Crude extracts of a multiply peptidase-deficient strain of Salmonella typhimurium contain an aminopeptidase that specifically removes N-terminal methionine from peptides. This activity shows pronounced specificity for the peptide's second amino acid. Methionine is removed from peptides with alanine, threonine, or glycine in this position but not when the second amino acid is leucine or methionine. The activity is stimulated by Co2l and is inhibited by EDTA.Mutations that lead to overproduction (up to He concluded that N-terminal modification is a stepwise process, with deformylation preceding the removal of methionine by an aminopeptidase. Attempts to isolate this methionine-specific aminopeptidase have not been successful, however. Vogt (6) purified and characterized an activity capable of rapid cleavage of Met-Ala-Ser. He concluded that the specificity of this enzyme is not consistent with its involvement in N-terminal methionine removal. In addition, mutants of E. coli or Salmonella lacking this enzyme grow normally (7,8). Earlier reports of a ribosomal peptidase activity (9) almost certainly are based on the artifactual association of the enzyme described by Vogt with the ribosome fraction (6). Another attempt to identify a methionine-specific aminopeptidase led to the isolation of an enzyme that appears to have specificity for methionine but hydrolyzes no substrates larger than dipeptides (10).Clearly, a major problem in identifying a methioninespecific aminopeptidase in cell extracts is the presence of several broad-specificity enzymes capable of hydrolyzing N-terminal methionine peptides. We have shown that at least four such enzymes are present in crude extracts of Salmonella typhimurium and E. coli (7,8,11). We have also isolated mutants that lack all of these activities and observed that these mutants, although greatly restricted in their capacity to use peptides as amino acid sources, still use certain Nterminal methionine peptides (8,11). This paper makes use of these mutant strains to identify a methionine-specific aminopeptidase and to isolate mutants that substantially overproduce this activity. The substrate specificity ofthis enzyme is entirely consistent with the proposal that it is involved in the removal of N-terminal methionine from newly synthesized proteins.
A new carrier molecule, NH2OCH2CO-(Gly)3-[Lys(H-Ser-)]5-Gly-OH, has been synthesized to facilitate the preparation of protein conjugates of defined structure. Special features are as follows: (i) (aminooxy)-acetyl as a terminal group, which reacts specifically to form an oxime bond under very mild conditions with an aldehyde group placed on a protein in a prior step; (ii) a spacer group of three Gly residues; and (iii) a set of five Lys residues, each of which is acylated with a Ser residue. A second form of the carrier molecule, HCO-m-C6H4CH = NOCH2CO-(Gly)3-[Lys(H-Ser)]5-Gly-OH, was also prepared. This form possesses a terminal aldehyde group which permits site-specific attachment by formation of a hydrazone bond to the carboxyl termini of polypeptide chains which have been modified enzymatically with carbohydrazide in a prior step. Once the carrier is linked to protein in one of the above ways, i.e. through formation of either an oxime or hydrazone bond, the Ser residues of the carrier (but not of the protein) may be oxidized by very mild periodate treatment to generate aldehyde groups. Drugs possessing a hydrazide group (e.g. methotrexate gamma-hydrazide or desacetylvincaleukoblastine hydrazide) may then be conjugated via hydrazone formation to the aldehyde groups of the carrier. A cluster of five drug molecules may thus be attached to a single site on a protein, giving a relatively homogeneous product in spite of the high drug conjugation ratio. Synthesis of the carrier, formation of a pentadrug-protein conjugate, and wider implications of the chemistry are presented.
A two-step approach to the production of well-defined protein conjugates is described. In the first step, a linker group, carbohydrazide, having unique reactivity (a hydrazide group) is attached specifically to the carboxyl terminus by using enzyme-catalyzed reverse proteolysis. Since the hydrazide group exists nowhere else on the protein, specificity is assured in a subsequent chemical reaction (formation of a hydrazone bond) of the modified protein with a molecule (chelator, drug, or polypeptide) carrying an aldehyde or keto group. The product is sufficiently stable at neutral pH, no reduction of the hydrazone bond being necessary for the hydrazones described. Protein modification is thus restricted to the carboxyl terminus and a homogeneous product results. With insulin as a model, conditions are described for producing such well-defined conjugates in good yields. The use of other linker groups besides carbohydrazide, and applications of these techniques to antibody fragments, are discussed.
A small computer program has been written to investigate the effect of increasing the mass resolution to very high values (lo6) on the shape of mass spectral peaks of small to medium-sized proteins. Previous work in this area has generally limited discussion to mass resolutions of a few thousand. Now that mass spectra of proteins are obtainable with a mass resolution exceeding one million, it is important to be able to conveniently generate theoretical spectra at the same resolution.
A site-specific immunoconjugate was prepared between an F(ab')2-like fragment of the monoclonal anti-CEA murine IgG1 A5B7 and a mutant of the dimeric enzyme carboxypeptidase G2 possessing an N-terminal Thr in place of Ala. First an aldehyde was introduced at the N-terminus of the enzyme by mild periodate oxidation and a residue of carbohydrazide was specifically introduced at the C-terminus of the truncated heavy chain of the F(ab')2-like fragment by reverse proteolysis. Then the two modified proteins were conjugated by the formation of a hydrazone bond between the hydrazide and the aldehyde groups. The conjugate obtained retained both enzymic activity and antigen-binding capacity. The antigen-binding capacity was better than that of a similar conjugate made conventionally by random reaction with side chains.
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