The cytotoxic peptide from honeybee venom, melittin, and a synthetic peptide analogue of it lyse human erythrocytes in a biphasic process. The kinetics of the lysis in 0.30 M sucrose, 0.01 M sodium phosphate, pH 7.30 at 4 degrees C were investigated. Our results show that melittin rapidly binds to the outer surface of the erythrocyte membrane, and the surface-bound monomers produce transient openings through which approximately 40 hemoglobin molecules can escape. Concomitantly, the melittin loses its ability to effect the process, presumably by translocation through the bilayer. The half-life for this process is 1.2 min. In a much slower process, dimers of this internalized melittin again produce transient membrane openings in a steady state. On a molar basis, the synthetic peptide analogue produces a fast process comparable to that caused by melittin, but is more efficient in the slow phase. Escape of hemoglobin and of carbonic anhydrase through the openings is diffusion controlled. These results suggest that the functional units necessary for the activity of melittin-like cytotoxic peptides are a 20 amino acid amphiphilic alpha-helix with a hydrophobic:hydrophilic ratio greater than 1 and a short segment with a high concentration of positive charges.
COMPOSITIONAL ANALYSIS The basic compositional data used in our analysis were taken from a recent critical compilation by Scanu and Kruski (6). The reported data are based on dry weight and thus they do not take into consideration the possible presence of water in the lipoprotein particle. In our analysis we also assumed the absence of significant amounts of structural water inside lipoproteins. Minor components, such as free fatty acid, carbohydrates, vitamins, and unidentified compounds, were ignored because, at best, they amount to not more than 5% of the total weight (6) and are likely not to play an essential structural role. For very low density lipoprotein (VLDL) and chylomicrons Scanu and Kruski presented only average compositional data. VLDL and chylomicrons are, in fact, particles of rather broad size distribution, and several authors reported compositional data on their subfractions (2, 7). These additional data on subfractions were also used wherever appropriate. Table 1 summarizes the weight percentage compositions of the major constituents of all lipoprotein classes.From the molecular weight and the weight percentage composition, assuming no structural water, one can calculate the number of molecules of each component in a lipoprotein particle ( Table 2). The following molecular weights were used for the calculations: triglycerides, 850; phospholipid, 775; cholesterol ester, 650; and cholesterol, 387. For the protein content it was not useful to calculate the number of protein molecules per particle because the various lipoprotein classes contain a variety of apoproteins of different molecular weights. For comparison it was more suitable to calculate the number of amino acid residues per particle, assuming an average residue weight of 100. Assuming that all lipoproteins are spherical particles, one can calculate from the molecular weight and the density of the particle the radius of an equivalent sphere r in units of A, also shown in Table 2. Since for the average VLDL and chylomicrons no reliable molecular 'weight data were available, we calculated the molecular weight from the experimentally determined average particle radii, 200 A and 600 A, respectively. Triglyceride and cholesterol ester compositionThe major components of lipoproteins can be divided into nonpolar lipids (triglycerides and cholesterol esters) and polar constituents (phospholipids, cholesterol, and protein). If hydrophobic interactions play an important part in organizing the components, then one would expect the existence of a hydrophobic phase, as it has already been suggested for VLDL and 837
A vector projection method is proposed to predict the cleavability of oligopeptides by extended-specificity site proteases. For an enzyme with eight specificity subsites the substrate octapeptide can be uniquely expressed as a vector in an 8-dimensional space, whose eight bases correspond to the amino acids at the eight subsites, P4, P3, P2, P1, P1', P2', P3', and P4', respectively. The component of such a characteristic vector on each of the eight bases is defined as the frequency of an amino acid occurring at a given site. These frequencies were derived from a set of octapeptides known to be cleaved by HIV protease. The cleavability of an octapeptide can then be estimated from the projection of its characteristic vector on an idealized, optimally cleavable vector. The high ratio of correct prediction vs. total prediction for the data in both the training and the testing sets indicates that the new method is self-consistent and efficient. It provides a rapid and accurate algorithm for analyzing the specificity of any multi-subsite enzyme for which there is no coupling between subsites. In particular, it is useful for predicting the cleavability of an oligopeptide by either HIV-1 or HIV-2 protease, and hence offers a supplementary means for finding effective inhibitors of HIV protease as potential drugs against AIDS.
Spectrophotometric methods are used to explore the kinetics of ester hydrolysis by alpha-chymotrypsin.
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