relevance of this protein kinase in reinitiating the protein synthesis process during germination is discussed.     

Liming induced stimulation of the amino acid metabolism in mycorrhizal roots of Norway spruce (Picea abies [L.] Karst.)

Localization and activity of three enzymes involved in the amino acid metabolism of ectomycorrhizas were investigated within an interdisciplinary experiment performed in a mature Norway spruce stand in Southern Germany (Höglwald). The enzymes NAD-glutamate dehydrogenase and aspartate aminotransferase were present in root cells, whereas aminopeptidase was found in mycorrhizas of Norway spruce such as Piceirhiza nigra and those with the fungi Cenococcum geophilum, Elaphomyces sp., Russula ochroleuca and Tylospora sp. Mycorrhizas growing in the humus layer contained about double the amount of protein found in those taken from the upper mineral soil (0–5 cm).Acid irrigation of the soil had no effect on the activity of any of the investigated enzymes, soluble protein or total N-contents irrespective of whether roots were taken from the organic layer or from the upper mineral soil. Liming, however, stimulated the activity of the three enzymes in mycorrhizas of the organic layer (O_f+O_h) whereas it had no effect on the activity of the investigated enzymes of mycorrhizas in the upper mineral soil. This effect is attributed to increased contents of soluble organic nitrogen compounds in the soil of the limed plots as compared to the unlimed plots.     

What studies of turbellarian embryos can tell us about the evolution of development mechanisms

In spiralian embryos determination of the axes of bilateral symmetry is associated with D quadrant specification. This can occur late through equal cleavage and cell interactions (conditional specification) or by the four-cell stage through unequal cleavage and cytoplasmic localization (autonomous specification). Freeman & Lundelius (1992) suggest that in spiralian coelomates the former method is ancestral and the latter derived, with evolutionary pressure to shorten metamorphosis resulting in early D quadrant determination through unequal cleavage and appearance of adult features in the larvae. Because of the key phylogenetic position of the turbellarian platyhelminthes, understanding the method of axis specification in this group is important in evaluating the hypothesis. Polyclad development, with equal quartet spiral cleavage, is believed to represent the most primitive condition among living turbellarians and has been examined experimentally in Hoploplana inquilina. Blastomere deletions at the two and four-cell stage produce larvae that are abnormal in morphology and symmetry, indicating that early development is not regulative, and also establish that the embryo does not have an invariant cell lineage. Deletions of micromeres and macromeres at the eight-cell stage indicate that cell interactions are involved in dorso-ventral axis determination, with cross-furrow macromeres playing a more significant role than non-cross-furrow cells. The results support the idea that conditional specification is the primitive developmental mode that characterized the common ancestor of the turbellarians and spiralian coelomates. Evolutionary trends in development in polyclads and other turbellarian orders are discussed.     

Preparation of immobilized enzyme with high activity using affinity tag based on proteins A and G

Affinity tag AG consisting of immunoglobulin G (lgG)-binding domains of protein A from Staphylococcus aureus (EDABC) and those of protein G from Streptococcus strain G148 (C2C3) were used to facilitate immobilization of beta-galactosidase (betagal) from Escherichia coli. Poly(methylmethacrylate/N-isopropylacrylamide/methacrylic acid) [P(MMA/NIPAM/MAA)] and poly(styrene/N-isopropylacrylamide/methacrylic acid) [P(St/NIPAM/MAA)] latex particles, which show thermosensitivity, were used as support materals to prepare affinity adsorbents. Human gamma-globulin (HgammaGb), whose major fraction is lgG, was used as an affinity ligand and was covalently immobilized onto the both latex particles by the carbodiimide method under various conditions. A fusion protein, AGbetagal, was immobilized at pH 7.3 by the specific binding of affinity tag to these affinity adsorbents. The amount of adsorbed AGbetagal per unit amount of immobilized HgammaGb, namely, efficiency of ligand utilization, was strongly affected by the type of latex particles and pH value for HgammaGb immobilization. The efficiency of ligand utilization was maximum in the affinity adsorbents prepared at pH 6.0 to 7.0, and that in the HgammaGb-P(MMA/NIPAM/MAA) latex particles was high. This result could be explained by the conformation and orientation of immobilized HgammaGb molecules. Immobilized AGbetagal retained approximately 75% of its activity in solution and the binding is stable enough to allow repeated use. These results clearly demonstrate that combination of the affinity tag AG and the affinity adsorbents, based on the thermosensitive latex particles, offers a simple and widely applicable method for preparation of immobilized enzyme with high activity. (c) 1995 John Wiley & Sons, Inc.     

Protein fractionation using electrostatic interactions in membranel filtration

One of the critical factors limiting the development of membrane systems for protein fractionation has been the poor selectivity that has generally been obtained with these membrane devices. We have demonstrated that it is possible to dramatically improve the selectivity of available membrane systems by exploiting the different electrostatic interactions between the two proteins and the membrane. The separation factor for the albumin-hemoglobin system could be increased to more than 70 simply by reducing the salt concentration and adjusting the pH to around 7 (near the isoelectric point of hemoglobin). This very high selectivity was a direct result of the strong electrostatic exclusion of the charged albumin from the membrane pores under these conditions. This high selectivity makes it possible to very effectively separate these albumin-hemoglobin mixtures using membrane filtration, and this was demonstrated experimentally using both a simple batch filtration process and a continuous diafiltration system. The hemoglobin recovery in the diafiltration experiment was greater than 70% after a 3-diavolume filtration, with the Hb purification factor being around 100 under these conditions. These results clearly demonstrate the potential of membrane systems for the fractionation of proteins even with very similar molecular weights. (c) 1995 John Wiley & Sons, Inc.     

Changes in chorion proteins induced by the exudate released from the egg cortex at the time of fertilization in the teleost, Oryzias latipes

The chorion of unfertilized medaka Oryzias latipes eggs consists of two major proteins (77–73 and 49 kDa) and a minor 150 kDa protein. Upon fertilization, these major chorion proteins are polymerized to insoluble high molecular weight proteins via the temporary formation of several new proteins (132, 114, 62 and 61 kDa). Increasing chorion toughness is closely related to the formation of high molecular weight proteins and the increasing insolubility of the chorion proteins. The changes in chorion proteins and hardening could be induced in vitro in isolated chorions by an egg exudate, which includes cortical alveolar contents. The effects of temperature and pH on the egg exudate-induced changes in chorion proteins were examined in the present study. The major proteins could be digested by proteolytic enzymes. The 49 kDa protein was PAS-positive. Analysis with polyclonal antibodies against the major proteins demonstrated that the temporarily formed 62 and 61 kDa proteins were derived from the 77–73 kDa protein and that higher molecular weight proteins, newly formed in the process of chorion hardening, contained the same epitopes as did the 77–73 and 49 kDa proteins. The results suggest that the changes in chorion proteins of the medaka egg at the time of fertilization can be induced by an enzyme(s) released from the egg cortex into the perivitelline space.     

Selective dimerization of cysteines in glycopeptides and phosphopeptides

Summary To study the influence of phosphorylation and oxidation on the repeat domains of human Tau protein, we faced the challenge to selectively dimerize two cysteine-containing peptides in the presence of a nearby phosphate group. To this end, we were able to demonstrate the utility of a selective dimerization approach by forming disulfide bonds in unprotected phosphopeptides and extended the methodology to unprotected glycopeptides. Activation of one cysteine of a peptide chain with 2,2-dithiodipyridine and coupling this thiopyridyl-peptide to another peptide chain, containing an unprotected cysteine residue, yielded the mixed dimers in high purities and reasonable yields. Phosphate or sugar side chains on either peptide component remained unaffected during the activation and dimerization processes. While for mixed dimers the activated peptides were isolated by chromatography, homodimers were obtained by a simple one-pot reaction after 1 h. We demonstrate that cysteines can be dimerized in unprotected phosphopeptides and glycopeptides, without any side reactions affecting these posttranslational modifications.Abbreviations DCM dichloromethane - DMF N,N-dimethylformamide - DTP 2,2-dithiodipyridine - Fmoc 9-fluorenylmethyloxycarbonyl - HPLC high-performance liquid chromatography - MALDI matrix-assisted laser desorption/ionization - MS mass spectrometry - NFT neurofibrillary tangles - PHF paired helical filaments - PKC protein kinase C - RP reversed phase - human Tau protein - TFA trifluoroacetic acid Parts of this paper were presented at the 24th European Peptide Symposium in Edinburgh, Scotland, U.K., September 8–13, 1996.     

Strategies for the identification and purification of ligands for orphan biomolecules

The isolation of related genes with evolutionary conserved motifs by the application ofpolymerase chain reaction-based molecular biology techniques, or from database searchingstrategies, has facilitated the identification of new members of protein families. Many of theseprotein molecules will be involved in protein–protein interactions (e.g. growth factors,receptors, adhesion molecules), since such interactions are intrinsic to virtually every cellularprocess. However, the precise biological function and specific binding partners of these novelproteins are frequently unknown, hence they are known as orphan molecules.Complementary technologies are required for the identification of the specific ligands orreceptors for these and other orphan proteins (e.g., antibodies raised against crude biologicalextracts or whole cells). We describe herein several alternative strategies for the identification,purification and characterisation of orphan peptide and protein molecules, specifically thesynergistic use of micropreparative HPLC and biosensor techniques.