Arnold Nordwig

Invertebrate collagens: Isolation, characterization and phylogenetic aspects

Abstract Acid-soluble collagens were extracted from the sea anemone Actinia equina and from the liver fluke Fasciola hepatica and purified by repeated reprecipitation. They share the following properties with collagens from highly developed animals: digestibility by collagenase; important characteristics of amino acid composition (one-third glycine, high imino acid content, presence of hydroxyproline and hydroxylysine); a firmly attached hexose moiety; sedimentation as an extremely sharp boundary in the native state; a high negative value for the specific optical rotation; the ability of forming cross-striated fibrils and segments as seen in the electron microscope. The molecular weight of the collagen from Actinia is in accord with that of vertebrate collagen (310,000). For the collagen isolated from Fasciola a higher value (500,000) was obtained. The subunits of the latter collagen were very labile and could not be characterized in the ultracentrifuge or by disc-electrophoresis. The length and the highly differentiated cross-striation pattern of the long spacing segments from the two invertebrate collagens were found to be identical with those of segments from calf skin collagen and carp swim bladder collagen. This indicates invariance of the tertiary and quaternary structures of the collagen molecule during evolution, and probably reflects also invariance of fundamental features of the amino acid sequence. Two further evolutionary aspects of collagen are discussed. Evidence for only one α-subunit was found in collagen from Actinia , whereas two or three are present in the collagens of highly developed animal phyla. This apparently confirms a hypothesis on the divergent evolution of the collagen α-chain gene. The thermal stability of the collagen molecule in solution depends on the environmental conditions of the animal from which the collagen was prepared. There is no relationship between this property, however, and the phylogenetic stage of the animal.

Investigations on collagen proline hydroxylation in a cell-free system.

Abstract 1. 1. The inhibition of the hydroxylation process converting proline to hydroxy proline was studied in a cell-free system. Enzyme source was the soluble fraction of a homogenate of calf-embryo skin. A neutral-salt-soluble, purified collagen preparation served as substrate. During its biosynthesis this substrate incorporates a precursor of the molecule which contains proline labelled with 14C and is free of hydroxyproline. Some of the investigations were duplicated using synthetic polypeptides having collagen-like structure as substrate. 2. 2. The hydroxylating enzyme is inhibited by chelating compounds such as 2,2′-dipyridyl, o-phenanthroline or EDTA. Some of the results obtained with the cell-free system differ from those obtained with intact cells. 3. 3. Experiments on reactivation of the inhibited enzyme indicate that Fe2+ is indispensable for the activity of the hydroxylase. 4. 4. Both the native and the denatured form of the collagen substrate are hydroxylated to the same extent. It thus appears that the primary structure of collagen is responsible for the selective action of the hydroxylase. 5. 5. The procedure of Juva and Prockop 1 for determining small amounts of radioactive hydroxyproline in the presence of labelled proline by specific oxidation to pyrrole was slightly modified. Assay of biosynthetic formation of hydroxyproline may now be performed more simply and rapidly.

Specificities of protocollagen hydroxylases from different sources.

Abstract 1. 1. Protocollagen hydroxylase from calf embryo skin is able to hydroxylate only proline residues in position I of synthetic ( l -Pro-Gly- l -Pro) n substrates. 2. 2. The corresponding enzyme from earthworm body wall is less specific. It converts proline residues in both position 1 and 3 of the polymeric tripeptide substrates to hydroxyproline. In both positions a high amount of 3- l -Hyp is synthesized besides the 4-isomer. 3. 3. When protocollagen is used as a substrate, 3-hydroxyproline is formed by both hydroxylases. The amounts are close to the values of 3-Hyp found in the collagens of the respective tissues from which these enzymes were extracted. 4. 4. Hydroxylase preparations from both sources lose half of their activity when once frozen and thawed. The original level of activity may be restored by addition of α-ketoglutarate.

Nonspecific collagenolytic activity: hydrolysis of native collagen by a mold protease.

Abstract A proteolytic enzyme from culture fluids of Aspergillus oryzae was purified by a multistep procedure including carrier-free electrophoresis and DEAE-Sephadex chromatography. It was a homogenous protein, judging from results obtained by disc electrophoresis, immunoelectrophoresis and end-group determination. The enzyme hydrolysed native collagen at a slow rate. Electron microscopic investigations of the digest revealed triple-helical fragments remaining after degradation of the collagen molecule at its N -terminal end. The results thus contribute to a current discussion on collagenolysis by proteolytic enzymes other than specific collagenases.