Biochemistry
-
The primary subsite specificities of human leukocyte elastase, cathepsin G, porcine pancreatic elastase, rat mast cell proteases I and II, bovine chymotrypsin A alpha, and the protease from strain V-8 of Staphylococcus aureus have been mapped with a series of tripeptide thiobenzyl ester substrates of the general formula Boc-Ala-Ala-AA-SBzl, where AA represents one of 13 amino acids. In addition, the effects of a P2 Pro and P4 methoxysuccinyl and succinyl groups were investigated. In an attempt to introduce specificity and/or reactivity into the substrate Boc-Ala-Ala-Leu-SBzl(X), the 4-chloro-, 4-nitro-, and 4-methoxythiobenzyl ester derivatives were studied. ⋯ A very reactive rat mast cell protease substrate, Boc-Ala-Ala-Leu-SBzl(NO2), was also found. The S. aureus V-8 protease was the most specific enzyme tested since it hydrolyzed only Boc-Ala-Ala-Glu-SBzl. Substituents on the thiobenzyl ester moiety of Boc-Ala-Ala-Leu-SBzl resulted in decreased KM values with human leukocyte elastase and rat mast cell protease I when compared to the unsubstituted derivative.(ABSTRACT TRUNCATED AT 250 WORDS)
-
The present study on the fatty acid binding protein, purified from pig heart and studied by three independent techniques (electron spin resonance, circular dichroism, and polyacrylamide gel electrophoresis), suggests that the protein self-aggregates and exists in at least four distinct molecular species. This plurality is demonstrated by the presence of four bands after electrophoretic migration at pH 7.2 and by three transitions of molar ellipticity theta 225 that depend on protein concentration. ⋯ A general equation for the curve is formulated, and the characteristic constants are evaluated by a nonlinear least-squares fit. The experimental results and their interpretation in quantitative terms lead to a theoretical evaluation of the importance of this new property of self-aggregation of the protein on the activity of membrane-bound model enzymes which are fatty acid or acyl coenzyme A dependent.
-
Elastin contains a number of cross-linking amino acid residues such as desmosine and isodesmosine which are primarily hydrophobic in character, but have a positively charged pyridinium ring. These cross-linking residues are formed by the action of lysyl oxidase upon Lys residues in tropoelastin, a precursor of elastin. A series of tetrapeptide 4-nitroanilides which contain Lys and a series of modified lysine residues were synthesized. ⋯ Except for two substrates with P2 hydrophobic residues (Bz and Pic), PP elastase was less reactive toward the substrates containing model desmosine residues than toward MeO-Suc-Ala-Ala-Pro-Val-NA. The data support the hypothesis that HL elastase cleaves elastin selectively ner cross-linking residues. The results also indicate that HL elastase binds tightly to these regions and would be poorly effective toward regions of elastin or tropoelastin which contain Lys residues.
-
We have studied elongation of SV40 DNA F1 by E. coli RNA polymerase looking specifically at the length of the transcript as a function of time. By running the transcription reactions at 18 degrees C with limited enzyme and adding heparin or rifampicin after elongation has started, we have achieved almost exclusive initiation from the SV40 DNA preferred promotor size [Zain, B. S., Weissmann, S. ⋯ In the type of experiments performed here, those pause sites had continuation relaxation times greater than 45 s at 37 degrees C. This implies that regardless of the nature of a pause, p will cause at least some termination at all hesitation sites with a relaxation time greater than 45 s. All the results are discussed in terms of a kinetic model for the termination of elongation.
-
The proteins of Drosophila melanogaster embryonic ribosomes were separated into seven groups (A80 through G80) by stepwise elution from carboxymethylcellulose with lithium chloride at pH 6.5 by procedures previously described [Chooi, W. Y., Sabatini, L. M., MacKlin, M. ⋯ Five proteins had no detectable contamination, and in each of the others the impurities were no greater than 9%. The amino acid composition of the individual purified proteins was determined. The molecular weights of the proteins were estimated by polyacrylamide gel electrophoresis in NaDodSO4.