The 26S Proteasome in Garlic (Allium sativum):  Purification and Partial Characterization

• Published in: Journal of agricultural and food chemistry Vol. 52; no. 11; pp. 3350 - 3355
• Main Authors: Malik, Mazhar N, Spivack, Warren D, Sheikh, Ashfaq M, Fenko, Michael D
• Format: Journal Article
• Language: English
• Published: Washington, DC American Chemical Society 02.06.2004
• Subjects: Agronomy. Soil science and plant productions; Allium sativum; Amino Acid Sequence; amino acid sequences; Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; bulbs; Chemical constitution; Cloning, Molecular; complementary DNA; DNA, Plant - chemistry; Economic plant physiology; enzyme activity; Enzymes and enzyme inhibitors; Fundamental and applied biological sciences. Psychology; garlic; Garlic - enzymology; Hydrolases; molecular cloning; Molecular Sequence Data; molecular weight; nucleotide sequences; Peptide Hydrolases - chemistry; Peptide Hydrolases - genetics; Peptide Hydrolases - isolation & purification; polyacrylamide gel electrophoresis; Protease Inhibitors - pharmacology; Proteasome Endopeptidase Complex; protein subunits; proteinases; proteolysis; purification; Spinacia oleracea; Multicatalytic endopeptidase complex; Monocotyledones; Purification; Enzyme; Characterization; Peptidases; Liliaceae; mass fingerprint analysis; prosome; Angiospermae; Hydrolases; Spermatophyta; Garlic; Molecular biology; Molecular cloning; multicatalytic protease; Allium sativum; polymerase chain reaction
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf035309r

The 26S proteasome (multicatalytic protease complex, MPC) was purified from fresh garlic cloves (Allium sativum) to near homogeneity by ion exchange chromatography on DEAE-sephacel, gel filtration on Sepharose-4B, and glycerol density gradient centrifugation. Two α-type (20S proteasome “catalytic core”) subunits were identified by the direct sequencing of peptide fragments (mass fingerprint analysis, Mass Spectrometry Lab, Stanford University) or the sequencing of a cloned cDNA generated using a garlic cDNA library as the template; these subunits were found to have a high homology to those from other plants. Polyacrylamide gel electrophoresis under denaturing conditions separated the garlic MPC into multiple polypeptides having molecular masses in the range of 21−35 (components of the 20S catalytic core) and 55−100 kDa (components of the 19S regulatory units). The banding pattern of the garlic MCP is similar to that of spinach and rat liver with minor differences in some components; however, polyclonal antibodies against mammalian proteasomes failed to significantly stain the enzyme from garlic. This is the first work to identify the garlic proteasome. Keywords: Allium sativum; multicatalytic protease; prosome; purification; molecular cloning; mass fingerprint analysis; polymerase chain reaction