Mammalian prolyl-tRNA synthetase corresponds to the approximately 150 kDa subunit of the high-M(r) aminoacyl-tRNA synthetase complex.

The high-M(r) aminoacyl-tRNA synthetase complex previously purified from sheep liver differed from those isolated from several other mammalian sources by the absence of prolyl-tRNA synthetase activity and the presence of glutamyl tRNA synthetase as a polypeptide of 85 kDa instead of 150 kDa. Using a milder extraction procedure that minimizes proteolysis, we now report the isolation of a sheep liver complex that contains both prolyl-tRNA synthetase activity and the 150-kDa polypeptide. The correspondence between prolyl-tRNA synthetase and the 150-kDa polypeptide, inferred from the results of several approaches reported in this study, was further demonstrated by showing that antibodies to a free form of sheep liver prolyl-tRNA synthetase generated by endogenous proteolysis, specifically reacted with the 150-kDa components of the complexes from sheep and rabbit, but failed to react with the previously purified complex from sheep that contained neither prolyl-tRNA synthetases activity nor the 150-kDa component. Moreover, we show that the 150-kDa polypeptide is also recognized by antibodies to the 85-kDa polypeptide previously assigned to glutamyl-tRNA synthetase. The possibility that the largest subunit of the mammalian high-M(r) complexes may be a bifunctional protein encoding both glutamyl- and prolyl-tRNA synthetase activities is considered and discussed in light of the recently published sequence of the corresponding polypeptide from HeLa cells. In accordance with this prediction, we show that the amino acid sequence of the carboxyl-terminal moiety of this bifunctional polypeptide shows significant similarity to the sequence of prolyl-tRNA synthetase from Escherichia coli.     

Purification of transfer RNA (m5U54)-methyltransferase from Escherichia coli. Association with RNA

tRNA (m5U54)-methyltransferase (EC 2.1.1.35) catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to transfer ribonucleic acid (tRNA) and thereby forming 5-methyluridine (m5U, ribosylthymine) in position 54 of tRNA. This enzyme, which is involved in the biosynthesis of all tRNA chains in Escherichia coli, was purified 5800-fold. A hybrid plasmid carrying trmA, the structural gene for tRNA (m5U54)-methyltransferase was used to amplify genetically the production of this enzyme 40-fold. The purest fraction contained three polypeptides of 42 kDa, 41 kDa and 32 kDa and a heterogeneous 48-57-kDa RNA-protein complex. All the polypeptides seem to be related to the 42/41-kDa polypeptides previously identified as the tRNA (m5U54)-methyltransferase. RNA comprises about 50% (by mass) of the complex. The RNA seems not to be essential for the methylation activity, but may increase the activity of the enzyme. The amino acid composition is presented and the N-terminal sequence of the 42-kDa polypeptide was found to be: Met-Thr-Pro-Glu-His-Leu-Pro-Thr-Glu-Gln-Tyr-Glu-Ala-Gln-Leu-Ala-Glu-Lys- . The tRNA (m5U54)-methyltransferase has a pI of 4.7 and a pH optimum of 8.0. The enzyme does not require added cations but is stimulated by Mg2+. The apparent Km for tRNA and S-adenosyl-L-methionine are 80 nM and 17 microM, respectively.     

Structural and nonstructural proteins of a rabbit parvovirus

The structural and nonstructural polypeptides of a rabbit parvovirus (RPV) (F-7-9 strain) were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The virion contained three polypeptide components, A (molecular weight, 96,000), B (85,000), and C (75,000). A part of the polypeptide C was cleaved into the smaller-molecular-weight polypeptide C' by proteolysis during purification steps. The major polypeptide C together with C' constituted about 87% of the total viral proteins, and the minor polypeptides, A and B, constituted 4 and 9%, respectively. The structural polypeptides of empty particles were similar in size and composition to those of the virion, but the content of the C' polypeptide was very low. When rabbit kidney cell cultures were infected with RPV, the C polypeptide was detected as early as 15 h postinfection, whereas A and B were first demonstrated at 18 h. The C' polypeptide was not detected for 44 h. In addition to the three structural polypeptides, at least three nonstructural polypeptides, E, F, and G, were demonstrated in the RPV-infected cells. Polypeptide E (molecular weight, 49,000), detected mostly in cytoplasm, seemed to be a cellular protein. The F (25,000) and G (22,000) polypeptides seemed to be virus-coded proteins since they were precipitated with the anti-RPV rabbit immunoglobulin. According to partial proteolysis and peptide mapping, the F and G polypeptides shared the same peptide components.     

Synaptonemal complex proteins

Synaptonemal complexes were isolated from rate spermatocytes for the purpose of biochemical and morphological analysis. Several monoclonal antibodies were elicited against purified synaptonemal complexes to study the composition and assembly of these structures. Four classes of antibodies could be discriminated according to the polypeptides that they recognize on Western blots of purified synaptonemal complexes, namely antibodies recognizing (i) a 190-kDa polypeptide; (ii) a 30- and a 33-kDa polypeptide; (iii) two polypeptides with molecular weights of about 120 kDa; and (iv) polypeptides with molecular weights of 66-55 kDa. The localization of these antigens within spermatocytes was analyzed light microscopically, by means of the immunoperoxidase technique and ultrastructurally, by immunogold labelling of surface-spread spermatocytes. The 66- to 55-kDa polypeptides are not confined to synaptonemal complexes; rather, these polypeptides appear to be chromosomal components. The 190-, 30-, and 33-kDa polypeptides make part of the lateral elements of paired as well as unpaired segments of synaptonemal complexes. The 120-kDa polypeptides were localized on the inner edge of the lateral elements, specifically in paired segments of synaptonemal complexes. The distribution of the 190-, 120-, 30-, and 33-kDa polypeptides within the testis was analyzed by immunofluorescence staining of cryostat sections. All these polypeptides turned out to be specific for nuclei of zygotene up to and including diplotene spermatocytes. Only in some early spermatids could the 190-, 30-, and 33-kDa polypeptides be detected, presumably in remnants of synaptonemal complexes.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of a receptor for acidic and basic fibroblast growth factor from embryonic chick

A receptor for acidic and basic fibroblast growth factors (aFGF and bFGF, respectively) was isolated from 7-day embryonic chick. Chromatography of solubilized membrane proteins on wheat germ agglutininagarose and aFGF-Sepharose yielded three major polypeptides migrating at 150, 70, and 45 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. These polypeptides were eluted from aFGF-Sepharose with either 1.0 M NaCl or 100 micrograms/ml heparin, but were not retained on underivatized Sepharose. Cross-linking of 125I-aFGF or 125I-bFGF to either crude membrane preparations or to purified fractions yielded a 165-kDa complex, suggesting the existence of a 150-kDa FGF receptor after subtraction of approximately 15 kDa for 125I-FGF. Addition of excess aFGF or bFGF competed for binding of either 125I-aFGF or 125I-bFGF to FGF receptor preparations. Purified FGF receptor fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Immobilon membranes, and incubated with 125I-aFGF or 125I-bFGF in order to identify FGF-binding polypeptides. Bound 125I-aFGF and 125I-bFGF were displaced by aFGF and bFGF, but not epidermal growth factor, consistent with the identification of the 150-kDa polypeptide as a receptor for acidic and basic FGF. Treatment of purified FGF receptor fractions with N-glycanase demonstrated that the 150-kDa polypeptide contained approximately 10 kDa of N-linked oligosaccharide. The apparent molecular mass of the 150-kDa polypeptide was unaffected by treatment with heparitinase, indicating that the 150-kDa polypeptide is not a heparan sulfate proteoglycan. Together, these data suggest that the 150-kDa polypeptide is a FGF receptor that may mediate the biological activities of aFGF and bFGF.     

Purification and characterization of two distinct complexes of assembly polypeptides from calf brain coated vesicles that differ in their polypeptide composition and kinase activities

The binding and assembly of clathrin triskelions on vesicle membranes seem to be mediated by certain assembly polypeptides (Keen, J.H., Willingham, M.C., and Pastau, I.H. (1979) Cell 16, 303-312). These assembly polypeptides were further purified into two distinct complexes using hydroxylapatite chromatography. Peak 1 consists of two major bands of 98 and 112 kDa, two minor bands of 103 and 118 kDa, and a polypeptide of 46 kDa. Peak 2 consists of one major band of 100 kDa, two minor bands of 103 and 115 kDa, and a polypeptide of 50 kDa. Both complexes have a native molecular mass of 290 kDa as determined by gel filtration. Each 290-kDa complex contains two polypeptides of 98-118/100-115 kDa and two polypeptides of 46/50 kDa. The 46-kDa polypeptide is not phosphorylated, whereas the 50-kDa polypeptide is. Both peaks contain 50-kDa kinase-like activity. Time courses of the 50-kDa phosphorylation show that the activity in peak 1 saturates much faster than the activity in peak 2; there may be two 50-kDa kinase activities in coated vesicles. A kinase that phosphorylates the polypeptides in 98-118-kDa group is present in peak 1 but not in peak 2. Both peaks assemble clathrin triskelions into cages under conditions in which the clathrin alone would not assemble. Both rotary shadowed and negatively stained preparations of these reassembled cages as well as the purified complexes were examined by electron microscopy. Thus, two complexes have been identified that differ in their polypeptide composition and kinase activities, but are similar in their ability to assemble clathrin triskelions into cages.