Cloning and expression in Escherichia coli of a rat hepatoma cell cDNA coding for 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase.

In liver, the 470-residue bifunctional enzyme 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2/FBPase-2) catalyses the synthesis and degradation of fructose 2,6-bisphosphate, a potent stimulator of glycolysis. In rat hepatoma (HTC) cells, this enzyme has kinetic, antigenic, and regulatory properties, such as insensitivity to cyclic AMP-dependent protein kinase and lack of associated FBPase-2 activity, that differ from those in liver. To compare the sequence of the HTC enzyme with that of the liver enzyme, we have cloned the corresponding fully-coding cDNA from HTC cells. This cDNA predicts a protein of 448 residues in which the first 32 residues of liver PFK-2/FBPase-2 including the cyclic AMP target sequence have been replaced by a unique N-terminal decapeptide. The rest of the protein is identical with the liver enzyme. An N-terminally truncated recombinant peptide of 380 residues containing the PFK-2 and FBPase-2 domains was expressed in Escherichia coli as a beta-galactosidase fusion protein. It was recognized by anti-PFK-2 antibodies but its enzymic activities were barely detectable. In contrast, a cDNA fully-coding for the HTC enzyme could be expressed in E. coli as a beta-galactosidase-free peptide that exhibited both PFK-2 and FBPase-2 activities. This peptide had those PFK-2 kinetic properties of the HTC enzyme that differ from the liver enzyme. These data, together with immunoblot experiments, suggest that the lack of associated FBPase-2 activity in HTC cells results from a post-translational modification of the enzyme rather than from the difference in amino acid sequence. As well as this peculiar type of PFK-2/FBPase-2 mRNA, HTC cells also contained low concentrations of the liver-type mRNA. Unlike in liver, neither mRNA was induced by dexamethasone in these cells.     

Structure and activity of proteins from pathogenic fungi Phytophthora eliciting necrosis and acquired resistance in tobacco

The phytopathogenic fungi Phytophthora cryptogea and Phytophthora capsici cause systemic leaf necrosis on their non-host tobacco; in culture they release proteins, called cryptogein and capsicein, which elicit similar necrosis. In addition, both proteins protect tobacco against invasion by the pathogen Phytophthora nicotianac, the agent of the tobacco black shank, that is unable to produce such an elicitor. Cryptogein causes visible leaf necrosis starting at about 1 microgram/plant, whereas 50-fold as much capsicein is required for the same reaction. Capsicein induces protection even in near absence of leaf necrosis. The activities of both elicitors are eliminated upon pronase digestion. They are proteins of similar Mr (respectively 10,323 and 10,155) and their complete amino acid sequences were determined. They consist of 98 residues, with some internal repetitions of hexapeptides and heptapeptides. 85% identity was observed between both sequences: only two short terminal regions are heterologous, while the central core is entirely conserved. Secondary structure predictions, hydropathy and flexibility profiles differ only around position 15 and at the C-terminus; these modifications could play a role in the modulation of their biological activities. After a search of the sequence data bases, they appear to be novel proteins.     

Synthesis and properties of azidonitrophenyl pyrophosphate, a photoaffinity probe of the nucleotide binding sites of mitochondrial F1-ATPase

4-Azido-2-nitrophenyl pyrophosphate (azido-PPi) labeled with 32P in the alpha position was prepared and used to photolabel beef heart mitochondrial F1. Azido-PPi was hydrolyzed by yeast inorganic pyrophosphatase, but not by mitochondrial F1-ATPase. Incubation of F1 with [alpha-32P]azido-PPi in the dark under conditions of saturation resulted in the binding of the photoprobe to three sites, two of which exhibited a high affinity (Kd = 2 microM), the third one having a lower affinity (Kd = 300 microM). Mg2+ was required for binding. As with PPi [Issartel et al. (1987) J. Biol. Chem. 262, 13538-13544], the binding of 3 mol of azido-PPi/mol of F1 resulted in the release of one tightly bound nucleotide. ADP, AMP-PNP, and PPi competed with azido-PPi for binding to F1, but Pi and the phosphate analogue azidonitrophenyl phosphate did not. The binding of [32P]Pi to F1 was enhanced at low concentrations of azido-PPi, as it was in the presence of low concentrations of PPi. Sulfite, which is thought to bind to an anion-binding site on F1, inhibited competitively the binding of both ADP and azido-PPi, suggesting that the postulated anion-binding site of F1 is related to the exchangeable nucleotide-binding sites. Upon photoirradiation of F1 in the presence of [alpha-32P]azido-PPi, the photoprobe became covalently bound with concomitant inactivation of F1. The plots relating the inactivation of F1 to the covalent binding of the probe were rectilinear up to 50% inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of four herbicide-resistant mutants of Rhodopseudomonas viridis by genetic analysis, electron paramagnetic resonance, and optical spectroscopy

Herbicides of the triazine class block electron transfer in the photosynthetic reaction centers of purple bacteria and PSII of higher plants. They are thought to act by competing with one of the electron acceptors, the secondary quinone, QB, for its binding site. Several mutants of the purple bacterium Rhodopseudomonas viridis resistant to terbutryn [2-(methylthio)-4-(ethylamino)-6-(tert-butylamino)-s-triazine] have been isolated by their ability to grow photosynthetically in the presence of the herbicide. Sequence analysis of the genes coding for the L and M subunits of the reaction center showed that four different mutants were obtained, two of them being double mutated: T1 (SerL223----Ala and ArgL217----His), T3 (PheL216----Ser and ValM263----Phe), T4 (TyrL222----Phe), and T6 (PheL216----Ser). The residues L223 and L216 are involved in binding of QB, whereas L217 and L222 are not. M263 is part of the binding pocket of the primary quinone, QA. The affinity of the reaction centers for terbutryn and the electron transfer inhibitor o-phenanthroline, determined via the biphasic charge recombination after one flash, is decreased for all mutants. The affinity for ubiquinone 9 is also decreased, except in T1. Characterization by EPR spectroscopy showed that the QB.-Fe2+ signal of T4, having a g = 1.93 peak, is different from the signals obtained with the wild type and the other mutants but very similar to those of Rhodospirillum rubrum and PSII. The results obtained by the combination of these different techniques are discussed with respect to the three-dimensional structure of the wild type and the mode of binding of ubiquinone, terbutryn, and o-phenanthroline as determined by X-ray structure analysis.     

Effects of phospholipases,proteases and neuraminidase on γ-hydroxybutyrate binding sites

Summary -Hydroxybutyric acid (GHB) is a natural compound of mammalian brain synthesized from GABA. The characteristics of its synthesis, transport, release, distribution and turnover, in addition to the presence of a high affinity binding site for this substance in brain are in favor of a modulator role for GHB. The effects of hydrolytic enzymes on the specific binding capacity of GHB have been studied in the present work. Phospholipases A₂ and C, neuraminidase and Pronase markedly decrease GHB binding to crude synaptosomal membranes from rat brain. This effect is time and enzyme concentration dependent. Trypsin, under the conditions employed, is less active. The inhibitory effects of phospholipases is correlated with phospholipid hydrolysis. Lysophospholipids, in the absence of bovine fatty acid free serum albumin partially inhibit GHB binding. The action of neuraminidase has been followed by sialic acid release and modifications of the ganglioside profile. The effects of phospholipase C and of neuraminidase are completely different to those on GABA binding sites. These results represent further data concerning the molecular existence of specific GHB binding sites on rat brain membranes.Abbreviations GHB -hydroxybutyrate - LPC L--lysophosphatidylcholine - LPE Lysophosphatidylethanolamine - PC Phosphatidylcholine - PE Phosphatidylethanolamine - BSA Bovine Serum Albumin     

Light-temperature interactions on the growth of Antarctic diatoms

Summary The combined effect of various temperatures and light intensities on the growth of seven species of antarctic diatoms in culture has been studied. With the exception of Chaetoceros deflandrei whose thermal tolerance is fairly good, these obligatory psychrophils cannot survive in temperatures above 6° to 9° C. Their mean growth rate is relatively low, between 0.24 div d^–1 for Corethron criophilum and 0.63 div d^–1 for C. deflandrei. Regardless of light intensity, growth rate increased with the temperature to reach a maximum between 3° and 5° C. The highest rates were obtained between 115 and 220 mol m^–2 s^–1 with 0.38 div d^–1 for C. criophilum, 0.56 div d^–1 for Synedra sp. and between 0.71 and 0.88 div d^–1 for the other 5 species. A reduction in light intensity from 220 to 46 mol m^–2 s^–1 slowed growth by nearly 50%. These results suggest that the combined effect of temperature and light is one of the factors involved in the limitation of antarctic phytoplankton growth. The low temperatures of the environment do not permit rapid growth, which, even under optimal light conditions remains low. In addition, in the euphotic layer, the overall light energy available for algae is considerably reduced due to turbulence, a factor which exacerbates the reduced growth rate.     

Ultrastructure of the tube foot sensory-secretory complex in Ophiocomina nigra (Echinodermata,Ophiuridea)

Summary The ultrastructure of the epidermal layer of both the oral and arm podia of the brittle star Ophiocomina nigra is described. Despite external differences, little variation occurs in their internal structure. The podial epidermis, which is overlain by a three-layered cuticle, consists of five cell types: support, mucous, sensory, adhesive secretory and monociliated neurosecretory-like cells. Areas of specialisation are superimposed on this basic plan. These comprise four cells forming cohesive units, made up of two adhesive secretory, one sensory and one monociliated neurosecretory-like cells. The two adhesive secretory cells may be identical or vary in the structure of their secretory packets. The sensory cells are of the normal type bearing a short cilium with a 9+2 microtubular arrangement. The monociliated neurosecretory-like cells contain many small dense vesicles and a short sub-cuticular cilium of irregular microtubular structure. Together, they appear to form a sensory-secretory complex which functions in adhesion both for feeding and locomotion. A system in which the secretion of the monociliated neurosecretory-like cell may control adhesive secretion is proposed.     

Hydrolysis of alpha-human atrial natriuretic peptide in vitro by human kidney membranes and purified endopeptidase-24.11. Evidence for a novel cleavage site.

alpha-Human atrial natriuretic peptide (hANP) is secreted by the heart and acts on the kidney to promote a strong diuresis and natriuresis. In vivo it has been shown to be catabolized partly by the kidney. Crude microvillar membranes of human kidney degrade 125I-ANP at several internal bonds generating metabolites among which the C-terminal fragments were identified. Formation of the C-terminal tripeptide was blocked by phosphoramidon, indicating the involvement of endopeptidase-24.11 in this cleavage. Subsequent cleavages by aminopeptidase(s) yielded the C-terminal dipeptide and free tyrosine. Using purified endopeptidase 24.11, we identified seven sites of hydrolysis in unlabelled alpha-hANP: the bonds Arg-4-Ser-5, Cys-7-Phe-8, Arg-11-Met-12, Arg-14-Ile-15, Gly-16-Ala-17, Gly-20-Leu-21 and Ser-25-Phe-26. However, the bonds Gly-16-Ala-17 and Arg-4-Ser-5 did not fulfil the known specificity requirements of the enzyme. Cleavage at the Gly-16-Ala-17 bond was previously observed by Stephenson & Kenny [(1987) Biochem. J. 243, 183-187], but this is the first report of an Arg-Ser bond cleavage by this enzyme. Initial attack of alpha-hANP by endopeptidase-24.11 took place at a bond within the disulphide-linked loop and produced a peptide having the same amino acid composition as intact ANP. The bond cleaved in this metabolite was determined as the Cys-7-Phe-8 bond. Determination of all the bonds cleaved in alpha-hANP by endopeptidase-24.11 should prove useful for the design of more stable analogues, which could have therapeutic uses in hypertension.     

Presence of serum albumin in normal human epidermis: Possible implications for the nutrition and physiology of stratified epithelia

In this paper, using both immunofluorescence and protein biochemistry techniques, we present definitive evidence that plasma proteins such as albumin are present within normal human epidermis. This result confirms several previous reports supporting the idea that relatively large molecules can diffuse through the epidermal basement membrane into epidermis. Our results bring new insights for discussing how hydrophobic ligands or drugs present in the bloodstream and bound to plasmatic carriers can reach epidermal cells of all layers.Abbreviations CHAPS 3-[3-cholamidopropyl dimethylammonio] propane sulfonate - kD kilodaltons - BSA bovine serum albumin - 2ME 2-mercaptoethanol - DTT dithiothreitol - SDS sodium dodecyl sulfate - pI isoelectric point - Mw molecular weight - Tris Tris-(hydroxymethyl) aminomethane - 1D one dimensional - 2D two dimensional - PAGE poly acrylamide gel electrophoresis - MEM Minimal Eagle's Medium