Abstract
ApoVLDL-II is a major apoprotein in avian very low density lipoproteins (Jackson, R. L., Lin, H.-Y., Chan, L., and Means, A.R. (1977) J. Biol. Chem. 252, 250-253). Partially purified apoVLDL-II mRNA was translated in vitro in a wheat germ system in the presence of various labeled amino acids. The product, designated preapoVLDL-II, was purified by immunoprecipitation and sodium dodecyl sulfate acrylamide gel electrophoresis. It was subjected to automated Edman degradation in a Beckman Sequencer. The signal peptide was found to be a 23-amino acid NH2-terminal extension of the mature protein with the following sequence: Met-Gln-Tyr-Arg-Ala-Leu-Val-Ile-Ala-Val-Ile-Leu-Leu-Leu-Ser-Thr-Val-Pro-Glu-Val-Cys-Ser-Lys where Lys is the NH2-terminal residue of mature apoVLDL-II. The abundance and distribution of the hydrophobic amino acid residues are very similar to those of other signal sequences and the average hydrophobicity for the 23 residues is -1227 cal/mol/residue. However, translocation of preapoVLDL-II would represent a unique case of vectorial migration of a protein through a membrane since apoVLDL-II is itself an apolipoprotein and binds lipid spontaneously.
Highlights
Membranes, the signal peptidoef apoVLDL-I1 is of considerable interest since the mature apoprotein, like many other eukaryotic apolipoproteins, spontaneously interacatsnd binds to lipids [7]
It was subjected to automated Edman degrada- Preparation of Cockerel Liver mRNA-The 4-week-old cockerels tion in a Beckman Sequencer
The studies of Milstein et al [1]and Blobel and Dobberstein ( 2, 3 )as well as others [4] indicate that secretory proteins are initially synthesized as intracellular precursor molecules which contain a highly hydrophobic NHP-terminal extension the supernatant fractionated on 8-cm cyclindrical12%polyacrylamide gels in 0.1%NaDodSOl by the method of Weber and Osborne [12].At the end of electrophoresis, gelsweresliced into 2-mmpieces, and radiolabeled peptides were eluted from the slices by incubation with 400plof10 mM sodium phosphate containing 0.1% Naof 15 t o 30aminoacids.ThisNHn-terminalpeptide ( DodS04, asmodified from the method of Strauss et al [13]
Summary
Membranes, the signal peptidoef apoVLDL-I1 is of considerable interest since the mature apoprotein, like many other eukaryotic apolipoproteins, spontaneously interacatsnd binds to lipids [7]. Purified apoVLDL-11mRNA was translated in vitro in a wheat germ system in the presence of various labeled amino acids.
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