Proteolytic Cleavage Product Research Articles

Molecular changes associated with proteolysis of bovine factor V by thrombin.

Native factor V contains two major polypeptide chains, h and 1. The molecular weights determined by gel electrophoresis in the presence of sodium dodecylsulfate and dithiothreitol (125 000 and 73 000) are in reasonable agreement with those obtained by gel filtration in 5 M guanidine-HC1 (125000 and 64000). Exposure of factor V to thrombin results in cleavage of the heavier chain to an altered form with a molecular weight of 87000. The other fragment of this proteolytic reaction appears to be a carbohydrate-rich piece, which migrates abnormally slowly on gel electrophoresis conducted under denaturing and reducing conditions. Both proteolytic cleavage products remain associated with the light chain during the purification of factor V. The 87000-Mr fragment is present in samples of factor V which are isolated by immunoprecipitation of blood obtained from a single animal by venous catheter. This finding suggests that some proteolysis may occur in vivo. In contrast, the molecular weight of the light chain is unaltered after thrombin proteolysis of either purified factor V or thrombin-treated plasma.

Assembly of bacteriophage lambda heads: Protein processing and its genetic control in petit λ assembly

Petit bacteriophage λ is a hollow λ head precursor which is found in λ-infected lysates, including lysates of phage λ carrying mutations in head genes. Wild-type petit λ has a protein composition similar to heads, except that it is missing pD § § Abbreviations used: pA, pB, etc., the protein products of genes A, B, etc; A − petit λ, B − petit λ, etc., petit λ purified from lysates of lysogenic cells in which the induced prophage carried an amber mutation in the indicated gene; groE − petit λ are obtained by induction of a groE mutant of E. coli lysogenic for wild-type λ. , a major component of heads. About 95% of the mass of petit λ is pE, the major structural protein of heads, and in addition it has proteins pB, h3, X1, and X2. Tryptic fingerprint analysis shows that h3 is a proteolytic cleavage product of pB, and previous experiments have shown that X1 and X2 are protein fusion products, closely related to each other and containing amino acid sequences of both pC and pE. Petit lambdas derived from infection by phages defective in genes A or D are indistinguishable from wild-type petit λ. B −, C −, or groE − defective petit lambdas show differences from wild-type in protein composition and in extent of protein processing. On the basis of the properties of mutant petit lambdas it is concluded that: (1) the protein processing reactions (cleavage of pB; fusion of pC with pE) occur on the petit λ structure; (2) cleavage of pB requires the functioning of genes C and groE but not A or D; (3) fusion of pC and pE requires gene groE but not A, B or D; (4) pNu3 participates directly in petit λ assembly but is lost from the structure by the time assembly is complete. Physical studies of petit λ show that wild-type, A −, B − and D − petit lambdas sediment at 150 S, while C − and groE − petit lambdas sediment at 190 S. Purified petit λ of either class has an ultraviolet absorption spectrum characteristic of pure protein.

Protein Fusion: A Novel Reaction in Bacteriophage λ Head Assembly

Parts of two phage-coded head proteins, pE and pC, become fused during bacteriophage lambda head assembly. pE is the main structural component of lambda heads and pC is a minor head protein that is not found as such in mature heads. The bond joining the two proteins appears to be covalent and is not a disulfide bond. Only a specific subset of the sequences of each protein is found in the fusion products, and these sequences are found in the products in equimolar amounts. Two nearly identical fusion products; X1 and X2, are detected; X2 is slightly smaller than X1 and appears to be a proteolytic cleavage product of X1. The fusion reaction probably takes place on a nascent head structure.

Persistence of Deoxyribonucleic Acid Polymerase I and Its 5′ → 3′ Exonuclease Activity in PolA Mutants of Escherichia coli K12

Abstract Partially purified enzyme preparations derived from a strain of Escherichia coli K12 with an amber mutation (polA1) in the DNA polymerase I gene contain approximately 1% as much polymerase I activity as the preparations from wild type strain purified by the same procedure. Two other polymerase I mutants, one temperature-sensitive (polA12) and the other nonsuppressible (polA5), also contain significant levels of polymerase I (12% and 35% of wild type, respectively). In the case of polA12, the polymerase I activity is abnormally thermolabile. The level of polymerase I observed in a given mutant is dependent, in part, on the pH and the template-primer used. Despite their low DNA polymerase I levels, these mutants possess nearly normal amounts of the 5' → 3' exonuclease activity that is known to be part of DNA polymerase I and is separable as a 2.8 S fragment by proteolytic cleavage. In the temperature-sensitive and nonsuppressible mutants, the 5' → 3' exonuclease and polymerase I activities cosediment at approximately 5.4 S just as in the wild type. In contrast, the polymerase I activity in the amber mutant sediments at 5.4 S, but the 5' → 3' exonuclease activity sediments at 2.8 S. These findings suggest that the residual DNA polymerase I activity sedimenting like the wild type enzyme in the polA1 mutants is the result of a low level read-through of the amber mutation, and that the 5' → 3' exonuclease represents the amber peptide, or a proteolytic cleavage product of it.