Partial primary structures of human and murine macrophage colony stimulating factor (CSF-1).

Approximately 40 amino-terminal residues and 20 internal residues of CSF-1 purified from the media of cultured human pancreatic carcinoma (MIA PaCa) cells and of cultured murine L cells have been identified. Results indicated that the two subunits in each molecule of biologically active CSF-1 are identical in their amino-terminal portions. The twelve amino-terminal residues of MIA PaCa CSF-1 were found to be identical to those of human-urinary CSF-1, suggesting that the polypeptide portions of the two human proteins may be identical. Approximately 75% of the amino acids identified in both MIA PaCa CSF-1 and murine CSF-1 were found to be common to both. No homology to other proteins was observed. This study suggests a subunit polypeptide Mr nearer to 17K than to 26K predicted from cDNA.

Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure.

Methionine aminopeptidase (MAP) catalyzes the removal of amino-terminal methionine from proteins. The Escherichia coli map gene encoding this enzyme was cloned; it consists of 264 codons and encodes a monomeric enzyme of 29,333 daltons. In vitro analyses with purified enzyme indicated that MAP is a metallo-oligopeptidase with absolute specificity for the amino-terminal methionine. The methionine residues from the amino-terminal end of the recombinant proteins interleukin-2 (Met-Ala-Pro-IL-2) and ricin A (Met-Ile-Phe-ricin A) could be removed either in vitro with purified MAP enzyme or in vivo in MAP-hyperproducing strains of E. coli. In vitro analyses of the substrate preference of the E. coli MAP indicated that the residues adjacent to the initiation methionine could significantly influence the methionine cleavage process. This conclusion is consistent, in general, with the deduced specificity of the enzyme based on the analysis of known amino-terminal sequences of intracellular proteins (S. Tsunasawa, J. W. Stewart, and F. Sherman, J. Biol. Chem. 260:5382-5391, 1985).

Biological properties and molecular biology of the human macrophage growth factor, CSF-1.

CSF-1 is a growth and differentiation factor for the production of mononuclear phagocytes from undifferentiated bone marrow progenitors. In addition to previously described effects on mature cells, we show here that CSF-1 stimulates the production by monocytes of interferon, tumor necrosis factor, and myeloid CSF that produces mainly mixed neutrophil-macrophage colonies in bone marrow culture. Pretreatment with CSF-1 also promotes resistance to viral infection and tumor cytotoxicity in murine peritoneal macrophages. Based on amino acid sequence data of purified human urinary and murine L cell CSF-1, we have cloned the complementary DNA (cDNA) from messenger RNA (mRNA) of the human CSF-1 producing MIA PaCa cell line. The cDNA specifies a 32 amino acid signal peptide followed by a protein of 224 amino acids. Several facts suggest, however, that one-third of the molecule at the C-terminal end is processed off intracellularly to derive the secreted growth factor. The gene is about 18 kilobases (kb) in length and contains 9 exons. Although there appears to be a single copy gene for CSF-1, cells expressing the factor contain several mRNA species, suggesting that the gene may have several functions or levels of regulation. High level expression of the recombinant protein will allow preclinical testing in several disease models for therapeutic efficacy that has been suggested from in vitro and in vivo biological properties of CSF-1.

Purification of human macrophage colony stimulating factor (CSF-1) from medium conditioned by pancreatic carcinoma cells.

Colony Stimulating Factor-1 has been purified to apparent homogeneity from the serum-free medium conditioned by cultured human pancreatic carcinoma cells which had been induced with phorbol myristate acetate. The purification scheme consisted of sequential steps of batchwise adsorption to calcium phosphate gel, adsorption to lentil lectin-Sepharose, binding to immobilized antibodies, hydrophobic interaction chromatography, and reversed-phase high-performance liquid chromatography. The purified glycoprotein was found to have a subunit molecular weight corresponding to the smallest of four species (approximately 40,000, 33,000, 28,000 and 23,000) which were observed when less purified preparations were examined.

Reversed-phase chromatography of interleukin-2 muteins.

Human recombinant interleukin-2 (IL-2) and related species have been characterized by chemical modifications, tryptic digestion, and cyanogen bromide digestion. The oxidation states of the cysteines and methionines in several IL-2 muteins have been determined. Reversed-phase high-performance liquid chromatography allowed us to distinguish the modifications in these muteins and to correlate retention behavior with their structure.

Molecular cloning of a complementary DNA encoding human macrophage-specific colony-stimulating factor (CSF-1).

Complementary DNA (cDNA) clones encoding human macrophage-specific specific colony-stimulating factor (CSF-1) were isolated. One cDNA clone codes for a mature polypeptide of 224 amino acids and a putative leader of 32 amino acids. This cDNA, which was cloned in the Okayama-Berg expression vector, specifies the synthesis of biologically active CSF-1 in COS cells, as determined by a specific radioreceptor assay, macrophage bone marrow colony formation, and antibody neutralization. Most of the cDNA isolates contain part of an intron sequence that changes the reading frame, resulting in an abrupt termination of translation; these cDNA's were inactive in COS cells. The CSF-1 appears to be encoded by a single-copy gene, but its expression results in the synthesis of several messenger RNA species, ranging in size from about 1.5 to 4.5 kilobases.

Nucleotide sequence of bacteriophage T4 gene 23 and the amino acid sequence of its product.

We have determined the nucleotide sequence of gene 23 of bacteriophage T4 by the methods of Maxam and Gilbert and of Sanger. The identities of approximately 80% of the amino acid residues of the major capsid protein which is encoded by gene 23 were determined additionally by Edman degradation of the intact protein and its peptides. Fifteen gene 23 amber mutation sites have been located within the sequence, and the 3' transcription termination site for genes 21, 22 and 23 has been identified.

Proteolysis of the major capsid protein T4 bacteriophage polyheads limited by quaternary structure.

Bacteriophage T4 carrying an amber mutation in gene 22 plus an amber mutation in gene 21 form aberrant, tubular structures termed rough polyheads, instead of complete phage when they infect Escherichia coli B. These rough polyheads consist almost entirely of the major capsid protein in its uncleaved form (gp23). When rough polyheads are treated under mild conditions with any of the five proteases, trypsin, chymotrypsin, thermolysin, pronase, or the protease from Staphylococcus aureus V8, the gp23 is rapidly hydrolyzed at a limited number of peptide bonds. In contrast, cleaved capsid protein (gp23) in mature phage capsids is completely resistant to proteolysis under the same conditions. A major project in this laboratory requires determining the primary structure of gp23, a large protein (Mr = 58,000) quite rich in those amino acids at which cleavages are achieved by conventional means. Recovery of peptides from the complex mixtures resulting from such cleavages proved to be extremely difficult. The limited proteolysis of gp23 in rough polyheads had yielded a set of large, easily purified fragments which are greatly simplifying the task of determining the primary structure of this protein.

Subunit structure of AMP-deaminase from chicken and rabbit skeletal muscle.

The AMP-deaminases from chicken and rabbit muscle have been investigated by techniques which include sedimentation equilibrium, sodium dodecyl sulfate gel electrophoresis, amino acid analysis, NH2- and COOH-terminal analyses, and tryptic peptide mapping. The molecular weights of the native chicken (276,000) and rabbit (271,000) enzymes obtained by sedimentation equilibrium studies are in good agreement with values of 276,000 (chicken) and 275,000 (rabbit) calculated from amino acid analyses. The enzymes were reduced, carboxymethylated, and treated with either maleic or succinic anhydride in the presence of 6 M guanidine hydrochloride. Sodium dodecyl sulfate gel electrophoresis of the chemically modified enzymes resulted in a single electrophoretic species having an apparent molecular weight of 85,000. This observation is consistent with previous studies on the nonacylated enzymes and suggests that the muscle AMP-deaminases from chicken and rabbit do not contain noncovalent linkages which are readily disrupted by a large increase in negative charge. NH2-terminal analyses by the method of Stark and Amyth as well as the dansyl technique, indicate that the NH2-terminal positions of these enzymes are blocked. The enzymes are also resistant to digestion with carboxypeptidases A or B (or both) in the presence of sodium dodecyl sulfate. The most distinctive feature of the amino acid compositions of both the chicken and rabbit AMP-deaminases is the presende of eight half-cystine residues per 69,000 g of protein. Tryptic digests of the S-14C-carboxymethylated proteins were fractionated by ion exchange chromatography and high voltage electrophoresis. Six and five radioactiviely labeled peptides were detected in the electrophoretograms of the chicken and rabbit enzymes, respectively. This observation and the number of ninhydrinposition spots, together with the physical data on the molecular weights of the native enzymes and their subunits, suggest that the AMP-deaminases from chidken and rabbit muscle consist of four identical or very similar polypeptide chains.