The DEV model1)-4) has been used to identify a functional region as five-amino-acid spatch, X=5. In this report, the size of X is investigated to identify 177-S as the active site of trypsin. The results obtained with X=3, 5 and 7 suggest that X=5 is the best choice for identification of trypsin 177-S. Comparing to linguistics, functional words in a protein have a distinct nature: two adjacent words sometimes overlap partly, from which the new meaning may be born.
Two unique characteristics of functional words in a protein are overlapping and repetition. It is found that these are responsible for the trypsin action around 171-D and 177-S. Synergism may involve these properties and its underlying mechanisms which are “additive and multiplicative mechanisms” are discussed. Research and development of agrochemicals or pharmaceuticals are discussed in relation to these mechanisms.
Trypsin catalysis that was analyzed by the DEV model is discussed from the standpoint of energetics. In trypsin, the nucleophilic activity of 177-S is enhanced by interaction with 40-H and 84-D, and the steric strain that will be caused by many kinds of structural fluctuation is gradually accumulated and stored as the internal energy of trypsin. A small amount of energy exerts the each fluctuation, which arises in a mild physical condition. We call this process a unit process. Such unit process consequently leads to the accumulation of free energy as well as to the activation of enzymatic reaction. The sophisticated nature of bio-catalysis is also discussed.
An anonymous human DNA fragment R16-2, which was isolated as a possible CpG island fragment by the segregation of partly melted molecules method (SPM), was found to contain a putative promoter region of the prostacyclin synthase gene. Based on this data, it is suggested that combined use of the SPM technique and the database match enables a fine characterization of the regulatory region of many genes.
Bromelain inhibitor VI from pineapple stem (BI-VI) is a unique double-chain cysteine proteinase inhibitor composed of an 11-residue light chain and a 41-residue heavy chain linked by disulfide bonds. The positions of six of the ten half cystines in the primary structure of BI-VI resemble those of the six half cystines in bovine pancreatic trypsin inhibitor (BPTI), a serine proteinase inhibitor which apparently shares some properties with BI-VI. These facts suggested that they might resemble in their three-dimensional structures. To clarify this point, the solution structure of BI-VI was elucidated using nuclear magnetic resonance spectroscopy and simulated annealing-based calculations. Thus, the inhibitor was shown to be composed of two distinct domains, each constructed by a three-stranded antiparallel β-sheet. Comparison with BPTI revealed conclusively that BI-VI is distinctly different in three-dimensional structure from BPTI.
Glutathione-independent prostaglandin D synthase is responsible for the biosynthesis of prostaglandin D2, an endogenous sleep-promoting substance, in the central nervous system of various mammals including humans. This enzyme is localized in the choroid plexus, leptomeninges, and oligodendrocytes of the central nervous system and is secreted into the cerebrospinal fluid as β-trace protein, a major constituent of human cerebrospinal fluid. Two monoclonal antibodies against human β-trace (1B7 and 10A3) were prepared by immunization of BALB/c mice with the recombinant protein expressed in Escherichia coli. Western blot analysis with human cerebrospinal fluid revealed that both 1B7 and 10A3 antibodies were immunoreactive toward a single protein at the same position as that of the purified β-trace (Mr=27, 000). A quantitative sandwich enzyme-linked immunosorbent assay was constructed with these two monoclonal antibodies. The assay system showed a linearity in the range from 0.06 to 4.00ng β-trace/well (100μl). The β-trace concentration was determined by the immunoassay to be 11.30±2.54μg/ml in human cerebrospinal fluid, 1.25±0.37μg/ml in the urine, and 0.27±0.01μg/ml in the serum.