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Biomedical subjects

F J Stevens

Publications and source records attributed to F J Stevens.

83 records · Page 5Linked to original sources
PubMed1981

Effects of self-association of ornithine aminotransferase on its physicochemical characteristics.

Previous work in this laboratory [e.g., Peraino, C., Bunville, L. G., & Tahmisian, T. N. (1969) J. Biol. Chem. 244, 2241--2249, and Morris, J. E., Peraino, C., & Strayer, D. (1974) Proc. Soc. Exp. Biol. Med. 147, 707--709] has shown that the molecular weight of ornithine aminotransferase (OAT) is concentration dependent. In the present study this property of OAT was further characterized by using sedimentation equilibrium centrifugation to determine the molecular weight of OAT in a range of enzyme concentrations. It was shown that OAT aggregates in a two-stage process as its concentration increases. The first stage involves the association of enzymatically active monomers into trimers, with association of the trimers into higher order aggregates occurring in the second stage. Decreasing the pH or raising the ionic strength enhances aggregation while raising the pH inhibits aggregation; however, the two-stage nature of the aggregation process was not affected by changes in pH and ionic strength. Kinetic analyses of purified enzyme showed that aggregation results in an increase in the kM for both substrates with the Vmax remaining constant, indicating that aggregation of monomers sterically hinders substrate binding. Increased Km values were also obtained for OAT sequestered in mitochondria from rats fed a high-protein diet to increase mitochondrial OAT levels. The higher Km values suggest that the elevation of OAT in vivo is accompanied by aggregation of the enzyme within the mitochondrion. We propose that the aggregation-dependent increase of Km in vivo has adaptive value in that it spares ornithine for use in the urea cycle.

Animals↗
PubMed1981

Some properties of D-mannose isomerase from Escherichia coli K12.

A second-stage mutant of Escherichia coli K12 designated as strain 806 grew faster on D-lyxose than the mutant strain 805 previously described. Both mutants produced constitutively a novel enzyme, D-mannose isomerase, but strain 806 produced twice as much as strain 805. The enzyme could fortuitously convert D-lyxose to D-xylulose, which is a normal intermediate in the D-xylose catabolic pathway. The purified enzyme consisted of four subunits each with a molecular weight of about 40 000. In 0.14 M-Na2SO4, the tetramer dissociated completely into dimers. While the tetramer Km values for D-mannose and D-lyxose were 80 mM and 300 mM, respectively, the dimer Km values for these two sugars were both 300 mM. The amino acid composition of the enzyme was also determined.

Aldose-Ketose Isomerases↗
PubMed1980

Self-association of human immunoglobulin kappa I light chains: role of the third hypervariable region.

Gel electrophoresis and molecular sieve chromatography were used to compare 17 different human kappa I type Bence Jones proteins including 5 for which the amino acid sequence is known. Although electrophoresis in the presence of NaDodSO4 showed uniformity of covalent dimer and monomer molecular weights, Sephadex chromatography under nondissociating conditions showed that monomers eluted with different apparent molecular weights. These differences were attributed to heterogeneity in light chain self-association; dimerization constants of the 17 proteins, calculated from a computer simulation of their behavior upon gel filtration, ranged from less than 10(3) to greater than 10(6) M-1. The variable region, more specifically the third hypervariable region, appears to be responsible for the variation in the dimerization constant. Association properties of light chains of known sequence suggest that the presence of an aromatic or hydrophobic residue at position 96 enhances dimer formation whereas a charged residue at that position results in light chains remaining stable monomers. The location of hypervariable residue 96 within the amino-terminal portion of the joining segment of the variable region suggests that the joining region may account for the variability of self-association of light chains and, moreover, that it has a function in determining the selective association of immunoglobulin polypeptide chains.

Amino Acid Sequence↗
PubMed1977

Demonstration of two 3,3'-diaminobenzidine oxidation reactions associated with photosynthetic membranes in anaerobic light-grown Rhodospirillum rubrum.

Photosynthetic membranes of anaerobic light-grown Rhodospirillum rubrum oxidized 3,3'-diaminobenzidine. When glutaraldehyde-treated cells were exposed to 3,3'-diaminobenzidine in the light aerobically, the oxidation appeared to occur by two systems. One reaction was stimulated by white light and the second required molecular oxygen. The O2-dependent activity was inhibited by KCN.

3,3'-Diaminobenzidine↗
PubMed1976

Growth on D-lyxose of a mutant strain of Escherichia coli K12 using a novel isomerase and enzymes related to D-xylase metabolism.

Escherichia coli K12 cannot grow on D-lyxose, but a mutant was isolated which can utilize D-lyxose as sole source of carbon and energy for growth. D-Lyxose is transported into the bacteria by the D-xylose permease. The mutant constitutively synthesizes a new isomerase which is not inducible in the parent strain under any of the conditions tested. This enzyme, whose native substrate appears to be D-mannose, fortuitously converts D-lyxose into D-xylulose. Its structural gene is located at around 85 min on the E. coli genetic map, away from other known isomerase genes. D-Xylulose is subsequently catabolized by the enzymes of the normal D-xylose metabolic pathway. D-Mannose isomerase was partially purified and some of its properties were examined.

Carbohydrate Epimerases↗
PubMedDate not supplied

Efficient recognition of protein fold at low sequence identity by conservative application of Psi-BLAST: application.

Based on a study involving structural comparisons of proteins sharing 25% or less sequence identity, three rounds of Psi-BLAST appear capable of identifying remote evolutionary homologs with greater than 95% confidence provided that more than 50% of the query sequence can be aligned with the target sequence. Since it seems that more than 80% of all homologous protein pairs may be characterized by a lack of significant sequence similarity, the experimental biologist is often confronted with a lack of guidance from conventional homology searches involving pair-wise sequence comparisons. The ability to disregard levels of sequence identity and expect value in Psi-BLAST if at least 50% of the query sequence has been aligned allows for generation of new hypotheses by consideration of matches that are conventionally disregarded. In one example, we suggest a possible evolutionary linkage between the cupredoxin and immunoglobulin fold families. A thermostable hypothetical protein of unknown function may be a circularly permuted homolog to phosphotriesterase, an enzyme capable of detoxifying organophosphate nerve agents. In a third example, the amino acid sequence of another hypothetical protein of unknown function reveals the ATP binding-site, metal binding site, and catalytic sidechain consistent with kinase activity of unknown specificity. This approach significantly expands the utility of existing sequence data to define the primary structure degeneracy of binding sites for substrates, cofactors and other proteins.

Algorithms↗
PubMedDate not supplied

Efficient recognition of protein fold at low sequence identity by conservative application of Psi-BLAST: validation.

A substantial fraction of protein sequences derived from genomic analyses is currently classified as representing 'hypothetical proteins of unknown function'. In part, this reflects the limitations of methods for comparison of sequences with very low identity. We evaluated the effectiveness of a Psi-BLAST search strategy to identify proteins of similar fold at low sequence identity. Psi-BLAST searches for structurally characterized low-sequence-identity matches were carried out on a set of over 300 proteins of known structure. Searches were conducted in NCBI's non-redundant database and were limited to three rounds. Some 614 potential homologs with 25% or lower sequence identity to 166 members of the search set were obtained. Disregarding the expect value, level of sequence identity and span of alignment, correspondence of fold between the target and potential homolog was found in more than 95% of the Psi-BLAST matches. Restrictions on expect value or span of alignment improved the false positive rate at the expense of eliminating many true homologs. Approximately three-quarters of the putative homologs obtained by three rounds of Psi-BLAST revealed no significant sequence similarity to the target protein upon direct sequence comparison by BLAST, and therefore could not be found by a conventional search. Although three rounds of Psi-BLAST identified many more homologs than a standard BLAST search, most homologs were undetected. It appears that more than 80% of all homologs to a target protein may be characterized by a lack of significant sequence similarity. We suggest that conservative use of Psi-BLAST has the potential to propose experimentally testable functions for the majority of proteins currently annotated as 'hypothetical proteins of unknown function'.

Algorithms↗
PubMedDate not supplied

Serologic crossreactions among primate immunoglobulins.

We have generated and characterized 50 murine monoclonal antibodies (mAb) specific for baboon IgG. We examined crossreactivity of these mAb to baboon IgM and immunoglobulin (Ig) of various other primates including human, chimpanzee, rhesus monkey, cynomolgus monkey, and African green monkey. Those mAB that crossreacted with human IgG were further examined using myeloma proteins for specificity to human Ig subclasses. One mAB crossreacted with all four human IgG subclasses and with human IgM. We further analyzed this reactivity utilizing Bence Jones proteins representative of various light (L) chain germline gene family products. This mAB reacted with Bence Jones proteins indicating the recognition of a kappa (k) L chain specificity associated with the kappa I, kappa III, and kappa IV subgroups, but not with kappa II. Based on the differences between kappa II germ line gene encoded L chains and the other kappa L chain subgroups, we ascribe this reactivity to six amino acids that define a discontinuous epitope.

Animals↗
PubMedDate not supplied

Fractionation of macromolecules in an alternating transverse electric field: simulation of the method.

An electric field of alternating polarity applied in a direction transverse to the direction of solute transport is used as the basis of a method for the separation of biological macromolecules. The method derives directly from the ability of an electric field to induce movement of a charged macromolecule and from the physics of laminar fluid flow; no adsorptive immobile phase component is involved. The method is simulated by computer for the case of solute molecules in a solvent flowing through a narrow chamber of rectangular cross section. A voltage differential of periodically reversed polarity generates an electric field orthogonal to the direction of solvent flow. Solute molecules repetitively traverse the solvent channel at rates determined by their electrophoretic mobility. During the transit across the channel, solute molecules are transported in the direction of solvent flow; at the channel wall, solvent velocity is negligible and solute transport is limited to that provided by transient diffusion into a mobile solvent zone. Molecules of different intrinsic electrophoretic mobility are separated. The computer model was used to illustrate the process and to demonstrate the 'tunability' of the method as a function of the oscillation frequency and voltage wave form. Because of this tunability, a single instrument can function as the equivalent of several different chromatographic systems. Because fractionation is effected by direct physicochemical phenomena rather than via interaction with chromatographic sites, variations in fractionation results arising from formation of polymers for gel electrophoresis, packing of chromatography columns, or deterioration of columns with use are avoided. This method may be of particular use for the purification of nucleic acid fragments and for the analysis of protein: nucleic acid interactions.

Chemical Fractionation↗
PubMedDate not supplied

[Evaluation of intramolecular interaction between complementary domains, connected with a flexible chain].

A method for the evaluation of the effective binding parameters for the interaction between two complementary domains connected with a flexible chain is suggested. The calculations are based on the assumption that the chain is absolutely flexible and does not hinder free relative diffusion of the domains in the solution but does not allow the domains to move away from each other further than the length of the chain. Then, if the distance between the connected domains and the affinity of the interaction between disconnected domains are known, the suggested method allows calculation so-called "local concentration" of the domains relative to each other. On this basis, it is possible to estimate the upper limit to the fractional content of domains in complex, which, when constrained by the linking polypeptide chain, may be much higher than implied by the absolute concentrations of the domains.

Macromolecular Substances↗