Isolation, purification, characterization, cloning and sequencing of N .alpha.-acetyltransferase

• US 4,966,848 A
• Filed: 12/14/1988
• Issued: 10/30/1990
• Est. Priority Date: 02/08/1988
• Status: Expired due to Fees

• Current Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Original Assignee: The General Hospital Corporation (Mass General Brigham, Inc.)
• Inventors: Smith, John A., Lee, Fang-Jen S.
• Primary Examiner(s): Weimar, Elizabeth C.
• Assistant Examiner(s): Paterson, Jr., Charles L.
• Application Number:
  US07/284,344
• Time in Patent Office: 685 Days
• Field of Search: 435/193
• US Class Current: 435/193
• CPC Class Codes: C12N 9/1029 transferring groups other than amino-acyl groups (2.3.1)
• CPC Class Codes: Y10S 435/942 Saccharomyces cerevisiae

Abstract

This invention is directed to N.sup.α -acetyltransferase with a molecular weight of about 180,000 daltons, said N.sup.α -acetyltransferase being composed of two subunit peptides having molecular weights of about 95,000 each, having enzyme activity greater than 100 wherein one unit of activity is defined as 1 pmol of acetyl residues incorporated into adrenocorticotropic hormone (ACTH) (amino acids 1-24) under standard assay conditions. This invention is further directed to a method for purifying the N.sup.α -acetyltransferase.

Claims

1. A substantially purified N.sup.α

-acetyltransferase having the following characteristics;

(a) a molecular weight of about 180,000;

(b) two subunit peptides having molecular weights of about 95,000 each;

(c) pH optimum 9.0;

(d) maximum specific activity at temperature from 30°

to 42°

C. wherein one unit of specific activity is defined as 1 pmol of acetyl residues incorporated into a substrate containing the first 24 amino acid residues of adrenocorticotropic hormone (ACTH) per mg of protein under standard conditions;

(e) inhibited by divalent cations Cu²⁺ and Zn²⁺ ; and

(f) an ability to acetylate a substrate peptide or protein molecule wherein said ability is specifically dependent on the amino-terminal residue of said substrate peptide or protein molecule.

2. The N.sup.α

-acetyltransferase of claim 1 having enzyme specific activity greater than 100 wherein one unit of specific activity is defined as 1 pmol of acetyl residues incorporated into a substrate containing the first 24 amino acid residues of adrenocorticotropic hormone (ACTH) per mg of protein under standard assay conditions.

3. The N.sup.α

-acetyltransferase of claim 1 having enzyme specific activity greater than 300 wherein one unit of specific activity is defined as 1 pmol of acetyl residues incorporated into a substrate containing the first 24 amino acid residues of adrenocorticotropic hormone (ACTH) per mg of protein under standard assay conditions.

4. The N.sup.α

-acetyltransferase of claim 1 having enzyme specific activity greater than 1000 wherein one unit of specific activity is defined as 1 pmol of acetyl residues incorporated into a substrate containing the first 24 amino acid residues of adrenocorticotropic hormone (ACTH) per mg of protein under standard assay conditions.

5. The N.sup.α

-acetyltransferase of claim 1 having enzyme specific activity greater than 7000 wherein one unit of specific activity is defined as 1 pmol of acetyl residues incorporated into a substrate containing the first 24 amino acid residues of adrenocorticotropic hormone (ACTH) per mg of protein under standard assay conditions.

6. A method for recovering the substantially purified N.sup.α

-acetyltransferase of claim 1 from a sample containing N.sup.α

-acetyltransferase comprising the following steps;

(a) recovering crude N.sup.α

-acetyltransferase from a sample containing said N.sup.α

-acetyltransferase;

(b) subjecting said crude N.sup.α

-acetyltransferase from step (a) to ion exchange chromatography to obtain active fractions of N.sup.α

-acetyltransferase wherein one unit of specific activity is defined as 1 pmol of acetyl residues incorporated into a substrate containing the first 24 amino acid residues of adrenocorticotropic hormone (ACTH) per mg of protein under standard assay conditions;

(c) applying said active fractions of N.sup.α

-acetyltransferase from step (b) to adsorption hydroxylapatite chromatography to obtain partially purified N.sup.α

-acetyltransferase;

(d) applying said active fractions of N.sup.α

-acetyltransferase from step (c) to ion exchange chromatography to obtain a more highly purified N.sup.α

-acetyltransferase; and

(e) purifying N.sup.α

-acetyltransferase to homogeneity by applying said partially purified n.sup.α

-acetyltransferase from step (d) to an affinity chromatography and eluting therefrom substantially purified N.sup.α

-acetyltransferase.

7. A substantially purified N.sup.α

-acetyltransferase with a molecular weight of about 180,000 daltons, said N.sup.α

-acetyltransferase being composed of two subunit peptides having molecular weights of about 95,000 each, having specific enzyme activity greater than 100 wherein one unit of specific activity is defined as 1 pmol of acetyl residues incorporated into a substrate containing the first 24 amino acid residues of adrenocorticotropic hormone (ACTH per mg of protein under standard assay conditions obtainable by a process comprising the steps of;

(a) recovering crude N.sup.α

-acetyltransferase from a sample containing said N.sup.α

-acetyltransferase;

(b) subjecting said crude N.sup.α

-acetyltransferase from step (a) to ion exchange chromatography to obtain active fractions of N.sup.α

-acetyltransferase wherein one unit of specific activity is defined as 1 pmol of acetyl residues incorporated into a substrate containing the first 24 amino acid residues of adrenocorticotropic hormone (ACTH) per mg of protein under standard assay conditions;

(c) applying said active fractions of N.sup.α

-acetyltransferase from step (b) to adsorption hydroxylapatite chromatography to obtain partially purified N.sup.α

-acetyltransferase;

(d) applying said active fractions of N.sup.α

-acetyltransferase from step (c) to ion exchange chromatography to obtain a more highly purified N.sup.α

-acetyltransferase; and

(e) purifying N.sup.α

-acetyltransferase to homogeneity by applying said partially purified N.sup.α

-acetyltransferase from step (d) to affinity chromatography and eluting therefrom substantially purified N.sup.α

-acetyltransferase.