Trypsin

The interaction of leech-derived tryptase inhibitor (LDTI) with bovine liver capsule tryptase (BLCT) and bovine trypsin has been studied using both thermo-dynamic and kinetic approaches. Several differences were detected: (i) the equilibrium affinity of LDTI for BLCT (Ka = 8.9 105 M–1) is about 600-fold lower than that for bovine trypsin (Ka = 5.1 108 M–1); (ii) LDTI be-haves as a purely non-competitive inhibitor of BLCT, while it is a purely competitive inhibitor of bovine tryp-sin. These functional data are compared with those previously reported for the LDTI binding to human tryptase, where tight inhibition occurs at two of the four active sites of the tetramer (Ka = 7.1 108 M–1). Amino acid sequence alignment of BLCT, human II-tryptase and bovine trypsin allows us to infer some possible structural basis for the observed functional differences.

Due to their unique fluorescent properties, quantum dots present a great potential for biolabelling applications; however, the toxic interactions of quantum dots with biopolymers are little known. The toxic interactions of glutathione-capped CdTe quantum dots with trypsin were studied in this paper using synchronous fluorescence spectroscopy, fluorescence emission spectra, and UV-vis absorption spectra. The interaction between CdTe quantum dots and trypsin resulted in structure changes of trypsin and inhibited trypsin's activity. Fluorescence emission spectra revealed that the quenching mechanism of trypsin by CdTe quantum dots was a static quenching process. The binding constant and the number of binding sites at 288 and 298 K were calculated to be 1.98 × 10(6) L mol(-1) and 1.37, and 6.43 × 10(4) L mol(-1) and 1.09, respectively. Hydrogen bonds and van der Waals' forces played major roles in this process.

Approximately 1000 lactobacillus strains were isolated and screened for the production of antimicrobial activity, using a target panel of spoilage organisms and pathogens. Only eight positive strains were found; two of these were studied in more detail. Lactobacillus salivarius M7 produces the new broad spectrum bacteriocin salivaricin B which inhibits the growth of Listeria monocytogenes, Bacillus cereus, Brochothrix thermosphacta, Enterococcus faecalis and many lactobacilli. A new atypical bacteriocin produced by Lact. acidophilus M46, acidocin B, combines the inhibition of Clostridium sporogenes with a very narrow activity spectrum within the genus Lactobacillus and was selected for further characterization. Acidocin B is sensitive to trypsin, heat-stable (80 degrees C for 20 min) and can be extracted from the culture supernatant fluid with butanol. Native acidocin B occurs as a large molecular weight complex (100 kDa), while with SDS-PAGE the partly purified activity migrates as...

Lactate dehydrogenase (LDH) isozyme 1 (B4), 5 (A4), and “X” (Testis or Sperm type) have been partially purified from mouse tissues.The following studies were carried out on the three isozymes: Km and optimum substrate concentration for pyruvate, α-oxo-butyrate, α-oxo-valerate, lactate, α-OH-butyrate, and α-OH-valerate, inhibition by substrate and product, effect of malate, N-(4-carboxy-2-hydroxyphenyl) maleimide, some citric acid cycle metabolites, urea, trypsin and pH.The mouse testicular LDH isozyme clearly differs from the common lactate dehydrogenases and from the tesficular isozymes from other species. It shows distinct sensitivity to inhibition by substrate or product whether the direct (pyruvate lactate) or reverse reactions are studied. There is no effect of increasing concentrations of pyruvate or lactate on the direct reaction, while a clear inhibition by lactate or pyruvate is demonstrated on the reverse reaction. Citric acid cycle metabolites, specially malate and succinate, inhibit the direct reaction catalyzed by the “X” isozyme.These peculiar characteristics suggest a high degree of specialization for the principal testicular isozyme of lactate dehydrogenase.

M. ZAMFIR, R. CALLEWAERT, P.C. CORNEA, L. SAVU, I. VATAFU and L. DE VUYST.1999.Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6·5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram-negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell-free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat-stable (30 min at 121 °C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N-terminal end was blocked.

Two fractions showing proteolytic enzymes have been obtained from the latex of Synadenium grantii Hook, ‘f’, using gel-filtration and anion-exchange chromatographic techniques. Both these proteases have the same molecular mass of 76±2 kDa each. They exhibit maximal activity at pH 7.0 and at a temperature of 60 °C. They display stability over a pH range from 5–10 and are also highly thermostable. Irreversible inhibition by PMSF indicates that they are serine proteases. In addition, histidine residues also appear to play an important role in catalysis as evidenced by inhibition with DEPC. They also exhibit similarity with respect to pH and temperature optima, kinetic properties and thermal stability.

Enzyme-cleaved antibodies are used widely for the treatment of envenoming. Such products should comprise only ‘highly pure’ immunoglobulin fragments since Fc or other contaminating protein fragments or their aggregates may lead to side effects. The digestion of ovine antiserum and its purified IgG were investigated using pepsin and trypsin. Trypsin was effective at digesting purified IgG but unsuitable for the direct digestion of serum. In contrast, pepsin was highly effective at digesting all unwanted serum components to low molecular weight (≤13 kDa) fragments while leaving the ~100-kDa F(ab′)2 intact. The optimum pH for pepsin digestion was between 3.25 and 3.50. The effects of salt concentration and pH on the digestion products were investigated by size exclusion chromatography under various conditions, which revealed a pH-dependent aggregation of some of the low molecular weight Fc and non-IgG fragments. These high molecular weight aggregates were not shown by SDS-PAGE. Unwanted low molecular weight fragments could be removed simply by diafiltration with a 30-kDa nominal molecular weight cutoff membrane and piperazine buffer (containing 150 mM NaCl, pH 6), leaving an F(ab′)2 solution contaminated only with some pepsin and a small amount of the aggregated low molecular weight fragments. These highly acidic contaminants were then removed easily using an anion exchange column and the F(ab′)2 produced following a subsequent concentration step was essentially free from pepsin and aggregates with a purity of over 96% and a yield of 19.3 g F(ab′)2/l serum. This novel, high yield method for processing serum to highly pure F(ab′)2 avoids salt precipitation and centrifugation and should be suitable for large-scale production.