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T M Minich

Publications and source records attributed to T M Minich.

4 recordsLinked to original sources
PubMed1984

Covalent binding efficiency of the third and fourth complement proteins in relation to pH, nucleophilicity, and availability of hydroxyl groups.

The binding of [3H]glycerol and [3H]putrescine to C3 was studied in a fluid-phase system using trypsin as the C3 convertase. The binding of glycerol showed little variation in the pH range between 6.0 and 10.0. The binding of putrescine (pKa = 9.0) is rather ineffective below pH 7.5 but becomes more efficient as the pH of the reaction mixture increases. These results agree with the contention that the final step of the binding reaction is the transfer of the acyl group of the exposed thio ester of C3 to a nucleophile since the nucleophilicity of hydroxyl groups is rather independent of pH whereas only the unprotonated form of amino groups is nucleophilic. The inefficient reaction of amino groups with the exposed thio ester of C3 is also supported by the study of the inhibitory activity of serine and its two derivatives, N-acetylserine and O-methylserine, to the binding of [3H]glycerol to C3. N-Acetylserine showed an inhibitory activity equivalent to that of serine, whereas O-methylated serine showed only minimal activity. It can be concluded, therefore, that serine reacts with the thio ester of C3 by its hydroxyl group but not by its alpha-amino group. The ability of the alcohol group of various alkanes to inhibit the binding of [3H]glycerol to C3 was also studied. The primary alcohols inhibit the binding reaction with an efficiency that is similar to glycerol, and there are no significant differences in the binding efficiencies of methanol, ethanol, 1-propanol, and 1-butanol.(ABSTRACT TRUNCATED AT 250 WORDS)

Alcohols↗
PubMed1984

Natural release of covalently bound C3b from cell surfaces and the study of this phenomenon in the fluid-phase system.

Covalently bound C3b is released from cell surfaces (EAC1423 and zymosan-C3b) on incubation under physiologic conditions. The release of C3b from cell surfaces occurs by the cleavage of the covalent bond. Sodium dodecyl sulfate (SDS) abolishes the release, thereby indicating the requirement of the native structure of C3b in this process. The phenomenon of release of C3b from cell surfaces has also been observed in the fluid-phase system by using C3b-[3H]glycerol. The kinetics of the release of [3H]glycerol from C3b-[3H]glycerol were studied at 37 degrees C in 0.15 M phosphate buffer, pH 7.4. The first-order rate constant was found to be 0.028 +/- 0.003 hr-1. The release does not take place in either 8 M urea or 6 M guanidine hydrochloride, at pH 7.4. Under alkaline conditions, the rate of release is unaffected in the presence of SDS, indicating that the release in this pH range is not dependent on the native structure of the protein. From the Arrhenius plot in the temperature range 18 to 37 degrees C, an apparent activation energy for the hydrolysis reaction of 21.2 kcal/mol was calculated. The release phenomenon is exclusive for ester-linked complexes, as inferred by the absence of release of [3H]threonine from C3b-[3H]threonine, wherein the linkage is of the amide type. The presence or absence of the C3a portion of the molecule has no effect on the rate of release. The modification of the -SH group of C3i-/C3b-[3H]glycerol alters the rate of hydrolysis of the ester bond between C3i/C3b and [3H]glycerol. Protease inhibitors (PMSF, benzamidine HCl, and DFP) do not alter the rate of release, indicating that the hydrolysis reaction is not due to trace amounts of contaminating proteases. Thus, it appears that some chemical group(s) of C3i/C3b is (are) involved in the intramolecular hydrolysis of the ester bond between C3i/C3b and small molecules. This phenomenon may play an important role in the release of C3b from receptive surfaces once the biologic functions that require covalently bound C3b have been mediated.

Alkylation↗
PubMed1981

Binding reaction between the third human complement protein and small molecules.

The covalent binding reaction of the third complement protein (C3) to receptive surfaces is thought to proceed by the following mechanism. An internal thioester [Tack, B. F., Harrison, R. A., Janatova, J., Thomas, M. L., & Prahl, J. W. (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 5764-5768; Law, S. K., Lichtenberg, N. A., & Levine, R. P. (1980b) Proc. Natl. Acad. Sci. U.S.A. 77, 7194-7198], which is usually hidden within the C3 molecule, is exposed upon proteolytic activation of C3 to C3b* (the hypothetical conformation of C3b which has the capacity to bind to receptive surfaces and small molecules). The exposed thioester is accessible to attack by hydroxyl groups on receptive surfaces. An acyl transfer reaction takes place, leading to the binding of C3b to the receptive surfaces via an ester linkage [Law, S. K., Lichtenberg, N. A., & Levine, R. P. (1979) J. Immunol. 123, 1388-1394]. We have used a fluid-phase system to demonstrate the specific binding of different small molecules to the labile binding site of C3. The small molecules include glycerol, different hexose monomers, sucrose, raffinose, and four amino acids. These molecules bind to C3b with different efficiencies, indicating that there is an order of preference of C3b* for these molecules. In certain cases, the small molecules bind to C3b via ester linkages (e.g., glucose); in others, the bond is an amide linkage (e.g., lysine). We have also studied the concentration dependence of the binding of small molecules to C3b. The binding is consistent with the following reaction scheme: (Formula: see text).

Amino Acids↗