The 26S proteasome in garlic (Allium sativum): purification and partial characterization.
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 alpha-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.
Malik
Mazhar N
MN
Department of Neuropharmacology, New York State Institute for Basic Research in Developmental Disabilities, 1050 Forest Hill Road, Staten Island, New York 10314, USA.
Spivack
Warren D
WD
Sheikh
Ashfaq M
AM
Fenko
Michael D
MD
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018744:DNA, Plant / Q000737:chemistry; D005737:Garlic / Q000201:enzymology*; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex
D018744:DNA, Plant; D011480:Protease Inhibitors; D010447:Peptide Hydrolases; D046988:Proteasome Endopeptidase Complex; C061553:ATP dependent 26S protease