Purification and characterisation of a polymorphic low M(r) bovine muscle cysteine proteinase inhibitor: structural identity with fatty-acid-binding proteins.
Three low molecular mass cysteine proteinase inhibitors were purified from a bovine skeletal muscle crude extract using a three-step procedure. The crude extract was first subjected to gel filtration on a Sephadex G100 column which separated five active fractions (F-I to F-V). Three papain inhibitors, P1, P2 and P3, were fractionated from the F-V fraction by chromatofocalisation on a poly buffer exchanger column. Purification was completed by chromatography on a Mono Q column. After SDS-PAGE, the three inhibitors showed only one band with an M(r) of 14,300. P1, P2 and P3 appeared to be highly resistant to temperature (40-90 degrees C), pH (3-10), reducing agents (5-50 mM) and to be specific for cysteine proteinases since no activity was detected against either serine or aspartyl proteinases. Although to a varying extent, P1, P2 and P3 inhibited papain, cathepsin B and cathepsin L. Analysis of the peptide mixtures of these inhibitors by RP-HPLC after hydrolysis with CNBr or aspartly endoproteinase N together with their amino acid composition revealed that P1, P2 and P3 cysteine proteinase inhibitors are isoforms of the same protein. As their N-terminal ends were blocked, partial sequence of some of these peptides was determined. Computer search in protein identification resources did not reveal any homology of these sequences with proteinase inhibitors of known primary structure. In contrast, they matched well with different parts of the total sequence of a fatty acid binding protein isolated from bovine heart. This homology was supported by the ability of these inhibitors to bind long chain fatty acids.(ABSTRACT TRUNCATED AT 250 WORDS)
Zabari
M
M
Muscle Biochemistry Unit, SRV, INRA de Theix, Saint-Genès-Champanelle, France.
Berri
M
M
Rouchon
P
P
Zamora
F
F
Tassy
C
C
Ribadeau-Dumas
B
B
Ouali
A
A
D000478:Alkylation; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D015853:Cysteine Proteinase Inhibitors / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D050556:Fatty Acid-Binding Proteins; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000737:chemistry*; D009363:Neoplasm Proteins; D010084:Oxidation-Reduction; D010206:Papain / Q000037:antagonists & inhibitors*; D013379:Substrate Specificity
D002352:Carrier Proteins; D015853:Cysteine Proteinase Inhibitors; D050556:Fatty Acid-Binding Proteins; D005231:Fatty Acids, Unsaturated; D009363:Neoplasm Proteins; D010206:Papain; C013641:parinaric acid