Search

Chicken apocytochrome c gene with correct reading frame was easily cloned through excision by polymerase chain reaction of the intron in the genomic clone of chicken cytochrome c gene, and was successfully overexpressed in Escherichia coli by cloning into expression vector pET-3d under the control of T7 promoter. Expressed protein can amount to as high as 40% of the total protein and mainly presents as inclusion body. Purification of chicken apocytochrome c from the inclusion body and characterization by SDS-PAGE, isoelectric focusing electrophoresis, and amino acid analysis showed that the purified apocytochrome c is identical to that prepared from chicken heart cytochrome c by chemically depletion of heme.

In contrast to horse heart apocytochrome c, the chicken one showed quite different folding propensity as titrated by NaCl at different pH. At pH 2.0, folding behaviour of both apocytochrome c are essentially similar; while at pH higher than 4.0, chicken heart apocytochrome c has much enhanced propensity to fold and aggregate, as was shown by circular dichroism spectra, intrinsic fluorescence and non-denatured polyacrylamide gel electrophoresis. Hydrophobic chromatography demonstrated much higher hydrophobicity of chicken heart apocytochrome c, thus strongly suggested that it is the hydrophobic interaction that stabilize the 'Molten Globule' like, partially-folded structure of chicken heart apocytochrome c at neutral pH.

The effect of Ca2+ on the glucose transporter of human erythrocytes was investigated. The results showed that extracellular Ca2+ had no effect. But, the glucose transport of erythrocytes was markedly inhibited due to the increase in cytoplasmic Ca2+ concentration by addition of ionophore A23187. The Ca2+ inhibition exhibited a dose-dependent manner with an apparent half maximal concentration of 250 microM and could not be recovered by 10 mM EGTA. Unlike Ca2+, Mg2+ did not affect the glucose transporter.

Sarcoplasmic reticulum (SR) vesicles with (1000 folds) or without transmembrane Ca2+ gradient have been prepared. Different fluorescence probes (DPH, TMA-DPH and n-AS), were used to determine the effect of transmembrane Ca2+ gradient on the lipid fluidity both in outer and inner layer of Ca(2+)-ATPase-containing SR vesicles. The results showed that transmembrane Ca2+ gradient could significantly decrease the fluidity of the inner layer of SR membrane, while no obvious change was monitored in the outer layer. This may be deduced that Ca(2+)-ATPase might be modulated mainly by the transmembrane Ca2+ gradient-mediated alteration of physical state of phospholipid in the inner layer of SR membrane.

Chemically-prepared chicken heart apocytochrome c with different unfolded states could be obtained during the renaturation process. They exhibited distinct circular dichroism patterns designated as Apo C1 (random coiled), Apo C2 (less ordered) and Apo C3 (more ordered). This characteristic is unique to chicken heart apocytochrome c while compared with its counterparts from Candida krusei, tuna heart or horse heart and promises the emergence of much more detail of correlation of translocation with unfolded states of apocytochrome c. When chicken heart apocytochrome c was subjected to a translocation assay in vitro using trypsin-enclosed large unilamellar vesicles from soybean phospholipids, the ability of the protein to penetrate into the liposomes was found to follow the order of Apo C1Apo C2Apo C3. Conformational alterations of Apo C1, Apo C2 and Apo C3 in association with soybean phospholipid vesicles shown by circular dichroism measurement demonstrated that Apo C1 bound to phospholipids existed in a more loosely folded conformation than Apo C2 and Apo C3. We propose that a more flexible structure of apocytochrome c following the interaction with phospholipids is required for its efficient translocation across the bilayer.

The translocation ability of two different species of apocytochrome c(horse heart, Candida krusei) across the soybean phospholipid vesicles decreased in the order: C. kruseihorse heart. Theoretical calculations of the amphiphilicity of their N- and C-terminal alpha-helix formation and the release experiment using calcein-enclosed soybean phospholipid vesicles showed that there was no direct relevance between their import ratio and the amphiphilic helicity. On the other hand, taking the advantage of circular dichroism technique, a more loosely folded structure of C. krusei following the interaction with soybean phospholipid vesicles was observed which should be responsible for the difference in translocation rate.