Partial molar volumes of the amino acid side-chains of proteins in aqueous solution: some comments on their estimation using partial molar volumes of amino acids and small peptides.

The partial molar volumes of some amino acid side-chains were determined in two recent studies [Makhatadze, G.I., et al. (1990) Biopolymers 30, 1001, and Reading, J.F. & Hedwig, G.R. (1990) J. Chem. Soc. Faraday Trans. 86, 3117] using partial molar volume data, V02, in aqueous solution at 25 degrees C for some peptides of sequence Gly-X-Gly, where X is an amino acid. These side-chain partial molar volumes are critically compared with those obtained using V02 data for amino acids. It is concluded that side-chain partial molar volumes calculated using V02 data for the tripeptides are better estimates of side-chain partial molar volumes in proteins than are those determined using V02 data for amino acids.     

Partial molar volumes of polypeptides and their constituent groups in aqueous solution over a broad temperature range

The partial molar volumes of various compounds that model protein constituent groups, such as tripeptides (Gly-X-Gly, where X = Gly, Ala, Val, Leu, Ile, Pro, Met, His, Ser), homopeptides (Glyn, n = 3,4,5), and simple organic analogues of amino acid side chains (methanol, acetamide, propanamide, acetic acid, propanoic acid, n-butanamine, n-butanamine nitrate, n-propylguanidine nitrate, 4-methylphenol), have been determined in aqueous solution with a vibrational densimeter in the temperature range of 5-85 degrees C. The partial molar volumes of amino acid side chains and the peptide unit were estimated from the data obtained. Assuming additivity of component groups, the partial molar volumes of polypeptide chains of several proteins over a broad temperature range were calculated. The partial molar volume functions of four proteins (myoglobin, cytochrome C, ribonuclease A, lysozyme) were compared with those determined experimentally for the unfolded and native forms of these proteins. It has been shown that the average deviation of the calculated functions from the experimental ones does not exceed 3% over the temperature range studied.     

Partial molar heat capacities of the peptides glycylglycylglycine, glycyl-L-alanylglycine and glycyl-DL-threonylglycine in aqueous solution over the temperature range 50 to 125 degrees C

Partial molar heat capacities of the three tripeptides glycylglycylglycine, glycyl-L-alanylglycine and glycyl-DL-threonylglycine in aqueous solution at the temperatures 50, 75, 100 and 125 degrees C have been determined using differential flow calorimetry. The results have been used to estimate the contributions to the partial molar heat capacities of peptides of the alanyl and threonyl side-chains. These side-chain contributions are compared with those reported in the literature.     

Calculation of the partial specific volumes of proteins in concentrated salt, sugar, and amino acid solutions

A method for calculating the isopotential partial specific volumes of proteins in concentrated salt, sugar, and amino acid solutions has been developed. It is based on the finding that the preferential hydration of the protein in these solutions is relatively independent of the concentration of the additive and is proportional to the specific surface area of the proteins, i.e., to the ratio of the total accessible surface area to molecular weight. Agreement between the calculated and experimental values was satisfactory, indicating the reliability of the proposed method. These calculations show that the isopotential partial specific volume increases greatly with the concentration of the additive, in particular in the case of Na2SO4, (NH4)2SO4 and sucrose, and for smaller proteins.     

Partial molar heat capacities of the side chains of some amino acid residues in aqueous solution. The influence of the neighboring charges

Partial molar heat capacities of the side chains of some amino acid residues (Ala, Val, Leu, Ile, Ser) have been determined over a broad temperature range from calorimetric heat capacity measurements of the corresponding tripeptides and cyclodipeptides. The data obtained are compared with those determined earlier from the heat capacities of analog compounds. It is shown that in amino acids and even tripeptides of the Gly-X-Gly type, the influence of the end charges on the heat capacity of the side chain is rather significant even in buffered solutions of high ionic strength.     

Protein volumes and hydration effects. The calculations of partial specific volumes, neutron scattering matchpoints and 280-nm absorption coefficients for proteins and glycoproteins from amino acid sequences

Amino acid sequences, carbohydrate compositions and residue volumes are used to compare critically calculations of partial specific volumes v, neutron scattering matchpoints and 280-nm absorption coefficients with experimental v values for proteins and glycoproteins. The v values that are obtained from amino acid densitometry underestimate experimental v values by 0.01-0.02 ml/g while the v values from crystallographic volumes overestimate the experimental v values by 0.04-0.05 ml/g. An intermediate consensus volume set of amino-acid-residue volumes is proposed in order to predict experimental v values using sequence information. The method is extended to carbohydrates and glycoproteins. Neutron scattering matchpoints can be calculated from crystallographic residue volumes on the basis of the non-exchange of 10% of the main-chain NH protons. Crystallographic results on protein-bound water are used to account for the experimental values of v and matchpoints. Finally, 280-nm absorption coefficients, A1%, 1 cm 280, of 5-27 are found to be well predicted by the Wetlaufer procedure based on the totals of Trp, Tyr and Cys residues. Average errors are +/- 0.7, and the experimental A(1%,1cm)280 values can be larger than the predicted values by 3%.     

Measurement of amino acid metabolism derived from [1-13C]glucose in the rat brain using13C magnetic resonance spectroscopy

To clarify the unique characteristics of amino acid metabolism derived from glucose in the central nervous system (CNS), we injected [1-¹³C]glucose intraperitoneally to the rat, and extracted the free amino acids from several kinds of tissues and measured the amount of incorporation of¹³C derived from [1-¹³C]glucose into each amino acid using¹³C-magnetic resonance spectroscopy (NMR). In the adult rat brain, the intensities of resonances from¹³C-amino acids were observed in the following order: glutamate, glutamine, aspartate, -aminobutyrate (GABA) and alanine. There seemed no regional difference on this labeling pattern in the brain. However, only in the striatum and thalamus, the intensities of resonances from [2-¹³C]GABA were larger than that from [2,3-¹³C]aspartate. In the other tissues, such as heart, kidney, liver, spleen, muscle, lung and small intestine, the resonances from GABA were not detected and every intensity of resonances from¹³C-amino acids, except¹³C-alanine, was much smaller than those in the brain and spinal cord. In the serum,¹³C-amino acid was not detected at all. When the rats were decapitated, in the brain, the resonances from [1-¹³C]glucose greatly reduced and the intensities of resonances from [3-¹³C]lactate, [3-¹³C]alanine, [2, 3, 4-¹³C]GABA and [2-¹³C]glutamine became larger as compared with those in the case that the rats were sacrificed with microwave. In other tissues, the resonances from [1-¹³C]glucose were clearly detected even after the decapitation. In the glioma induced by nitrosoethylurea in the spinal cord, the large resonances from glutamine and alanine were observed; however, the intensities of resonances from glutamate were considerably reduced and the resonances from GABA and aspartate were not detected. These results show that the pattern of¹³C label incorporation into amino acids is unique in the central nervous tissues and also suggest that the metabolic compartmentalization could exist in the CNS through the metabolic trafficking between neurons and astroglia.Abbreviations NMR nuclear magnetic resonance - GABA -aminobutyrate - GFAP glial fibrillary acidic protein Special issue dedicated to Dr. Bernard W. Agranoff.     

Host growth temperature and a conservative amino acid substitution in the replication protein of pPS10 influence plasmid host range.

pPS10 is a replicon isolated from Pseudomonas syringe pv. savastanoi that can be established at 37 degrees C efficiently in Pseudomonas aeruginosa but very inefficiently in Escherichia coli. The establishment of the wild-type pPS10 replicon in E. coli is favored at low temperatures (30 degrees C or below). RepA protein of pPS10 promotes in vitro plasmid replication in extracts from E. coli, and this replication depends on host proteins DnaA, DnaB, DnaG, and SSB. Mutant plasmids able to efficiently replicate in E. coli at 37 degrees C were obtained. Three of four mutants whose mutations were mapped show a conservative Ala-->Val change in the amino-terminal region of the replication protein RepA. Plasmids carrying this mutation maintain the capacity to replicate in P. aeruginosa and have a fourfold increase in copy number in this host. The mutation does not substantially alter the autoregulation mediated by RepA. These results show that the physiological conditions of the host as well as subtle changes in the plasmid replication protein can modulate the host range of the pPS10 replicon.     

Repeating patterns of amino acid residues in the suequences of some high sulphur proteins from α-keratin

The amino acid sequences of sulphur-rich proteins derived from the matrix substance of wool keratin have been analysed for internal and external homologies, and the nature of the repeating patterns residues has been investigated in the proteins termed B2A and BIIIA3. The Fourier transform method was used to identify preferred positions in the pentapeptide periodicity that exists in these materials and the structural and functional implications of the results are discussed.     

Evolution of amino acid frequencies in proteins over deep time: inferred order of introduction of amino acids into the genetic code

To understand more fully how amino acid composition of proteins has changed over the course of evolution, a method has been developed for estimating the composition of proteins in an ancestral genome. Estimates are based upon the composition of conserved residues in descendant sequences and empirical knowledge of the relative probability of conservation of various amino acids. Simulations are used to model and correct for errors in the estimates. The method was used to infer the amino acid composition of a large protein set in the Last Universal Ancestor (LUA) of all extant species. Relative to the modern protein set, LUA proteins were found to be generally richer in those amino acids that are believed to have been most abundant in the prebiotic environment and poorer in those amino acids that are believed to have been unavailable or scarce. It is proposed that the inferred amino acid composition of proteins in the LUA probably reflects historical events in the establishment of the genetic code.     

Aldocyanoin microspheres: Partial amino acid analysis of the microparticulates formed from simple reactants under various conditions

Summary The work of Kenyon and Nissenbaum on aldocyanoin microspheres was repeated and extended. It was determined that the microspheres contained amino acids and that specific amino acids could be incorporated into the microspheres by adding the requisite aldehyde or ketone precursor to the model mixture. Microsphere formation was found to be dependent on the availability of oxygen. Under anaerobic conditions of synthesis, no micro-spheres formed in the time allotted and the amino acid composition of the macromolecular material was simple. Microparticulate material synthesized by C. Folsome using a quenched spark technique was analyzed and found to contain amino acids that had a qualitative composition similar to both a Miller-Urey discharge and the Kenyon-Nissenbaum microspheres.     

The anomalous effect of some ACTH-fragments missing the amino acid sequence 1-10 on the corticosteroidogenesis in purified isolated rat adrenal cells

The corticosteroidogenicity of ACTH-derived peptides was tested in a purified isolated rat adrenal cell system. The activity was related to the activity of standard ACTH (synthetic hACTH; potency fixed at 100). The peptides ACTH NH2 and ACTH showed steroidogenicity at pharmacological doses (potencies: 0.00067 and 0.00032, respectively). None of the peptides tested inhibited or potentiated the ACTH-induced steroidogenesis at low doses (0.5-50 000 pg). The results suggest the presence of a second center within the ACTH molecule capable of inducing steroidogenesis.     

Proteins of calcified endoskeleton: II partial amino acid sequences of endoskeletal proteins and the characterization of proteinaceous organic matrix of spicules from the alcyonarian, Synularia polydactyla

Calcified organic substances in the skeleton contain a protein-polysaccharide complex taking a key role in the regulation of bio-calcification. However, information concerning the matrix proteins in alcyonarian and their effect on calcification process is still unknown. For this reason, we have studied the organic matrix of endoskeletal spicules from the alcyonarian coral, Synularia polydactyla, to analyze the proteins with their sequences and investigate the functional properties by a molecular approach. The separated spicules from the colony were identified by scanning electron microscope (SEM). The soluble organic matrix comprised 0.04% of spicule weight. By recording decline of pH in the experimental design, the inhibitory effect of the matrix on CaCO3 precipitation was revealed. Prior to electrophoresis, our analysis of proteins extracted from the soluble organic matrix of the spicules revealed an abundance of proteins in molecular weight. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the preparations showed seven bands of proteins with an apparent molecular mass of 109, 83, 70, 63, 41, 30 and 22 kDa. The proteins were electrophoresed on Tricine-SDS-PAGE after electro-elution treatment, and then transferred to polyvinylidene difluoride (PVDF) membranes and their N-termini were sequenced. Two bands of proteins of about 70 and 63 kDa successfully underwent N-terminal amino acid sequencing. For the detection of calcium binding proteins, a Ca2+ overlay analysis was conducted on the extract by 45Ca autoradiography. The 109 and 63 kDa calcium binding proteins were found to be radioactive. Periodic acid schiff staining indicated that 83 and 63 kDa proteins were glycosylated. An assay for carbonic anhydrase, which is thought to play an important role in the process of calcification revealed low level of the activity. These findings suggest that the endoskeletal spicules of alcyonarian corals have protein-rich organic matrices, which might be related to the calcification process.