The origin of kinetic cooperativity in prehiotic catalysts

A polyallylamine carrying long hydrophobic dodecyl groups and adenine residues as side chains (PALAD C₁₂) may be able to catalyze the hydrolysis ofN-carbobenzoxy-l-alaninep-nitrophenyl ester (N-Cbz-Ala) as well asp-nitrophenyl acetate (pNPA). The progress curve of hydrolysis of the former displays a long lag and apparently no steady state. After this transient the rate falls off due to the accumulation of the products. Conversely, the hydrolysis ofp-nitrophenyl acetate displays classical burst kinetics followed by a slow decline of the reaction rate. Theoretical considerations show that a steady state may be expected to occur only if the concentration of the free catalyst is very small during the reaction. This condition is sufficient to allow the rate of disappearance of the substrate to be equal to the rate of appearance of the products, which is precisely a condition for the existence of a steady state. If the catalyst is poorly active and has a loose affinity for its substrate and product, the measurement of a significant reaction rate will require a much larger concentration of the catalyst. Therefore, under these conditions, one cannot expect a steady state to occur. The mathematical expression of the error made in the steady-state assumption has been derived. This error increases with the catalyst concentration and decreases if the affinity of the substrate for the catalyst is high. Therefore the lack of steady state is associated with the affinity (or the dissociation) of the substrate and the product for the catalyst. When this affinity is low, the free concentration of the catalyst during the reaction is high and one cannot expect a steady state to occur. This is precisely what takes place with N-Cbz-Ala. A mathematical expression of the rate of hydrolysis of N-Cbz-Ala and of any reactant that displays this type of kinetics may be derived at the end of the transient when the rate is close to its maximum value. Under these conditions the rate cannot follow classical Michaelis-Menten kinetics and displays positive cooperativity. It may therefore be speculated that primordial template-like catalysts that were displaying a poor affinity for their substrates and products were already exhibiting apparent positive cooperativity in the kinetic reactions they were able to catalyze. Correspondence to: J. Ricard     

In Vivo and In Vitro Evidence for the Biosynthesis of Testosterone in the Telencephalon of the Female Frog

Abstract: Neurons and glial cells are capable of synthesizing various steroid hormones, but biosynthesis of testosterone in the CNS has never been reported. The aim of the present study was to demonstrate the synthesis of testosterone in the frog brain. The presence of 17β-hydroxysteroid dehydrogenase (17β-HSD)-like immunoreactivity was detected in a population of glial cells located in the telencephalon. Reversed-phase HPLC analysis of brain tissue extracts combined with radioimmunoassay detection revealed the presence of substantial amounts of testosterone and 5α-dihydrotestosterone (5α-DHT) in the telencephalon where 17β-HSD-positive cells were visualized. In male frogs, castration totally suppressed testosterone and 5α-DHT in the blood and in the rhombencephalon but did not affect the concentration of these two steroids in the telencephalon. Chemical characterization of testosterone in female frog telencephalon extracts was performed by coupling HPLC analysis with gas chromatography-mass spectrometry. Using the pulse-chase technique with [³H]pregnenolone as a precursor, the formation of a series of metabolites was observed, including dehydroepiandrosterone, androstenedione, testosterone, 5α-DHT, and estradiol. These data demonstrate the existence of an active form of 17β-HSD in the frog telencephalon, which is likely involved in testosterone biosynthesis within the brain.     

Insulin receptor substrate 1 binds two novel splice variants of the regulatory subunit of phosphatidylinositol 3-kinase in muscle and brain.

We have identified two novel alternatively spliced forms of the p85alpha regulatory subunit of phosphatidylinositol (PI) 3-kinase by expression screening of a human skeletal muscle library with phosphorylated baculovirus- produced human insulin receptor substrate 1. One form is identical to p85alpha throughout the region which encodes both Src homology 2 (SH2) domains and the inter-SH2 domain/p110 binding region but diverges in sequence from p85alpha on the 5' side of nucleotide 953, where the entire break point cluster gene and SH3 regions are replaced by a unique 34-amino-acid N terminus. This form has an estimated molecular mass of approximately 53 kDa and has been termed p85/AS53. The second form is identical to p85 and p85/AS53 except for a 24-nucleotide insert between the SH2 domains that results in a replacement of aspartic acid 605 with nine amino acids, adding two potential serine phosphorylation sites in the vicinity of the known serine autophosphorylation site (Ser-608). Northern (RNA) analyses reveal a wide tissue distribution of p85alpha, whereas p85/AS53 is dominant in skeletal muscle and brain, and the insert isoforms are restricted to cardiac muscle and skeletal muscle. Western blot (immunoblot) analyses using an anti-p85 polyclonal antibody and a specific anti-p85/AS53 antibody confirmed the tissue distribution of p85/AS53 protein and indicate a approximately 7-fold higher expression of p85/AS53 protein than of p85 in skeletal muscle. Both p85 and p85/AS53 bind to p110 in coprecipitation experiments, but p85alpha itself appears to have preferential binding to insulin receptor substrate 1 following insulin stimulation. These data indicate that the gene for the p85alpha regulatory subunit of PI 3-kinase can undergo tissue-specific alternative splicing. Two novel splice variants of the regulatory subunit of PI 3-kinase are present in skeletal muscle, cardiac muscle, and brain; these variants may have important functional differences in activity and may play a role in tissue-specific signals such as insulin-stimulated glucose transport or control of neurotransmitter secretion or action.     

Effects of gamma irradiation on the plasma membrane of suspension-cultured apple cells. Rapid irreversible inhibition of H+-ATPase activity

The effects of ionizing radiation, used in post-harvest treatment of fruit and vegetables. were investigated on cultured apple cells ( Pyrus malus L. cv. Royal Red) on a short-term period. Irradiation (2 kGy) induced an increase of passive ion effluxes from cells and a decrease of cell capacity to regulate external pH. These alterations are likely due to effects on plasma membrane structure and function and were further investigated by studying the effects of irradiation on plasma membrane H⁺-ATPase activity. Plasma membrane-enriched vesicles were prepared and the H⁺-ATPase activity was characterized. Irradiation of the vesicles induced a dose dependent inhibition of H⁺-ATPase activity. The loss of enzyme activity was immediate, even at low doses (0.5 kGy), and was not reversed by the addition of 2m M dithiothreitol. This inhibition may be the result of an irreversible oxidation of enzyme sulfhydryl moieties and/or the result of changes induced within the lipid bilayer affecting the membrane-enzyme interactions. Further analysis of the H⁺-ATPase activity was carried out on vesicles obtained from irradiated cells confirming the previous results. In vivo recovery of activity was not observed within 5 h following the treatment, thus explaining the decrease of cell capacity to regulate external pH.
This rapid irreversible inhibition of the plasma membrane H⁺-ATPase must be considered as one of the most important primary biochemical events occurring in irradiated plant material.     

Purification and Characterization of the Voltage-Dependent Anion-Selective Channel Protein from Wheat Mitochondrial Membranes

An approximately 29-kD protein was purified from the membrane fraction of wheat (Triticum aestivum cv Dganit) mitochondria by the utilization of standard liquid chromatography techniques. The protein, designated MmP29 for mitochondrial membrane protein having a molecular mass of approximately 29 kD, exhibited cationic properties in a buffering solution, adjusted to pH 7.5. This positive charge enabled its passage through a diethylaminoethyl column, without interaction with the positively charged matrix. Subsequently, this protein was separated from the remaining polypeptides by a preferential elution from a hydroxylapatite/celite mixed column. Reconstituted liposomes containing this protein were characterized as being permeable to 8-amino-naphthalene 1,3,6-trisulfonic acid disodium salt (Mr 445) but non-permeable to dextran fluorescein (Mr 40,000). Additionally, MmP29 was inserted into planar phospholipid membranes, and anion-selective, voltage-dependent channels were demonstrated. All of the MmP29 properties mentioned highly resemble voltagedependent, anion-selective channel (VDAC) proteins, suggesting that MmP29 is the mitochondrial outer membrane VDAC protein of wheat.     

Characterization of Xanthomonas campestris Pathovars by rRNA Gene Restriction Patterns

Genomic DNA of 191 strains of the family Pseudomonadaceae, including 187 strains of the genus Xanthomonas, was cleaved by EcoRI endonuclease. After hybridization of Southern transfer blots with 2-acetylamino-fluorene-labelled Escherichia coli 16+23S rRNA probe, 27 different patterns were obtained. The strains are clearly distinguishable at the genus, species, and pathovar levels. The variability of the rRNA gene restriction patterns was determined for four pathovars of Xanthomonas campestris species. The 16 strains of X. campestris pv. begoniae analyzed gave only one pattern. The variability of rRNA gene restriction patterns of X. campestris pv. manihotis strains could be related to ecotypes. In contrast, the variability of patterns observed for X. campestris pv. malvacearum was not correlated with pathogenicity or with the geographical origins of the strains. The highest degree of variability of DNA fingerprints was observed within X. campestris pv. dieffenbachiae, which is pathogenic to several hosts of the Araceae family. In this case, variability was related to both host plant and pathogenicity.     

Identification of a developmentally regulated sialidase inEimeria tenella that is immunologically related to theTrypanosoma cruzi enzyme

Sporozoites and merozoites of three species ofEimeria, E. tenella, E. maxima, andE. necatrix, that cause diarrhea in chickens worldwide, were examined for their expression of sialidase (SA) activity. The enzyme was found in three species, and the activity of merozoites was 10–20 times higher than that of sporozoites. The enzyme was resistant to degradation by proteases that are normally present in the intestine, a site inhabited by theEimeria parasites, and it was relatively resistant to heat, with optimum activity being at 40°C, which is within the range of temperature in the chicken intestine (40–43°C).E. tenella SA was immuniprecipitated by monoclonal and polyclonal antibodies raised against theTrypanosoma cruzi SA (TCSA), and enzyme activity was neutralized by these antibodies.E. tenella SA was identified by immunoblots as a doublet of molecular weight 190 000 and 180 000 using, as a probe, anti-TCSA antibodies and antibodies against a synthetic peptide (TR) derived from the long tandem repeat domain of TCSA. Binding of the monoclonal and polyclonal antibodies toE. tenella was completely blocked by TR, but not by an irrelevant peptide (BR). Therefore,E. tenella expresses a developmentally regulated SA that is structurally related to theT. cruzi counterpart. Because of the high SA activity in merozoites, and by analogy with other SA-producing microbes that inhabit mucin-rich epithelia, we suggest that theEimeria SA plays a role in desialylating intestinal mucins to reduce viscosity of the local environment and thereby facilitate parasite migration. The enzyme could also play a role in host cell-parasite interaction.Abbreviations SA sialidase (neuraminidase) - Neu5Ac N-acetylneuraminic acid - 4-MU-Neu5Ac 2-(4-methylumbelliferyl)--N-acetyl-d-neuraminic acid - BSA bovine serum albumin - PBS phosphate buffered saline - PMSF phenylmethylsulfonyl fluoride - PNA peanut agglutinin - Ab antibody - TCN-2 monoclonal antibody toT. cruzi sialidase, anti-Ars, monoclonal antibody top-azophenylarsonate - TCSA Trypanosoma cruzi sialidase