Characterization of native and recombinant peptidyl prolyl cis-trans isomerases derived from Methanococcus thermolithotrophicus based on cDNA sequence.

It is important to establish whether a recombinant protein is an authentic copy of the predicted cDNA sequence. In this study, recombinant protein for native peptidyl prolyl cis-trans isomerase (N-PPIase) and double-labeled (13C- and 15N-) protein (DL-PPIase) appeared on the sodium dodecyl sulfate (SDS) electropherograms as two bands for N-PPIase and four bands for DL-PPIase. Since the N-terminal amino acid residues of all bands were the same, we characterized these bands using the peptide mapping method and amino acid composition analysis. Peptide mapping of the proteins seemed to be almost identical but they could not reflect the whole amino acid sequences of the protein. The bands on the polyvinylidene difluoride (PVDF) membrane, electroblotted after SDS-polyacrylamide gel electrophoresis (SDS-PAGE), were hydrolyzed and their amino acid composition was analyzed using a highly sensitive 6-aminoquinolyl-N-hydroxysuccinimidyl carbamate (AQC) amino acid analysis and compared with the cDNA sequences for proteins. The matching score (sigma(T%-E%)2) for similarity of proteins was calculated by summation of the square difference between the theoretical (T%) and the experimental (E%) amino acid composition of the recombinant protein. The amino acid composition of all bands of both proteins showed more than 93% of the theoretical values. The major molecular weights of both proteins were 16812 and 17694 by electrospray ionization (ESI)-mass spectrometry. However, the purified proteins also contained minor compounds with Mr of 3721 for N-PPIase and 5285 for DL-PPIase. These compounds were considered to be nonpeptidyl products that comigrated with the protein. Similarities of the amino acid composition of the four bands were more than 98%. Our results indicate that AQC amino acid analysis is the most suitable method for characterization of a recombinant protein.

Identification of multidrug resistant protein 1 of mouse leukemia P388 cells on a PVDF membrane using 6-aminoquinolyl-carbamyl (AQC)-amino acid analysis and World Wide Web (WWW)-accessible tools.

Multidrug resistant protein 1 (MDR1) in a doxorubicin-resistant mouse leukemia cell line (P388/DOX) was identified using its amino acid composition combined with protein database searching (ExPASy and EMBL PROPSEARCH) via the World Wide Web. The proteins were separated by one-dimensional SDS-polyacrylamide gel electrophoresis, blotted onto a polyvinylidene fluoride membrane, and stained with Coomassie brilliant blue. A 160-kDa protein band was acid-hydrolyzed in the vapor phase (6 N HC1) and converted to 6-aminoquinolyl-carbamyl (AQC)-amino acids without extraction of the amino acids from the membrane. The amino acid composition of the protein was determined using the sensitive AQC-amino acid analysis method, improving our previously described method. The improved method involved using a Cosmosil 5C8-MS column instead of a Pegasil C8; replacement of the mobile phase A, constituent, 75 mM ammonium phosphate (pH 7.5), with 30 mM sodium phosphate buffer (pH 7.2); and slight modification of the separation program (9). All manipulations for protein hydrolysis and AQC derivatization were carried out in a hood using clean tools. This minimized contamination of amino acids at the low femtomolar level. A database search was carried out with bovine serum albumin as a calibration protein. MDR1 in P388/DOX was ranked first by both databases with high reliability (score 14 for ExPASy, distance 1.34 for EMBL).

Altered proteoglycan synthesis by micromelial limbs induced by 6-aminonicotinamide. Appearance of abnormal forms of cartilage-characteristic proteoglycan (PG-H).

Since administration of 6-aminonicotinamide (10 micrograms) to day-4 chick embryos in ovo was shown to induce micromelial limbs, biosynthesis of cartilage-characteristic proteoglycan-H (PG-H) as an important index of limb chondrogenesis was examined in day-7 normal and micromelial hind limbs by biochemical and immunological methods. (1) Metabolic labelling of the micromelial limbs with [6-3H]glucosamine and either [35S]sulphate or [35S]methionine, followed by analyses of labelled PG-H by glycerol density-gradient centrifugation under dissociative conditions, showed a marked reduction in the PG-H synthesis. (2) PG-H synthesized by the micromelial limbs was much lower than that synthesized by the normal limbs in the biosynthetic ratio of chondroitin sulphate to keratan sulphate and glycoprotein-type oligosaccharide, although no significant difference was observed in the immunological properties of these proteoglycans. (3) The degree of sulphation of chondroitin sulphates of PG-H was lowered in the micromelial limbs as judged by the increase of unsulphated disaccharide (delta Di-OS) released by chrondroitinase ABC digestion, although there were no significant differences between the normal and the micromelial limbs in the average molecular size (Mr = 38,000) of labelled chondroitin sulphates of PG-H. (4) Addition of beta-D-xyloside, an artificial initiator for chondroitin sulphate synthesis, to the micromelial limbs in culture recovered the incorporation of labelled glucosamine into chondroitin sulphate to that comparable with the normal control with beta-D-xyloside, although the incorporation of [35S]sulphate was lower in the micromelia than in the control with beta-D-xyloside. These results suggest that the reduction in the biosynthesis of the PG-H as well as the production of altered forms of PG-H induced by 6-aminonicotinamide during a critical period of limb morphogenesis may be an important factor for the micromelia.