Ring-Toss: Capping highly exposed tyrosyl or tryptophyl residues in proteins with β-cyclodextrin

We have used UV difference spectroscopy and fluorescence spectroscopy to study the perturbation by β-cyclodextrin of tyrosyl or tryptophyl residues located at each of the 10 variable consensus contact positions in the third domain of turkey ovomucoid. The goal was to monitor the accessibility of the side chain rings of these residues when located at these positions. The results indicated that the tyrosyl or tryptophyl rings are most highly exposed when located in the P₁ position followed by the P₄ position. It was possible to determine the association constants for β-cyclodextrin binding at these positions. When located at the P₂, P₅, P₆ and P₃′ positions, the rings of the tyrosyl or tryptophyl residues were exposed but less so than at the P₁ or P₄ positions. By contrast, when located at the P₁′, P₂′, P₁₄^′ and P₁₈^′ positions, the tyrosyl or tryptophyl residues were insufficiently exposed to be perturbed by β-cyclodextrin, although they reacted positively to dimethyl sulfoxide solvent perturbation. These findings indicate that β-cyclodextrin perturbation provides a convenient way to detect highly exposed tyrosyls or tryptophyls in proteins. Furthermore, we evaluated the ability of β-cyclodextrin to inhibit the interaction of turkey ovomucoid third domain variants with different P₁ residues. The results showed that the presence of β-cyclodextrin had little effect on the association constant when the P₁ residue was a glycyl residue, but greatly decreased the association constant when the P₁ residue was a tyrosyl or tryptophyl residue. Thus, β-cyclodextrin may be used to selectively modulate the interaction between proteinase inhibitors and their cognate enzymes.     

Production of acid proteinases by Humicola lutea mycelium immobilized in polyurethane sponge.

Humicola lutea 120-5 spores were entrapped in polyurethane sponge cubes and were cultivated inside the carrier to form an immobilized mycelium further used for production of acid proteinases in batch mode. A carrier—spore suspension ratio of 10:0.5 (wt) should be used to obtain optimal results. The polyurethane sponge-immobilized mycelium could be applied repeatedly, the enzyme activity secreted during the first 10 cycles being about the same as that produced by free cells. The advantages of immobilizing fungal cells by germinating conidia entrapped inside the supporting material are discussed.     

Evidence for a recent mutation giving rise to a truncated copy of a cysteine proteinase gene in Leishmania pifanoi

We have previously identified and characterized two amastigote-specific cysteine proteinases of Leishmania pifanoi. The slightly different isoforms of the more abundant proteinase are coded by a gene family of approximately 20 gene copies, that contain a C-terminal extension characteristic of cysteine proteinases of trypanosomatids. In this gene family, we have detected a copy that codes for a truncated form of this proteinase, lacking the C-terminal extension. Interestingly, when the deletion of a nucleotide that creates a stop codon causing this truncation is disregarded, the translated sequence gives rise to a divergent C-terminal extension that has many conserved amino acids when compared to Leishmania and Trypanosome, suggesting that a recent mutation led to the truncation.     

The flocculation of wine yeasts: biochemical and morphological characteristics in Kloeckera apiculata

The floc-forming ability of flocculent strains of Kloeckera apiculata, isolated from musts, was tested for susceptibility to proteinase and sugar treatments. Three different flocculation phenotypes were discriminated by protease digestion, whereas the inhibition of flocculation by sugars distinguished two definite patterns: one mechanism of flocculation involved a galactose-specific protein and the other a broad-specificity lectin. SEM and TEM observation of the cell surface of two different Kloeckera strains revealed fine fibrils and a diffuse structure at the point of contact in one strain, and thick masses of mucus on the cell wall of the other strain.