Roberto Marco

A convenient micromethod for the assay of primary amines and proteins with fluorescamine. A reexamination of the conditions of reaction.

Fluorescamine, a reagent capable of reacting with a wide variety of nucleophiles, only gives fluorescent products when reacted with primary amines. The results of a reexamination of the principal parameters affecting the reaction are presented with emphasis on practical aspects. In particular, the potential competition of other nucleophiles (secondary amines and thiols) and the effect of the pH on the reaction and on the fluorescence of the product are presented. Polyalcohols, due to their reversible interaction with fluorescamine, affect the velocity without interfering with its extent. The initial hydrolysis product of fluorescamine under mild alkaline conditions has been isolated and characterized. Although unreactive with most amines, it is shown that at pHs below 7 it will react with amines that show abnormal low pH-dependent reactivity with fluorescamine. Histidine is the only amino-acid that under these conditions will react giving a fluorescent product. Among other amino groups containing molecules tested, only polyamines, histamine, and N-amino terminal-free peptides have been found to give this type of reaction. The factors affecting the limit of sensitivity of the reaction are also studied. The very low, but significant, fluorescence of the blanks sets the actual limit of sensitivity of the reaction. A micromethod that reduces the amount of fluorescamine in the assay under conditions in which the reaction is quantitative is described. A similar micromethod can be used with the O-phtalaldehyde reaction. The particular application of fluorescamine in the assay of proteins is compared to other methods. Alkaline and acid hydrolysis are shown to decrease the variability of the reaction shown by different proteins, while increasing the sensitivity. The different practical alternatives in the assay of proteins with fluorescamine and O-phtalaldehyde are discussed.

Plant cell proliferation and growth are altered by microgravity conditions in spaceflight.

Seeds of Arabidopsis thaliana were sent to space and germinated in orbit. Seedlings grew for 4d and were then fixed in-flight with paraformaldehyde. The experiment was replicated on the ground in a Random Positioning Machine, an effective simulator of microgravity. In addition, samples from a different space experiment, processed in a similar way but fixed in glutaraldehyde, including a control flight experiment in a 1g centrifuge, were also used. In all cases, comparisons were performed with ground controls at 1g. Seedlings grown in microgravity were significantly longer than the ground 1g controls. The cortical root meristematic cells were analyzed to investigate the alterations in cell proliferation and cell growth. Proliferation rate was quantified by counting the number of cells per millimeter in the specific cell files, and was found to be higher in microgravity-grown samples than in the control 1g. Cell growth was appraised through the rate of ribosome biogenesis, assessed by morphological and morphometrical parameters of the nucleolus and by the levels of the nucleolar protein nucleolin. All these parameters showed a depletion of the rate of ribosome production in microgravity-grown samples versus samples grown at 1g. The results show that growth in microgravity induces alterations in essential cellular functions. Cell growth and proliferation, which are strictly associated functions under normal ground conditions, appeared divergent after gravity modification; proliferation was enhanced, whereas growth was depleted. We suggest that the cause of these changes could be an alteration in the cell cycle regulation, at the levels of checkpoints regulating cell cycle progression, leading to a shortened G2 period.

Distribution of hexokinase and glucokinase between parenchymal and non-parenchymal cells of rat liver

Phosphorylation is the first catalyzed step in glucose utilization by liver. The discovery in rat liver of a special isoenzyme for this reaction, the insulin dependent, high Km glucokinase by Vifiuela, Salas and Sols [I] , opened the way for a significant increase in the understanding of the regulation of glucose metabolism in liver. Nevertheless, the additional presence of constitutive, low Km hexokinase in rat liver was somewhat disturbing. Sols and coworkers [2,3] advanced the idea that the glucokinase, being an enzyme apparently unique to the liver, would be restricted to the parenchymal cells of this organ, while the common hexokinase found in homogenates could be contributed, at least mainly, by the mesenchymatous tissue. In order to test experimentally this hypothesis we have undertaken the isolation of parenchymal and non-parenchymal fractions from rat liver. The results indicate that indeed these two enzymes have essentially different cellular location in liver, the hepatocytes being virtually free of hexokinase.

Visualization under ultraviolet light enhances 100-fold the sensitivity of peroxidase-stained blots.

As described in this article, visualization and/or photography under uv light of 4-chloro-1-naphthol-developed, peroxidase-marked immunoblots allows an increase in sensitivity of more than 100 times over the apparent staining results observable under normal visible white light. This increase in sensitivity can be obtained with the minimal additional requirement of an uv lamp, with the actual chloronaphthol staining procedure remaining unaltered and thereby allowing the monitoring of specific reactions with much smaller quantities of antigen or antibodies. Substantial shortening of the procedure is another advantage, making it possible to complete in 20 min or even less a procedure usually requiring 3 to 6 h. The phenomenon depends on the uv absorption and the fluorescence quenching properties of the products of the peroxidase reaction. The absorption spectra of the membranes with or without peroxidase products indicate that an intermediate in the peroxidase reaction is responsible for the absorption under uv light. This intermediate accumulates under conditions where the final product absorbing in the visible light has not begun to be produced, thus explaining the large increase in sensitivity. The behaviors of three types of membranes, nitrocellulose, nylon, and Immobilon (PVDF), are compared. Due to its lower uv absorption, PVDF gives by far the best results, followed by nitrocellulose.

Troponin-T is a calcium-binding protein in insect muscle: in vivo phosphorylation, muscle-specific isoforms and developmental profile in Drosophila melanogaster

Two sets of muscle polypeptides showing calcium-binding capacity and intense labelling in vivo with 32P were purified and characterized from Drosophila melanogaster adult extracts. The polypeptides exhibit crossed immunoreactivity and share similar biochemical properties such as those involved in purification. They have been identified as isoforms of troponin-T (TnT) by sequence analysis of a cDNA clone isolated from an embryonic library. The two sets of TnT polypeptides correspond to the fibrillar and non-fibrillar muscle isoforms, respectively. The non-fibrillar muscle isoforms separate into two bands which are differentially expressed during development. Analysis of TnT isoforms in bee thoraces indicates that the expression of the fibrillar muscle isoform correlates with the acquisition of functional flight capability. In vivo labelling experiments reveal that the two TnT sets are readily phosphorylated. The Drosophila TnTs show calcium-binding properties by three different types of assays. Our results suggest that this property could be specific to insect TnTs and may be related to the long, extremely acidic polyglutamic carboxy-terminus present in these polypeptides, which does not occur in non-arthropod TnTs.

Embryogenesis and aging of Drosophila melanogaster flown in the space shuttle

This experiment was made possible by the support of the Comision Nacional de la Investigacion del Espacio (CONIE). Partial support of the CAICYT is also acknowledged.

Oxaloacetate metabolic crossroads in liver. Enzyme compartmentation and regulation of gluconeogenesis

Summary1.The subcellular distribution of the enzymes involved in oxaloacetate metabolism and certain related enzymes in the liver of normal fed and starved rats has been studied. A simplified fractionation procedure was developed in the course of this work. Pyruvate carboxylase and citrate synthase were found to precipitate exclusively associated to mitochondria.2.The precise location of the mitochondrial enzymes was studied in submitochondrial fractions. The existence of two distinct oxaloacetate metabolic crossroads in rat liver has been clearly established. One is located in the mitochondrial matrix and involves the oxaloacetate forming pyruvate carboxylase, the citrate synthase and the mitochondrial malate dehydrogenase and glutamic oxaloacetic transaminase. The second one is located in the cytosol and involves the phosphoenolpyruvate carboxykinase and the cytosolic malate dehydrogenase and glutamic oxaloacetic transaminase.3.The results of similar fractionation studies applied to rabbit liver indicate that in this animal phosphoenolpyruvate carboxykinase is located in the mitochondrial matrix. The other enzymes tested showed the same localization as in the rat. 4. The search in rat liver for a cytosolic activity from 3C to 4C gluconeogenic precursors has been negative. This result stresses the importance of the regulatory problems associated with the exit of oxaloacetate out of the mitochondria in gluconeogenesis. 5. The adaptive behaviour of the enzymes of the mitochondrial and cytoplasmic oxaloacetate crossroads has been studied in rat liver. Only phosphoenolpyruvate carboxykinase shows a marked increase in specific activity when the animals are fasted. 6. The kinetic properties of rat liver citrate synthase have been studied. NADPH inhibition is competitive against oxaloacetate while ATP inhibition is competitive against acetyl-CoA. 7. The Michaelis constants of the mitochondrial malate dehydrogenase and the kinetic constants of the mitochondrial glutamic oxaloacetic transaminase have been measured. 8. A model of the possible regulation of the metabolic flow at the oxaloacetate crossroads in gluconeogenesis in rat liver is proposed.

The stability of the yolk granules of Artemia. An improved method for their isolation and study

The yolk granules of Artemia behave as unstable structures during isolation, especially after hatching. A stringent dechorionization of the cysts is required for an easy homogenization and good extraction of the yolk granules. The use of Ficoll and the avoidance of high dilutions in the homogenization process allows the isolation in an intact state of a pure yolk granules fraction without nuclear contamination. Previously described methods for the isolation of yolk granules lead to low recoveries of these structures in preparations with nuclear contamination. Lipovitellin, the major yolk protein, can be used as a biochemical marker to monitor the recovery of yolk granules and the possible yolk contamination of other subcellular fractions. The isolation of nuclei is also improved, their double membrane preserved in an enriched fraction with less yolk contamination than usually obtained with previously described methods. The other major storage structure, the lipoid bodies, can be isolated intact in these conditions, whereas they are also disrupted in common homogenization media.

Allosteric properties of adipose tissue pyruvate kinase

Summary The pyruvate kinase of rat adipose tissue is strongly inhibited by physiological concentrations of alanine. This inhibition is counteracted by FDP + in the μmolar range of concentrations. These effects can be observed independently of the homogenization medium employed and in partially purified preparations of the enzyme. Aging of the preparations tends to produce the loss of the inhibitory and activatory effects, pointing to their allosteric nature.

Diversification and Independent Evolution of Troponin C Genes in Insects

Troponin C (TpnC), the calcium-binding subunit of the troponin regulatory complex in the muscle thin filament, is encoded by multiple genes in insects. To understand how TpnC genes have evolved, we characterized the gene number and structure in a number of insect species. The TpnC gene complement is five genes in Drosophilidae as previously reported for D. melanogaster. Gene structures are almost identical in D. pseudoobscura, D. suboboscura, and D. virilis. Developmental patterns of expression are also conserved in Drosophila subobscura and D. virilis. Similar, but not completely equivalent, TpnC gene repertoires have been identified in the Anopheles gambiae and Apis mellifera genomes. Insect TpnC sequences can be divided into three groups, allowing a systematic classification of newly identified genes. The pattern of expression of the Apis mellifera genes essentially agrees with the pattern in Drosophilidae, providing further functional support to the classification. A model for the evolution of the TpnC genes is proposed including the most likely pathway of insect TpnC diversification. Our results suggest that the rapid increase in number and sequence specialization of the adult Type III isoforms can be correlated with the evolution of the holometabolous mode of development and the acquisition of asynchronous indirect flight muscle function in insects. This evolutionarily specialization has probably been achieved independently in different insect orders.