Reiji Yoshinaka

Molecular species of collagen in the intramuscular connective tissue of fish

Abstract 1. 1. The major collagen of intramuscular connective tissue of carp, lizardfish, Japanese eel, sturgeon and spotted shark was found to be Type I collagen. 2. 2. The major collagen of lamprey intramuscular connective tissue was found to be different from the Type I collagen isolated from lamprey skin and to be similar to the invertebrate muscle collagen. 3. 3. Type III-like collagen was not present in a detectable amount in the intramuscular connective tissue of the fish examined. 4. 4. Type V collagen was widely distributed in the intramuscular connective tissue of the fish examined and conserved its amino acid composition during evolution from cyclostome to mamals more closely than Type I collagen.

Isolation of types I and V collagens from carp muscle

1. The major constituent of carp intramuscular connective tissue was found to be Type I collagen. 2. A collagen homologous to Type V collagen of higher vertebrate was also isolated from carp muscle. 3. Relative portion of Type V collagen was higher in carp muscle than in mammalian muscles.

Extracellular matrix formation by amebocytes during epithelial regeneration in the pearl oysterPinctada fucata

SummaryTo identify the cells which produce the extracellular matrix during bivalve wound healing, we observed epithelial regeneration inPinctada fucata and evaluated the ability of amebocytes to produce the matrix in vitro. Between days 1 and 3 after an ovary was implanted with abiotic material (a shell ball) via an incision, agranular amebocytes formed a sheath, consisting of 10–20 cell layers, between the implant and incised ovarian tissue. Extracellular matrix was deposited in the spaces between the amebocytes in the sheath. At the incised follicle, gonadal epithelial cells were attached to the newly formed matrix. When a mantle allograft (2 mm square) was implanted with abiotic material to bring them into close contact, epithelial cells emigrated from the allograft along the surface of the abiotic material where they attached to the newly formed matrix at the sheath of amebocytes. In vitro, agranular amebocytes formed a matrix composed of fibrils with a diameter of 20 nm during a 6-day culture period. Pepsin-digested extract of the cell layer forming the matrix gave protein bands with electrophoretic mobilities identical to α- and β-sized components of a collagen purified from this animal. The matrix exhibited immunoreaction to antiserum raised against the collagen and was stained by alcian bluc. Thus, the agranular amebocyte apparently has the ability to produce an extracellular matrix containing collagen and possibly proteoglycan(s).

Enzymatic characterization of anionic trypsin of the catfish (Parasilurus asotus).

An anionic trypsin, isolated from the pancreatic extract of the catfish (Parasilurus asotus), had a pH optimum of 8.3 for the hydrolysis of N-tosyl-L-arginine methyl ester. The enzyme was most stable at pH 6.0-8.5, and was stabilized by calcium ions. The enzyme was inhibited by typical trypsin inhibitors including some serine proteinase inhibitors. Km and kcat values of the enzyme for N-tosyl-L-arginine methyl ester and N-tosyl-L-lysine methyl ester were quite similar to those of bovine cationic trypsin.

Distribution of collagen in body muscle of fishes with different swimming modes

Abstract 1. 1. The collagen content in different parts of body muscle of fishes with different swimming modes, including anguilliform swimmers, Japanese eel and stone flounder; subcarangiform swimmer, red sea bream; carangiform swimmer, chub mackerel; thunniform swimmer, bluefin tuna, was examined in order to elucidate the biological significance of the muscle collagen of fish. 2. 2. The results indicate that the collagen content and its distribution pattern in body muscle are closely related to swimming modes of fishes and that the flexible body musculature comprises a high proportion of collagen.

Two genetically distinct types of collagen in kuruma prawn penaeus japonicus

Abstract 1. 1. At least three types of collagen was found in the muscle of kuruma prawn Penaeus japonicus . Two of them, called AR-I and AR-II collagens, were isolated from the pepsin-solubilized collagen. 2. 2. The subunit composition of AR-I collagen, major molecular species, was proved to be (α I) 3 homotrimer. Its amino acid composition was similar to that of Type V collagen rather than Type I collagen. 3. 3. AR-II collagen, minor molecular species, was found to have disulfide bonds in the molecule. Its amino acid composition was similar to that of AR-I collagen. The sodium dodecyl sulfate-polyacrylamide gel electrophoresis pattern of AR-II collagen suggested the complexes of its structure.

Content and partial characterization of collagen in crustacean muscle

Abstract 1. 1. The collagen content in the abdominal muscle of seven species including shrimp, prawn, lobster and squilla varied among the species ranging from 1.1 to 6.2% of total tissue protein and the content in pereiopod and thoracic muscles of four species of crab varied ranging from 0.2 to 0.8%. 2. 2. These results indicate that the musculature in flexible part comprises a high proportion of collagen. 3. 3. The major collagen from the crustacean muscle was found to be similar to Type V collagen from the vertebrate muscle with respect to the solubility and amino acid composition.

Isolation and partial characterization of a new alpha component of collagen from muscle of kuruma prawn Penaeus japonicus

Abstract 1. 1. A new α component, α2(Pr), was successfully isolated from the pepsin-solubilized collagen from the muscle of kuruma prawn. 2. 2. The component α2(Pr) was genetically distinct from components comprising Type AR-I and AR-II collagens by peptide mapping with proteases, amino acid analysis, and immunoblotting, and had high contents of leucine and hydroxylsine, and a low content of alanine. 3. 3. The effect of pepsin digestion on the molecule containing the α2(Pr) component was examined by using immunological techniques. The component α2(Pr) in intact form consisted of several types of components. Although they were all identical to each other in the helical region, each of them had a distinct form of non-helical region with a slight modification.

Immunohistochemical localization of genetically distinct types of collagen in muscle of kuruma prawn Penaeus japonicus

Abstract 1. 1. The location of genetically distinct types of collagen in muscular tissue of the kuruma prawn was examined using immunohistochemical techniques. 2. 2. Collagen was distributed not only in muscle connective tissues, which were classified into three forms, epimysium, perimysium and endomysium, but also in subcuticular membrane, which was mainly composed of two layers, hypodermis and subcuticular connective tissue. 3. 3. The α1(Pr) component existed in all connective tissues in the kuruma prawn muscle. Type AR-II collagen was distributed in all the connective tissues except for the hypodermis, while the α2(Pr) component existed in the thin connective tissues, the perimysium and endomysium, and in the hypodermis.

THE IMMUNOLOGICAL STUDY ON THE EFFECT OF PEPSIN DIGESTION ON GENETICALLY DISTINCT TYPES OF COLLAGEN IN MUSCLE OF KURUMA PRAWN PENAEUS JAPONICUS

Abstract 1. 1. The effect of pepsin digestion on each type of collagen from the muscle of kuruma prawn Penaeus japonicus was examined by immunological techniques. 2. 2. The major collagen (named type AR-I) in intact form comprised of several types of α-components. Although they were all identical to each other in the helical region, each of them had a distinct form of non-helical domain with a slight modification. 3. 3. The minor collagen (named type AR-II) in intact form had the large non-helical domain sensitive to pepsin digestion and disulfide bonds in its molecule. 4. 4. Another type of minor collagen was proved to be present in the prawn muscle.