Formation of tightly bound forms of annexins V and VI in rabbit skeletal muscle membranes isolated in the presence of barium ions.

After isolation of rabbit skeletal muscle membranes in the presence of Ba2+ or Ca2+, significant portions of annexin V and VI tightly bind to membranes and become inaccessible for chelating agents. Tightly bound annexin VI is virtually completely solubilized only after treatment with a buffer supplemented both with EGTA and detergent. The portion of tightly bound annexin V cannot be removed even by extraction with buffer containing both EGTA and detergent. In some cases, tightly bound annexin V or VI is detected even in the control (not treated with cations) membranes, thus indicating the possible formation of tight annexin--membrane complexes in situ. The addition of exogenous cations seems to promote only the accumulation of tightly bound annexins within the cell. After temperature-induced phase separation, annexin V and VI bound to the membranes isolated in the presence of Ba2+ or Ca2+ remains mainly in the aqueous phase, similarly to annexins isolated from the control membranes. Neither annexin partitions into the detergent-enriched phase. This indicates the absence of hydrophobicity change in comparison with the standard EGTA-soluble annexins.

Electrophoretic separation of high-molecular hydrophobic proteins: apolipoprotein B and other protein constituents of low-density lipoproteins.

Apolipoprotein B (apoB) and 40 different protein standards were submitted to sodium dodecyl sulfate-polyacrylamide gel electrophoresis under various conditions. The system selected is capable of fractionating well the apoB and its high-molecular-weight species formed after treatment of serum lipoproteins by malonic dialdehyde. The basis for good resolution of protein bands is the use of complex gels combining in one block the areas with constant (T = 3 and 10%) or continuously changing (gradient gel with T = 3-6%) concentrations of acrylamide. The procedure allows one to create the optimal conditions for simultaneous separation of both high- and low-molecular-weight polypeptides. The method was used to determine the relative molecular mass (Mr) of apoB. When apoB was isolated in the presence of proteolytic inhibitors, its Mr was found to be equal to 540,000 +/- 9500 or 500,000 +/- 5700, depending on the linear regression equation applied. The results are in good accordance with values calculated from the amino acid sequence of mature apoB.

Calcium-dependent phospholipid binding proteins associated with the membranes of rabbit skeletal muscle.

By using extraction in the presence of Ca2+ and Triton X-100 and then in the presence of EGTA without detergent, a set of Ca2+-dependent phospholipid binding proteins has been identified in the membranes of transverse tubules (T-tubules) and sarcoplasmic reticulum (SR), isolated from rabbit skeletal muscles. Longitudinal SR, junctional SR and T-tubule membranes yielded about 9, 14 and 3.3 micrograms of EGTA-soluble proteins per 1 mg of membrane protein, respectively. In the presence of 1 mM CaCl2, 68 and 33 kDa proteins of T-tubules and junctional SR as well as 30 kDa protein of T-tubules were shown to bind to liposomes made of 1:1 w/w mixtures of (i) phosphatidylcholine and (ii) phosphatidylserine, phosphatidic acid, or phosphatidyl ethanolamine. In the presence of EGTA, the above-mentioned proteins were mostly found in the supernatants. Binding of the proteins with liposomes consisting of pure phosphatidylcholine was negligible.

Short-chain alkanols and the functional efficiency of the Ca pump in the sarcoplasmic reticulum of rabbit skeletal muscles.

The effect of 10 low molecular mass alkanols on the activity of Ca-ATPase (EC 3.6.1.38), Ca uptake and Ca efflux as well as the functional efficiency of the Ca pump in the fragmented sarcoplasmic reticulum of rabbit skeletal muscles has been studied. Some alkanols, especially when taken at low concentration, have been found to stimulate the activity of the Ca pump and Ca-ATPase, namely tert-butanol, isopropanol and ethanol (from the group of hydrophilic alkanols), and pentanol, isopentanol and hexanol (from the more hydrophobic alkanols). Methanol (from the first group) and isobutanol, butanol and propanol (from the second) do not stimulate the Ca pump compared with the control. The specific effect of different alkanols cannot be explained in terms of a unitary mechanism based on 'fluidity' changes of the membrane. It is assumed that, at low concentrations, certain alkanols (or groups of related alkanols) are able to promote the specific transition of membrane proteins into the active state, whereas at higher concentrations all alkanols provide for the non-functional state of the proteins.

Apolipoprotein B: removal of lipids by sodium cholate and reassociation of a lipid-free apoprotein with dipalmitoyl phosphatidylcholine.

Apolipoprotein B (apoB) of human plasma low-density lipoprotein has been solubilized with sodium cholate added in an amount highly above its critical micellar concentration. During isolation by gel exclusion chromatography on Sepharose CL-4B, the apoB forms mixed micelles of protein and detergent that are free of endogenous lipids. The circular dichroic spectra of the sodium cholate-solubilized apoB indicate significant heterogeneity within the fractions obtained by gel chromatography. The peak position fraction of apoB taken from the column was used for reassociation with dipalmitoyl phosphatidylcholine (DPPC). A soluble apoB-DPPC complex has been prepared by incubation of apoB-sodium cholate and DPPC-sodium cholate solutions at 42 degrees C, followed with detergent removal by extensive dialysis in the presence of a XAD-2 ion-exchange resin. Data from negative-stain electron microscopy suggests the incorporation of solubilized apoB into single-bilayer phospholipid vesicles. Upon reassociation with phospholipid, a shift (to shorter wavelengths) occurs in the intrinsic fluorescence of the apoB, thus indicating a transfer of tryptophan residues to a more hydrophobic environment. Sodium dodecylsulfate-polyacrylamide electrophoresis gives a single band (apparent Mr 370,000) for apoB after solubilization, purification and interaction with the phospholipid.