Biomedical subjects

A I Yuan

Publications and source records attributed to A I Yuan.

7 recordsLinked to original sources

PubMed1992

Structure of alpha 2-macroglobulin-protease complexes. Methylamine competition shows that proteases bridge two disulfide-bonded half-molecules.

alpha 2-Macroglobulin (alpha 2M) forms several different covalent complexes with proteases. These include unusual forms in which more than one of the four identical subunits of alpha 2M are cross-linked by amide bonds to more than one lysyl amino group of the bound protease. The structure of these complexes and the question of how the identical subunits are arranged to form two protease binding sites are matters of current controversy. The 185-kDa subunits are arranged into two disulfide-bonded half-molecules which are, in turn, noncovalently associated. We have provided evidence that, in the major multivalent cross-linked form, proteases can span the two half-molecules, forming a covalently bonded tetramer [Wang, D., Yuan, A. I., & Feinman, R. D. (1984) Biochemistry 23, 2807-2811]. An alternative theory has recently been proposed in which the major high molecular weight form has two bonds to protease that are within half-molecules--a multivalent cross-linked dimer [Sottrup-Jensen, L., Hansen, H. F., Pedersen, H. S., & Kristensen, L. (1990) J. Biol. Chem. 265, 17727-17737]. To resolve this conflict, experiments were carried out to determine the structure of one of the high molecular weight bands (band 3) seen on SDS-PAGE. Band 3 has anomalous migration, corresponding to markers of apparent molecular mass of 550 kDa (between the tetramer and dimer). In the experiments described here, reactions of thrombin with alpha 2M were run in the presence of methylamine, which competes for one of the two thrombin-alpha 2M covalent bonds.(ABSTRACT TRUNCATED AT 250 WORDS)

Disulfides↗

PubMed1990

Effect of methylamine on the reaction of alpha 2-macroglobulin with enzymes.

The kinetics of reaction of alpha 2-macroglobulin (alpha 2M) with thrombin and with trypsin were studied in the presence and absence of methylamine. The rate of enzyme-induced thiol release was found to be the same whether or not amine was present. The result suggests that covalent bond formation and enzyme-catalyzed amine incorporation proceed via a common (enzyme-dependent) rate-determining step. The reaction of lysyl-modified enzymes (which show poor covalent binding with alpha 2M) was similarly unaffected by amine, indicating that enzyme-catalyzed steps were also rate determining for hydrolysis of the thiol ester. The products of the reactions were analyzed by native and denaturing gel electrophoresis. Methylamine did not affect the total binding of enzyme to alpha 2M but did cause a substantial decrease in covalent binding. Surprisingly, not all covalent complexes were affected by the presence of amine: complexes in which enzyme was covalently bound to one half-molecule increased compared to the reaction with no amine; complexes in which two half-molecules are cross-linked by two bonds to a single enzyme were substantially reduced, however. The results are consistent with a mechanism of reaction in which an enzyme-dependent step is rate determining. This step is accompanied by activation of two thiol esters. One of these reacts immediately with the bound enzyme (or may be hydrolyzed if the enzyme amine groups are blocked). The other activated center is capable of reaction with external nucleophiles such as methylamine.

Humans↗

PubMed1985

Kinetics of the reaction of thrombin and alpha 2-macroglobulin.

The kinetics of the reaction of alpha 2-macroglobulin (alpha 2M) with human thrombin were studied by recording the appearance of thiol groups spectrophotometrically and by measuring the distribution of protein species by denaturing non-reducing gel electrophoresis. The goals were to study the relation between the formation of various covalent enzyme-inhibitor complex species and the appearance of free thiol, and from the kinetic analysis, to try to characterize the chemical nature of the protein complexes. The kinetics of thiol-group release were observed to be biphasic, the early phase showing second-order behaviour, results consistent with previous reports in the literature. The observed second-order rate constant for thiol-group release was found to be faster than the second-order rate constant for the disappearance of the band corresponding to native alpha 2M on gel electrophoresis. This may be a reflection of the multiple products formed from the thioester. Alternatively, it is possible that covalent-bond formation is slower than some enzyme-induced change in the thioester centre, and this may be suggestive evidence for a reactive alpha 2M centre that does not contain an intact thioester. The kinetics of covalent-bond formation were found to be consistent with the internal cross-link of several alpha 2M chains by the bound proteinase, providing further evidence that the very-high-Mr species seen on gels may arise from dimers of the alpha 2M molecule held together by covalent bonds to the enzyme.

Electrophoresis, Polyacrylamide Gel↗

PubMed1984

Covalent thrombin-alpha 2-macroglobulin complexes. Evidence for bivalent cross-linking of inhibitor chains by a single enzyme molecule.

Complexes formed between thrombin and alpha 2-macroglobulin (alpha 2M) were studied by polyacrylamide gel electrophoresis. The results provide evidence for the existence of a recently proposed novel enzyme-inhibitor species in which a single thrombin molecule forms two or more covalent bonds to two or more different alpha 2M chains. At least one of several slowly migrating bands (greater than 375K on nonreduced gels) that have previously been observed in the literature but not well characterized can be assigned to the new species. The involvement of the lysyl amino groups of thrombin is shown by the observation that methylation of these groups reduces the higher molecular weight bands. In addition, increasing the thrombin:alpha 2M ratio causes a relative decrease in the higher molecular weight species, suggesting that these complexes arise by intramolecular reactions that are susceptible to competition by solution thrombin. The data provide support for our previous proposal [Wang, D., Yuan, A., & Feinman, R.D. (1983) Ann. N.Y. Acad. Sci. 421, 90-97] that the 260K band seen in reduced gels is composed of two proteolyzed inhibitor subunits linked to one thrombin molecule. This intersubunit link maintains the integrity of the alpha 2M in sodium dodecyl sulfate, accounting for the high molecular weight bands under nonreducing conditions. Comparison with a synthetically cross-linked alpha 2M molecule allows a tentative but not unambiguous assignment of one of the bands to this novel structure.

Cross-Linking Reagents↗

PubMed1983

Structure of alpha 2-macroglobulin-protease complexes.

The analysis of thrombin-alpha 2M reaction mixtures by two-dimensional SDS-PAGE has allowed us to assign several probable molecular species to the mixture of complexes formed. These include structures previously described in, or predicted from, the literature, as well as two types of novel species: Divalent cross-linking of two inhibitor chains by a single enzyme molecule. Very high molecular weight species that are attributed to intermolecular cross-linking of more than one inhibitor molecule. Species containing enzyme monovalently linked to an intact subunit are not supported by our data, but are not excluded and additional study will be required to determine if they exist.

Binding Sites↗

PubMed1982

Two major allelic forms of myosin light chain-1 in strains of normal and dystrophic chickens.

Evidence is presented from electrophoresis and peptide-mapping for the existence of two major allelic forms of myosin light chain-1 in the fast white muscle fibers of domestic chickens. One form predominates in birds of White Leghorn stock, the other in birds of New Hampshire Red stock. The two light chain-1 forms were invariant during development. Variability was not detected in light chains-2 or -3. The distribution of the two forms in two strains homozygous for the am gene for muscular dystrophy--Connecticut dystrophic and line 413--and their controls, White Leghorn and line 412, respectively, while clearly unrelated to avian dystrophy, emphasizes the heterogeneity in background genes of these non-inbred lines and indicates caution in their use in studies of avian dystrophy.

Alleles↗

PubMed1981

Abnormal myosin heavy chain variant associated with avian muscular dystrophy.

Avian muscular dystrophy is characterized by the degeneration of fast white skeletal muscle fibers, with onset during development. Using a one-dimensional peptide mapping technique, we have detected two forms of the myosin heavy chain in the fast white fibers of adult domestic chickens, one form characteristic of birds homozygous for muscular dystrophy, the other of their normal controls. Four dystrophic strains carrying the same gene for muscular dystrophy were examined. No differences were detected in the embryonic heavy chain peptide maps of normal and dystrophic chickens, consistent with the developmental onset of the condition. Differences were also absent from the peptide maps of heavy chains from slow red fibers, which are unaffected in dystrophy. No dystrophy-specific peptide map differences were detected in the three light chains. Analysis of peptide maps of rod and the heavy chain component of subfragment-1 from normal and dystrophic heavy chains indicates the presence of amino acid sequence differences in the two proteins.

Amino Acid Sequence↗