Purification and molecular cloning of the APO-1 cell surface antigen, a member of the tumor necrosis factor/nerve growth factor receptor superfamily. Sequence identity with the Fas antigen.

The APO-1 antigen as defined by the mouse monoclonal antibody anti-APO-1 was previously found to be expressed on the cell surface of activated human T and B lymphocytes and a variety of malignant human lymphoid cell lines. Cross-linking of the APO-1 antigen by anti-APO-1 induced programmed cell death, apoptosis, of APO-1 positive cells. To characterize the APO-1 cell surface molecule and to better understand its role in induction of apoptosis, the APO-1 protein was purified to homogeneity from membranes of SKW6.4 B lymphoblastoid cells by solubilization with sodium deoxycholate, affinity chromatography with anti-APO-1 antibody, and reversed phase high performance liquid chromatography. Each purification step was followed by an APO-1-specific solid phase enzyme-linked immunosorbent assay using the monoclonal antibody anti-APO-1. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the APO-1 antigen was found to be a membrane glycoprotein of 48-kDa. Endoproteinase-cleaved peptides of the APO-1 protein were subjected to amino acid sequencing, and corresponding oligonucleotides were used to identify a full-length APO-1 cDNA clone from an SKW6.4 cDNA library. The deduced amino acid sequence of APO-1 showed sequence identity with the Fas antigen, a cysteine-rich transmembrane protein of 335 amino acids with significant similarity to the members of the tumor necrosis factor/nerve growth factor receptor superfamily. The APO-1 antigen was expressed upon transfection of APO-1 cDNA into BL60-P7 Burkitt's lymphoma cells and conferred sensitivity towards anti-APO-1-induced apoptosis to the transfectants.     

Dissimilarity in protein chain elongation factor requirements between yeast and rat liver ribosomes.

Factor requirements for yeast and rat liver ribosomes were determined in several different reactions using either yeast or liver factors. In polymerization assays yeast ribosomes required a factor in addition to elongation factor 1 (EF-1) and elongation factor 2 (EP-2). The third factor (EF-3) requirement was observed with EFs from either yeast or liver for both poly(U)-directed polyphenylalanine synthesis and elongation of endogenous peptidyl-tRNA. No significant effect of EF-3 was observed with liver risomes in either assay. In contrast to results with polypeptide synthesis EF-3 was not required for EF-1 dependent binding of [3H]Phe-tRNA or the translocation-dependent formation of N-acetylphenylalanylpuromycin. Up to 2-fold stimulation of the binding reaction was observed with saturating levels of either yeast or liver EF-1. No effect of EF-3 was observed on ribosome-EF-2-GDP-fusidic acid complex formation. The data suggest that the yeast EF-3 may be a loosely bound ribosomal protein which is not required for a specific step in the elongation cycle but is involved in the coordination of the partial reactions required for polymerization.     

Purification and properties of a yeast protein kinase.

A protein phosphokinase (EC 2.7.1.1.37) was isolated from baker's yeast (Saccharomyces cerevisiae) after a 17,000-fold purification; the purified enzyme is homogeneous according to the criteria of gel electrophoresis and ultracentrifuge analysis. The enzyme has a high isoelectric point of ca. 9 and appears to exist as a monomer with a molecular weight of 42,000 plus or minus 1500. It is neither stimulated by cyclic 3',5'-AMP, -GMP, -CMP or -ump nor inhibited by the regulatory subunit of rabbit muscle protein kinase (Reimann, E. M., Walsh, D. A., and Krebs, E. G. (1971), J. Biol. Chem. 246, 1986). In the presence of divalent metal ions, preferably Mg-2+ or Mn-2+, the enzyme readily transfers the terminal phosphate group of ATP to phosvitin, alphaS1B- and beta a-casein and an NH2-terminal tryptic peptide derived from beta a-casein, but not to protamine, lysine, or arginine-rich histones or to yeast enzymes such as phosphorylase, phosphofructokinase, or pyruvate carboxylase; serine and polyserine were also inactive as phosphate acceptors. Km values of 0.17 mM for beta a-casein and 0.2 mMfor ATP were determined at 10 mM Mg-2+. The urified yeast protein kinase also catalyzes the reverse reaction, namely, the transfer of phosphate from fully phosphorylated beta a-casein or its NH2-terminal peptide to ADP resulting in the formation of ATP. AMP, GDP, UDP, and CDP did not serve as phosphate acceptors in this reaction. As observed by Rabinowitz and Lipmann (Rabinowitz, M., and Lipmann, F. (1960), J. Biol. Chem. 235, 1043) both reactions have different pHoptima with values of 7.5 for the forward reaction (phosphorylation of the proteins) and ca 5.2 for the formation of ATP; both are differently affected by salts. Phosphorylation of beta a-casein with [gamma-32-P]ATP followed by digestion of the labeled protein with trypsin indicated that all the radioactivity was exclusively introduced in an NH2-terminal peptide possessing the unique sequence: Glu-Ser(P)-Leu-Ser(P)-Ser(P)-Ser(P)-Glu-Glu...(Ribadeau-Dumas, B., Brignon, G., Grosclaude, F., and Mercier, J.-C. (1971), eur J. Biochem. 20, 264). By subjecting beta a-casein and its NH2-terminal peptide to the combined action of almond acid phosphatease and purified yeast protein kinase, it was determined that the phosphorylation and dephosphorylation reactions proceed randomly, i.e., all seryl phosphate residues are equally susceptible and that the rate of phosphorylation decreases drastically as the number of bound phosphate groups in the substrate diminishes.     

Molecular cloning and expression of a transformation-sensitive human protein containing the TPR motif and sharing identity to the stress-inducible yeast protein STI1.

A transformation-sensitive human protein (IEF SSP 3521) that is 2-fold up-regulated in SV40-transformed MRC-5 fibroblasts has been purified by two-dimensional gel electrophoresis, microsequenced, and cDNA cloned using oligodeoxyribonucleotides. The 2.1-kilobase cDNA encodes a 543-amino acid protein with a calculated molecular mass of 62.6 kDa and a calculated pI of 6.77. Expression of the cDNA in AMA cells using the vaccinia virus expression system followed by two-dimensional gel electrophoresis showed that the protein comigrated with IEF SSP 3521. The protein contains the tetratricopeptide repeat found in families of fungal proteins required for mitosis and RNA synthesis. In particular, the protein has 42% amino acid sequence identity to STI1, a stress-inducible mediator of the heat shock response in Saccharomyces cerevisiae. Northern blot analysis indicated that the 3521 mRNA is up-regulated in several transformed cells. Immunofluorescence studies using a polyclonal antibody raised against the purified protein revealed that the antigen is present mainly in the nucleus of SV40 transformed MRC-5 fibroblasts, while it localizes to the Golgi apparatus and small vesicles in their normal counterparts. The possible physiological role of IEF SSP 3521 is discussed in the light of the structural relationship with STI1.     

Purification, molecular cloning, and immunohistochemical localization of dipeptidyl peptidase II from the rat kidney and its identity with quiescent cell proline dipeptidase

We purified dipeptidyl peptidase II (DPP II) to homogeneity from rat kidney and determined its physicochemical properties, including its molecular weight, substrate specificity, and partial amino acid sequence. Furthermore, we screened a rat kidney cDNA library, isolated the DPP II cDNA and determined its structure. The cDNA was composed of 1,720 base pairs of nucleotides, and 500 amino acid residues were predicted from the coding region of cDNA. Human quiescent cell proline dipeptidase (QPP) cloned from T-cells is a 58-kDa glycoprotein existing as a homodimer formed with a leucine zipper motif. The levels of amino acid homology were 92.8% (rat DPP II vs. mouse QPP) and 78.9% (rat DPP II vs. human QPP), while those of nucleotide homology were 93.5% (rat DPP II vs. mouse QPP) and 79.4% (rat DPP II vs. human QPP). The predicted amino acid sequences of rat DPP II and human and mouse QPP possess eight cysteine residues and a leucine zipper motif at the same positions. The purified DPP II showed similar substrate specificity and optimal pH to those of QPP. Consequently, it was thought that DPP II is identical to QPP. Northern blot analysis with rat DPP II cDNA revealed prominent expression of DPP II mRNA in the kidney, and the order for expression was kidney > testis > or = heart > brain > or = lung > spleen > skeletal muscle > or = liver. In parallel with Northern blot analysis, the DPP II antigen was detected by immunohistochemical staining in the cytosol of epithelial cells in the kidney, testis, uterus, and cerebrum.     

Manganese-stimulated phosphorylation of a rat pancreatic protein: identity with elongation factor 2

To investigate the effect of Mn2+ on pancreatic protein phosphorylation, we incubated rat pancreatic cytosol in Tris buffer (pH 7.5) with [gamma-32P]ATP. Analysis using sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography revealed a single protein (p98), with an Mr of 98,000 and a pI of 6.4-6.5, which was phosphorylated in a dose-dependent manner by Mn2+. A threshold effect was observed at 35 microM, and maximal effect at 1.1 mM Mn2+. Ca2+ and calmodulin (CaM) did not cause p98 phosphorylation, but Mg2+ (10 mM) caused faint non-specific phosphorylation of p98. Ca2+ (0.03-3 mM) and CaM (1-10 micrograms/ml) significantly enhanced, whereas trifluoperazine (TFP) and Mg2+ inhibited Mn(2+)-stimulated p98 phosphorylation. Under the above incubation conditions, Mn(2+)-stimulated protein phosphorylation of p98 was also observed in isolated pancreatic acini, but not in cytosols from liver or kidney. Partial purification of p98 and amino acid sequencing of the protein band corresponding to p98 indicated complete sequence homology with rat elongation factor 2 (EF-2). Furthermore, the combination of Ca2+, Mg2+ and CaM, which is known to induce the phosphorylation of EF-2, mimicked the actions of Mn2+. Inasmuch as EF-2 is the major substrate for CaM-dependent protein kinase III (CaM-PK III), these studies suggest that in the pancreatic acinar cell Mn2+/CaM protein kinase activity is mediated via CaM-PK III and the Mn2+ participates in the regulation of this enzyme in the pancreas.     

Purification of human tumor cell autocrine motility factor and molecular cloning of its receptor.

Tumor autocrine motility factor (AMF) has been detected in and purified from serum-free conditioned medium of human HT-1080 fibrosarcoma cells. Under nonreducing conditions, AMF migrates in sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of 55 kDa but under reducing conditions as a band of 64 kDa. Two-dimensional polyacrylamide gel electrophoresis of the purified AMF resolved two groups of polypeptides with isoelectric points of 6.1 and 6.2 (majors), 6.35 and 6.4 (minors). Purified AMF stimulated HT-1080 cell migration in a dose-dependent fashion. The motility stimulation of the fibrosarcoma cells with AMF is associated with the phosphorylation of the AMF receptor, a 78-kDa cell surface glycoprotein (gp78), suggesting protein kinase participation in migratory signal transduction. The gene encoding gp78 was cloned from an HT-1080 fibrosarcoma complementary DNA library. The deduced sequence encodes a polypeptide of 323 amino acids. The nucleotide and predicted amino acid sequence of the gp78 reveals significant homology with the human suppressor/oncogene p53 protein.     

Limited proteolysis of yeast elongation factor 3. Sequence and location of the subdomains

Elongation factor 3 (EF-3) is an ATPase essential for polypeptide chain synthesis in a variety of yeasts and fungi. We used limited proteolysis to study the organization of the subdomains of EF-3. Trypsinolysis of EF-3 at 30 degrees C resulted in the formation of three fragments with estimated molecular masses of 90, 70, and 50 kDa. Yeast ribosomes protected EF-3 and the large fragments from further degradation. ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments. The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding. Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of EF-3. Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA. Neither tRNA nor rRNA altered the tryptic cleavage pattern. The proteolytic products were sequenced by mass spectrometric analysis. EF-3 is blocked NH(2)-terminally by an acetylated serine. The 90-, 70-, and 50-kDa fragments are also blocked NH(2)-terminally, confirming their origin. The 50-kDa fragment (Ser(2)-Lys(443)) is the most stable domain in EF-3 with no known function. The 70-kDa fragment (Ser(2)-Lys(668)) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain. The primary ribosome binding site is located near the loosely structured carboxyl-terminal end.