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PubMed1982

Peptide mapping of peroxisomal catalase and its precursor. Comparison to the primary wheat germ translation product.

To investigate possible structural modifications of catalase during its biogenesis and packaging into peroxisomes, we have labeled three species of catalase with [35S]methionine: the wheat germ cell-free translation product, the extraperoxisomal precursor made in vivo in rat liver, and mature peroxisomal catalase. These three species have identical mobilities in sodium dodecyl sulfate polyacrylamide gels, when analyzed separately or in mixtures. Tryptic digestion yields 10 [35S]Met-labeled peptides from each, which are indistinguishable when mapped in two dimensions by electrophoresis and chromatography. Partial proteolyses of the three species in sodium dodecyl sulfate gels yielded identical fragmentation patterns. The primary translation product of catalase was labeled with formyl[35S] methionine; its size was indistinguishable from the subunit of mature catalase. Its radioactivity appeared in dansyl methionine if and only if it was deformylated prior to dansylation. These results demonstrate that within the limits of the methods, catalase undergoes no covalent modification during its uptake into peroxisomes and its subsequent maturation to a tetrameric hemoprotein.

Animals↗
PubMed1983

The architecture of the animal fatty acid synthetase. I. Proteolytic dissection and peptide mapping.

The vertebrate fatty acid synthetase complex consists of two, apparently identical, subunits which contain seven catalytic centers and an acyl carrier site. As a prelude to studying the functional organization of this system, a comprehensive proteolytic analysis of the chicken fatty acid synthetase subunit has been carried out. Of 24 proteases tested, seven (chymotrypsin, elastase, trypsin, Myxobacter protease, subtilisins A and B, and kallikrein) were found to be capable of inflicting specific cleavages in the native enzyme. The proteolytic profiles were analyzed with respect to both the kinetics and the size of the fragments generated. The relationships between the various fragments were further delineated by combinatorial digestions. All of the data points to the fact that the subunits are indeed identical. The cleavage patterns indicate that the fatty acid synthetase subunit is primarily arranged into three major domains of Mr 127,000, 107,000, and 33,000, since these fragments are commonly produced by most of the proteases. The latter peptides may be further subdivided and mapped by the action of individual enzymes into a number of distinct regions: the 127,000 domain into fragments of Mr 60,000, 45,000, and 23,000; the 107,000 domain into fragments of Mr 14,000, 56,000, 21,000, and 15,000; all aligned accordingly. This proteolytic map serves as a reference for the controlled dissection of the fatty acid synthetase complex, thereby allowing determination of the location of functional centers within the parent polypeptide.

Animals↗
PubMed1983

Peptide mapping of the human transferrin receptor in normal and transformed cells.

Human transferrin receptor protein from various human cell lines in tissue culture (phytohemagglutinin-activated lymphocytes, normal embryonic fibroblasts (WI-38), a choriocarcinoma (Be Wo), an epidermoid carcinoma (KB), and a metastatic breast carcinoma (MCF-7)) was metabolically labeled with [35S]methionine and purified by two methods. The first method involves affinity chromatography of the transferrin receptor with a transferrin-linked Sepharose 4B resin. The second method involves direct immunoprecipitation of the receptor protein with a goat polyclonal antibody raised against placental transferrin receptor. Additionally, transferrin receptor from normal gestational placenta was similarly radiolabeled with [35S]methionine and subsequently purified with transferrin-bound resin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the reduced receptor isolated by both purification techniques revealed an Mr = 94,000 protein to be present in all cells assayed for this study. Partial proteolytic digestion with Staphylococcus aureus protease followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed identical mapping patterns of the Mr = 94,000 protein isolated from each cell line, irrespective of the isolation technique employed to purify the receptor. These findings indicate an identical peptide structure of the human transferrin receptor in cells with a differentiated function for iron metabolism, in normal cells undergoing mitosis, and in neoplastic cells in long term culture, within the limits of detectability of the proteolytic digestion maps.

Cell Division↗
PubMed1981

A comparison of proteins among various influenza B virus strains by one-dimensional peptide mapping.

The major virus-specific proteins (HA, NA, NP, NS1 and M) of five different isolates of influenza B virus (B/Lee/40, B/Osaka/2/70, B/Yamagata/1/73, B/Aomori/1/76 and B/Yamagata/26/77) were compared by limited proteolysis with Staphylococcus aureus V8 protease and subsequent polyacrylamide gel electrophoresis. The peptide patterns of matrix (M) proteins from all five strains were virtually identical. The nucleoproteins (NP) as well as the non-structural proteins (NS1) were also very similar among strains although the peptides of B/Lee/40 could be distinguished from those of the strains isolated from 1970 to 1977. In contrast, the peptides from haemagglutinin (HA) glycoproteins were largely different even among the strains isolated later than 1970. It therefore appears that the HA glycoproteins of influenza B virus are more changeable than any of the non-glycosylated proteins. Furthermore, it was found that the maps of HA1 were markedly different among strains while the maps of HA2 were very similar, which suggests that the structural changes in the HA polypeptide occur preferentially in the HA1 portion. The neuraminidase (NA) glycoproteins also showed strain-dependent differences in their mapping patterns.

Electrophoresis, Polyacrylamide Gel↗
PubMed1997

Enzymatic cleavage and HPLC peptide mapping of proteins.

Detailed procedures are described for successfully digesting reasonably small quantities (i.e., usually > 10 pmol) of proteins with a variety of proteases and for then isolating the resulting peptides by reverse-phase HPLC. Since sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) appears to be the current method of choice for final purification of proteins for structural analysis, special attention is given to carrying out in-gel proteolytic digests on SDS-PAGE-separated proteins that have usually been stained with Coomassie Blue. A compilation of data from nearly 200 "unknown" samples is used to help provide realistic expectations with respect to the results that are likely to be obtained from carrying out in-gel proteolytic digests on large numbers of proteins.

Amino Acids↗