title: A cysteine-activated protease isolated from Todarodes pacificus squid degrades collagen below its denaturation temperature.
abstract: Collagen purified from the mantle muscle of the Japanese common squid, Todarodes pacificus, showed autodegradation during incubation under acidic conditions at 25 degrees C, without the addition of exogenous enzymes. This suggests that the collagenolytic proteases bind to collagen tightly through the steps of collagen preparation. Collagenolytic activity also was detected in a crude extract of mantle muscle, and leupeptin and E-64 were observed to inhibit collagenolytic activity within the collagen fraction and muscle extract. We purified these collagenolytic cysteine proteases by leupeptin column chromatography and cellulose acetate membrane electrophoresis. Optimal enzymatic activity was observed at pH 3.5, and collagenolytic activity was completely suppressed at neutral or alkaline pH. The purified enzymes were 28 kDa and 25 kDa in size, and both had gelatinolytic activity, as detected by gelatin zymography, and cut the specific site of denatured collagen alpha chain. The purified enzymes degraded squid collagen at 25 degrees C, which is 2.5 degrees lower than the temperature at which squid collagen normally denatures; however, the proteases were ineffective at 20 degrees C. Interestingly, the isolated proteases were capable of digesting both squid and bovine gelatin. In this article, we describe collagenolytic cysteine proteases that bind to the collagen of Todarodes pacificus, thereby digesting it by attacking microunfolding regions generated by incubation 2-3 degrees C below the denaturation temperature.
authors.affiliation: Nippi Research Institute of Biomatrix, Tokyo, Japan.
authors.forename: Yohko
authors.initials: Y
authors.lastname: Miura-Yokota
authors.forename: Youco
authors.initials: Y
authors.lastname: Matsubara
authors.forename: Tetsuya
authors.initials: T
authors.lastname: Ebihara
authors.forename: Shunji
authors.initials: S
authors.lastname: Hattori
authors.forename: Shinkichi
authors.initials: S
authors.lastname: Irie
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D003094:Collagen / Q000737:chemistry / Q000378:metabolism*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000502:physiology*; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology; D049832:Decapodiformes / Q000737:chemistry / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007976:Leupeptins / Q000494:pharmacology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009132:Muscles / Q000737:chemistry / Q000201:enzymology; D049831:Octopodiformes / Q000737:chemistry; D011489:Protein Denaturation; D012908:Snails / Q000737:chemistry; D013045:Species Specificity; D013696:Temperature
chemical_list: D015853:Cysteine Proteinase Inhibitors; D007976:Leupeptins; D003094:Collagen; D003546:Cysteine Endopeptidases; D007930:Leucine; C032854:leupeptin; C024974:E 64