Biochemical kinetics of skeletal actosubfragment-1 at high subfragment-1 concentrations.

The actomyosin ATPase activity of skeletal myosin subfragment-1 (S-1) is typically studied by keeping the S-1 concentration low and varying the actin concentration. General agreement exists over the kinetic data observed. Another way of studying the ATPase activity is to keep the actin concentration low and vary the S-1 concentration. The picture that has emerged is that the maximal ATPase rate (per micromolar actin), Vamax, is several fold greater than the Vsmax measured at fixed S-1. Likewise, the apparent activation constant Kam is several fold weaker than KATPase. In addition it is found that Kam, henceforth Kam(At), varies with the total actin concentration At, but controversy continues over the actin dependence of Vamax. Of particular interest is the fact that the Lymn-Taylor and refractory state models could not account for the data. Here we have repeated studies on the ATPase activity at fixed actin concentration in an attempt to determine if the current models for the actin activated myosin ATPase activity can account for both the constant actin and constant S-1 data simultaneously, or if these data imply that new kinetic models need be postulated. We conclude that the current kinetic models can account for the data

Physicochemical properties of alkali-treated oat globulin

Oat globulin dispersions (10% w/v) were incubated at an initial pH of 9.8 at 25, 37, and 55°C over a period of 96 h. Turbidity increased at 25 and 37°C with the formation of insoluble aggregates and decreased at 55°C with little precipitation. The free SH content decreased progressively with time, while the surface hydrophobicity was increased at 25°C and decreased at 37°C. Differential scanning calorimetry showed progressive increases in denaturation temperature and decreases in width at half-peak height. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated formation of soluble aggregates at 55°C and degradation of oat globulin polypeptides at 25 and 37°C, possibly due to proteolysis by protease(s) coextracted with the protein. No significant racemization of amino acids was observed in the alkali-treated protein.link_to_subscribed_fulltex

Molecular and Microstructural Studies of Thermal Denaturation and Gelation of β-Lactoglobulins A and B

The thermal properties of β-lactoglobulins (β-lg) A and B at pH 3, 5, 7, and 8.6 were studied by differential scanning calorimetry. Fourier transform infrared spectroscopy was used to monitor changes in the secondary structure of the proteins when heated from 25 to 95 °C. The microstracture of β-lg A and B gels made from 10% (w/v) protein solutions by heating at 90 °C for 30 min was studied by scanning and transmission electron microscopy. β-Lg B had greater thermal stability and required more energy to denature than β-lg A; denaturation of β-lg B was also more cooperative. Infrared spectroscopy showed that β-lg B had a higher proportion of β-sheet than the A variant at pH 3 and 5. At pH 7 and 8.6 the secondary structures of the two variants were similar. At all four pH values, aggregation bands (1682 and ∼1622 cm -1) were observed when the proteins were heated. Electron microscopy showed that the gel matrix of β-lg B at both acid and alkaline pH was made up of larger aggregate structures than β-lg A. The aggregates formed by both variants were large (1-2 μm) and globular at acid pH but much smaller (nanometer range) and amorphous at alkaline pH. This information provides a useful model for studying the relationship between protein structure and function.link_to_subscribed_fulltex

Plasmodium falciparum: Effects of Membrane Modulating Agents on Direct Binding of Rhoptry Proteins to Human Erythrocytes

We studied the effects of membrane modulation on the interaction of Plasmodium falciparum rhoptry proteins of 140/130/110 kDa (Rhop-H) with human and mouse erythrocytes. Cells treated with 2-(2-methoxyethoxy)ethyl-8-(cis-2-n-octylcydopropyl)octanoate, myristoleyl alcohol, and proteins extracted with sublytic concentrations of membrane solubilizing detergents were used in erythrocyte binding assays. Protein binding was evaluated by immunoblotting using Rhop-H- and SERA-specific antisera, 1B9, K15, and 5E3, respectively. Protein binding to liposomes prepared with dipalmitoyl-L-α-phosphatidylcholine (DPPC) or dilauroyl-L-α-phosphatidylcholine (DLPC) was also examined. Our results show that erythrocyte membrane modulation markedly enhanced direct Rhop-H binding to intact human erythrocytes. Binding of SERA to intact human erythrocytes appeared unaffected. Both DPPC and DLPC liposomes had similar Rhop-H and SERA protein binding activities. However, binding to DLPC liposomes was reduced. Rhop-H and SERA extracted with the detergents octanoyl-N-methylglucamide, decanoyl-N-methylglucamide, sodium deoxycholate, and 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulfonate bound directly to intact human erythrocytes, probably by partitioning hydrophobically into the membranes. Sodium carbonate treatment demonstrated a nonintegral association of Rhop-H with the erythrocyte membrane during invasion. Membrane modulation may expose cryptic phospholipid binding sites in the bilayer. © 1995 Academic press, Inc