Studies of ribosomal proteins of yeast species and their hybrids gel electrophoresis and immunochemical cross-reactions

Summary The cytoplasmic ribosomal proteins (r-proteins) of seventeen yeast species of the genera Saccharomyces and Kluyveromyces were analyzed by one-dimensional gradient polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate. The electrophoretic patterns of cytoplasmic r-proteins from different species display extensive differences in both the 40S and the 60S subunit. Relatedness of species suggested by r-protein patterns correlates well with that based on DNA/DNA homology (Bicknell and Douglas 1970). Immunochemical cross-reactions and antibiotic susceptibility tests were also used to compare different species.Analyses of r-proteins from two different interspecific hybrids showed that their ribosomes were hybrid, containing r-proteins from both parents. These findings are discussed in relation to the evolution of yeast species and the regulation of expression of r-proteins in cucaryotes.     

A sensitive and rapid immunochemical method for quantitation of proteins

A sensitive and rapid ELISA for quantitation of seed globulins is described. This method employs conjugation of pigeon pea (Cajanus cajan) globulin antibodies and the enzyme peroxidase together with dextran. Using this conjugate, proteins as low as 0.1 ng were detected. Dextran conjugate has a ten-fold greater efficiency of quantitating pigeon pea globulins than the commercial goat anti-rabbit IgG conjugate, and is three-fold more efficient than pigeon pea globulin IgG peroxidase conjugate. The method can be conveniently adapted for quantitation of other proteins also.     

Overrepresentation of interactions between homologous proteins in interactomes

It is well proved that the probability that a protein interacts with itself is higher than that it interacts with another protein. It has been recently shown that the probability of interaction is also higher for proteins with significant sequence similarity. In this paper we show that proteins sharing identical PFAM domains interact more often than expected by chance in Saccharomyces cerevisiae and Escherichia coli. We also analyze the variety of domain interfaces used by homologous proteins to interact and show that the overrepresentation of interactions between homological proteins is not caused by small number of pairs of identical "sticky domains" shared between interacting proteins.     

Similarity between average distance maps of structurally homologous proteins

A similarity between average distance maps (Kikuchiet al., 1988a)—that is, predicted contact maps of two tertiary structurally homologous proteins—is examined. Comparisons of shapes of average distance maps (we refer to this as ADM) are made by superpositions of ADMs for two homologous proteins. Also, we compare shapes of actual contact maps for the pair of proteins. We search a optimal superposition mode of each pair of maps showing that two proteins are most similar. It is concluded that two ADMs are also similar when actual tertiary structures between two proteins show similarity. A criterion for similarity of maps is also proposed. The possibility of application of this method to detect weak homology between protein structures is discussed.     

ELISA measurement of immunochemical cross-reactions among Lathyrus lectins as an assessment of their phylogenetical relationship

The immunochemical cross-reactions among Lathyrus and other Vicieae lectins have been quantified by using an ELISA (enzyme-linked immunosorbent assay) technique, in order to assess their phylogenetical relationship. The data are consistent with those arising from the comparison of both the amino acid compositions of the lectins and the amino acid sequences of their light and heavy subunits. They confirm that two-chain lectins from the tribe of Vicieae are phylogenetically closely related.     

Immunological cross-reactions between heterologous hemopexins.

1. The degree of immunological identity of the hemopexin of 64 mammals and non-mammals is determined by double diffusion in agar and two radioimmunoassays, employing antisera produced with the human or the rabbit protein. 2. Antigenic determinants are shared by the Eutherian mammals but not by the non-Eutherian mammals and lower animals. 3. The hemopexins of man and apes appear to be identical, whereas those of Old World monkeys lack one antigenic determinant, and New World monkeys lack at least two antigenic determinants.     

A method of detecting conserved and variable segments in the amino acid sequences of homologous proteins

A set of aligned homologous protein sequences is divided into two groups consisting of m and n sequences. Each group contains sequences from the most related organisms. Value of the position dissimilarity of proteins from different groups of m and n sequences is defined as a number of mismatches in comparison of all possible m X n pairs of amino acid residues in the position (each from different group) divided by m X n. Ten position average of dissimilarity values is plotted vs. the first position number. Area of the figure between the profile of dissimilarity values and its mean value line characterizes the overall irregularity of amino acid substitutions along the protein sequences. If the area is greater than the average area for 1000 random profiles by more than two standard deviation units, the profile extrema containing the "surplus" of area are cut off. The cut-off stretches are likely to be variable and constant regions. If necessary, each of stretches may be separately tested and statistically estimated using a standard size sample of artificial protein families. Intergroup comparison of protein sequences reveals high overall irregularity of amino acid substitutions and identifies variable and conservative regions for all considered families of proteins: phospholipases A2, aspartate aminotransferases, alpha-subunits of Na+, K(+)-ATPase, L- and M-subunits of photosynthetic bacteria photoreaction centre, human rhodopsins.     

On the stochastic model for estimation of mutational distance between homologous proteins

Summary A set of simple equations is derived which gives the relationship between the observed amino acid differences per 100 codons and the evolutionary distance per 100 codons using Holmquist's stochastic model of molecular evolution.Contribution No. 910 from the National Institute of Genetics, Mishima, Shizuoka-ken 411 Japan.     

Heat capacity-independent determination of differential free energy of stability between structurally homologous proteins

Under the assumption of equivalent heat capacity values, the differential free energy of stability for a pair of proteins midway between their thermal unfolding transition temperatures is shown to be independent of DeltaC(p) up to its cubic term in DeltaT(m). For model calculations reflecting the nearly 30 degrees C difference in T(m) for the adenylate kinases from the arctic bacterium Bacillus globisporus and the thermophilic bacterium Geobacillus stearothermophilus, the resultant error in estimating DeltaDeltaG by the formula 0.5 [DeltaS(T(m1))(1)+DeltaS(T(m2)) (2)] DeltaT(m) is less than 1%. Combined with the analogous thermal unfolding data for the adenylate kinase from Escherichia coli, these three homologous proteins exhibit T(m) and DeltaS(T(m)) values consistent with differential entropy and enthalpy contributions of equal magnitude. When entropy-enthalpy compensation holds for the differential free energy of stability, the incremental changes in T(m) values are shown to be proportionate to the changes in free energy.     

A theoretical method for distinguishing between soluble and membrane proteins

A method for distinguishing between membrane and soluble proteins in an amino acid sequence was developed, using only two parameters associated with the hydrophobicity: the average hydrophobicity and the power spectral density of period longer than 30 residues. The power spectral density was calculated by a maximum entropy method of Fourier transformation. Membrane proteins could be distinguished from soluble proteins with a distinction rate as high as 97%. This fact strongly suggests that the morphology of proteins, i.e., membrane or soluble forms, is determined thermodynamically through the hydrophobicity of polypeptides.     

Demonstration of immunochemical identity between the synaptic vesicle proteins synaptin and synaptophysin/p38

The synaptic vesicle proteins synaptin and synaptophysin/p38 were shown to be immunochemically identical. Western immunoblot analysis of Triton X-100 extracts from rat brain showed that polyclonal polyspecific anti-synaptin antibodies and monoclonal antibody SY38 against synaptophysin both reacted with a band of 38 kDa. In two-dimensional immunoblots of chromaffin granule membranes from bovine adrenal medulla anti-synaptin and anti-synaptophysin antibodies also recognized the same component. Finally, in a Western immunoblotting experiment SY38 reacted with an immuno-isolated synaptin antigen.     

A duplex approach for immunochemical staining and typing of proteins in Western blots

In this study, we describe a detection system for the indirect detection of vaccinia virus by DNA analysis. The system uses quartz crystal microbalance (QCM) as the detection technique and polymerase chain reaction (PCR) for amplification. Different immobilization strategies for the capture probe on the quartz chip are studied. For the QCM detection of hybridisation, the influence of the structure and length of target DNA is analyzed. For the detection of DNA from an amplification product, an efficient denaturation procedure is developed. On the basis of these investigations, vaccinia virus DNA is detected with only a low number of amplification rounds and a short analysis time. Specificity can be clearly shown. To enhance the signal strength and to have a further proof of specificity, a gold nanoparticle-tagged enhancer sequence can be used.