Protein and Peptide Letters - Volume 9, Issue 6, 2002

The results in this study show that the rhodamine fluorophore can be specifically conjugated to Angiotensin II at Lys3 residue (substituted for a Val) without altering the biological activity of the parent compound. The conjugated peptide was characterized using HPLC, mass spectrometry, and N-terminal sequencing. The rhodamine-Angiotensin II binds effectively to AT1 receptor and gets internalized in clathrin coated vesicles by endocytosis. These results clearly suggest the usefulness of fluorophoreconjugated peptides in studies such as, ligand-receptor binding, and ligand-receptor complex internalization, for drug delivery using cell receptors and as an alternative to peptide hormone radioimmunoassays.

HP (2-20) (AKKVFKRLEKLFSKIQNDK) is the antibacterial sequence derived from N-terminus of Helicobacter pylori Ribosomal Protein L1 (RPL1). It has a broad-spectrum microbicidal activity in vitro that is thought to be related to the membrane-disruptive properties of the peptide. Based on the putative membrane-targeted mode of action, we postulated that HP (2-20) might be possessed virus-cell fusion inhibitory activity. To develop the novel virus-cell fusion inhibitory peptides, several analogues with amino acid substitution were designed to increase or decrease only net hydrophobic region. In particular, substitution of Gln and Asp for hydrophobic amino acid, Trp at position 17 and 19 of HP (2-20) (Anal 3) caused a dramatic increase in virus-cell fusion inhibitory activity without hemolytic effect.

To investigate the effect of net positive charge, α-helicity and hydrophobicity of the peptides on antibacterial activity, we designed three kinds of cecropin A (CA) derivatives. Compared to CA, F3 with the highest net positive charge exhibited similar or slightly weaker antibacterial activity (MIC: 3.13∼6.25 μM). F1 showed lower antibacterial activity than that of F3, even though it has higher hydrophobicity and α-helicity than F3. This result indicates that the net positive charge of cecropin A-like peptides appears to be a more important factor in antibacterial activity than α-helicity and hydrophobicity. The two peptides F1 and F2 possessed almost similar antibacterial activity, but F2 showed very lower activity in the membrane disruption than F1, suggesting other factors are involved in the antibacterial activity of the peptides as well as peptide-lipid interaction.

A model for the cytoplasmic domain of the M2 proton channel of influenza A virus was formulated based primarily on the cytoplasmic domain of the Vpu protein of HIV-1 using sequence similarity and structure prediction techniques. The model consists of a pair of antiparallel helices followed by a strand parallel to the first helix. Structural analogies with other proteins contribute support for features of the model and suggest ways in which the M2 cytoplasmic domain can interact with other viral and cellular factors.

Treatment of human serum with ammonium sulfate fraction (0-50%) of Alocasia macrorhiza tuber extract resulted in precipitation at neutral pH. The precipitate was dissolved at pH 10.5 and chromatographed on Sephadex G-100 column. Two protein peaks were resolved. While the first peak represented α2-macroglobulin and haptoglobin, the second peak accounted for specific Alocasia protein. Incidentally the Alocasia protein was shown to be responsible for selective and specific precipitation of α2-macroglobulin and haptoglobin from serum. Thus the plant protein in its pure form or in crude stage could be used for the rapid isolation of two of the prominent a2-globulins.

Caspases, Asp-specific cysteine protease, cleave proteins upon apoptosis. To identify and characterize new caspase substrate in the nucleus, the proteome of the rat liver extracts was analyzed after the treatment with caspases. One of the identified proteins was KSRP / FBP2 that is preferentially cleaved by caspase-3 and -7 at two sites after Asp102 and Asp183. The second site was cleaved only in the protein produced in cells, but not in in vitro translated protein. These results indicate that more than the primary sequence may be important for the recognition by caspases.

In the development of cell-hybrid biomaterials, the functional activity of cells depends on the selective binding of cells to artificial ligands on the biomaterials. The extracellular matrix (ECM) is the most important ligand for cell activity. ECM is known to contain collagen, one of whose constituents is gelatin. Although natural gelatin has good cell attachment properties, the melting point of gelatin hydrogel is lower than body temperature. Thus, non-chemically cross-linked gelatin hydrogel is not a biomaterial that is used for prostheses. In the present study, we report the preparation of acyl-gelatin hydrogels with high melting point (>37°C) and high affinity for hydrophobic surfaces for easy handling for transportation and adhesion activities on the hydrophobic surfaces. In addition, the doubling time of endothelial cells on the coated cell culture plate was faster than that of natural gelatin owing to the higher adhesion activity of acyl-gelatin. The results clearly demonstrated that the acyl-gelatin acted as an interface that enabled cell adhesion to artificial materials surfaces.

A simple and concomitant esterification method for the synthesis of methyl, ethyl, t-butyl, benzyl, and 9-fluorenylmethyl esters of Fmoc- / Boc- / Z-β-homoamino acids employing Fmoc- / Boc- / Z-α- aminodiazoketones by Wolff rearrangement is described. The method offers good yield with purity.

Temporin A, 18 analogs, and a cecropin A-temporin A hybrid peptide were tested with antibiotic sensitive and resistant bacteria, fungi, human erythrocytes, and in clotting assays.Several peptides were active in these assays, and some analogs (D-TA, W1-TA, and Con-L4,G10) may be useful lead compounds for further antibiotics development.The activity of temporin A was found to be dependent upon several of its structural features, including amino acid composition and sequence, chirality, helicity, and positive charge.

A direct and continuous spectrophotometric method was developed for determining arginine kinase (phosphoarginine synthesis) activity. Protons are produced during the phosphoarginine synthesis course, so adding the complex acid-base indicator to this solution and monitoring the decrease of absorbance of the solution at 575 nm will follow the arginine kinase activity. For this condition, one activity unit of arginine kinase was defined as 1 μmol H+ produced in 1 minute in the enzymatic reaction catalyzed by arginine kinase.

Human augmenter of liver regeneration has been expressed in Escherichia coli, purified and crystallized. The crystals belong to space group C222, with unit-cell parameters a=51.7 Å, b=78.8 Å, c=63.7 Å. Diffraction data were collected to 2.80 Å with a completeness of 99.9% (99.9% for the last shell), a Rsym value of 0.092(0.236) and an I / σ(I) value of 6.2(2.7).