Dataset Information

Purification and partial sequencing of myristoyl-CoA:protein N-myristoyltransferase from bovine brain.

ABSTRACT: The enzyme myristoyl-CoA:protein N-myristoyltransferase (NMT; EC 2.3.1.97) catalyses the transfer of myristic acid to the N-terminal glycine residue of cell and viral proteins. In this report the purification and partial sequencing of this enzyme from bovine brain is described. Using a combination of ammonium sulphate precipitation, chromatography on DEAE-Sepharose and affinity chromatography on CoA-agarose the enzyme was purified some 40-fold. Size-exclusion chromatography of this material in the presence of myristoyl-CoA yielded two peaks of enzyme activity with apparent molecular masses of 66 kDa and 43 kDa. Chromatography of the CoA-affinity-purified material on MONO-S followed by size-exclusion chromatography in the presence of myristoyl-CoA resulted in the isolation of the large form of the enzyme purified 3000-fold. Analysis by SDS/PAGE of this material showed a major 60 kDa silver-stained band. Similar analysis of the 43 kDa enzyme fraction from the same separation showed that this fraction contained several proteins including a major component with an apparent molecular mass of 49 kDa. Attempts at N-terminal sequencing of the 66 kDa form of the enzyme were unsuccessful and therefore this material was digested with trypsin and the resulting peptides separated by reverse-phase h.p.l.c. N-terminal protein sequencing of these peptides yielded sequences which show sequence similarity to those of yeast N-myristoyl-transferase.

SUBMITTER: McIlhinney RA

PROVIDER: S-EPMC1132288 | biostudies-other | 1993 Mar

REPOSITORIES: biostudies-other

ACCESS DATA

Similar Datasets

Project description:Soluble extracts prepared from bovine thymus contain an angiotensin-I-phosphorylating activity that is activated several-fold by high concentrations of NaCl. Fractionation of this protein-tyrosine kinase activity by chromatography on DEAE-cellulose yields a major diffuse peak of activity. The enzymes responsible for this activity are found at much higher levels in extracts from bovine thymus than from bovine spleen. The peak of activity from the DEAE-cellulose column can be further separated into two major peaks by chromatography on heparin-agarose. The second peak to elute from the heparin-agarose column was previously purified through several chromatographic steps to yield a 40 kDa protein-tyrosine kinase (p40). We have now partially purified the early-eluting peak of kinase activity by chromatography on columns of butyl-agarose, protamine-agarose and Sephacryl S200. The enzyme was identified following covalent modification with 5'-fluorosulphonylbenzoyladenosine (FSBA) by reactivity with anti-FSBA antibodies. This procedure labelled a series of 52-56 kDa proteins. These proteins were also recognized by polyclonal anti-peptide antibodies raised against the C-terminal 33 amino acids of p56lck, a major T lymphocyte protein-tyrosine kinase. Peptide maps of the partially purified enzyme were identical to maps generated from p56lck obtained from LSTRA cells. These data suggest that bovine thymus p56lck is responsible for the activity found in the early-eluting peak from heparin-agarose. Antibodies raised against a peptide corresponding to amino acids 39-64 of p56lck, a sequence found near the N-terminus, recognized the slower-migrating, but not the faster-migrating, form of the enzyme, indicating that a fraction of the protein had been proteolysed near the N-terminus during purification. The partially purified bovine enzyme exhibited a restricted substrate specificity in vitro and did not readily phosphorylate human erythrocyte band 3, casein or histone, but was able to phosphorylate acid-treated enolase. The dilute enzyme present in fractions eluting from chromatography columns was unable to catalyse an autophosphorylation reaction. Autophosphorylation could be detected in more concentrated enzyme samples and was readily observed in immune-complex assays. The phosphorylation of angiotensin I by bovine thymus p56lck was weakly activated by polyionic compounds such as heparin and polylysine, and was strongly activated by high concentrations of NaCl.

Dataset Information

Purification and partial sequencing of myristoyl-CoA:protein N-myristoyltransferase from bovine brain.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets