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Occurrence and properties of a thermostable protease secreted by sea-urchin embryos.

ABSTRACT: 1. A trypsinogen-activating enzyme is distinct from the chorion-dissolving enzyme of the sea-urchin blastula, but is secreted with it. 2. This ;trypsinogenase' does not attack N-alpha-toluene-p-sulphonylarginine methyl ester, and is not inhibited by concentrations of di-isopropyl phosphorofluoridate or 7-amino-1-chloro-3-toluene-p-sulphonamidoheptan-2-one that are 98% effective against trypsin. It requires a small amount of Ca(2+) for activity, but is inhibited by more, from as little as 1mm. 3. Enzymic activity is associated with a wide range of particle weights, some apparently as high as 15x10(6). The mean particle weight can be decreased by sonication. 4. Fresh trypsinogenase in the higher range of particle weights appears to be remarkably stable to heating, e.g. retaining full activity after boiling for 15min.

SUBMITTER: Barrett D

PROVIDER: S-EPMC1178830 | biostudies-other | 1970 Mar

REPOSITORIES: biostudies-other

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Project description:The sea urchin Paracentrotus lividus (Lamarck, 1816) is a keystone herbivore in the Mediterranean Sea due to its ability to transform macroalgal-dominated communities into barren areas characterized by increased cover of bare substrates and encrusting coralline algae, reduced biodiversity and altered ecosystem functions. P. lividus is also an excellent animal model for toxicology, physiology and biology investigations having been used for more than a century as a model for embryological studies with synchronously developing embryos which are easy to manipulate and analyze for morphological aberrations. Despite its importance for the scientific community, the complete genome is still not fully annotated. To date, only a few molecular tools are available and a few Next Generation Sequencing (NGS) studies have been performed. Here we aimed at setting-up an RNA extraction method to obtain high quality and sufficient quantity of RNA for NGS from P. lividus embryos at the pluteus stage. We compared five different RNA extraction protocols from four different pools of plutei (500, 1000, 2500 and 5000 embryos): TRIzol®, and four widely-used Silica Membrane kits, GenElute™ Mammalian Total RNA Miniprep Kit, RNAqueous® Micro Kit, RNeasy® Micro Kit and Aurum™ Total RNA Mini Kit. The quantity of RNA isolated was evaluated using NanoDrop. The quality, considering the purity, was measured as A260/A280 and A260/230 ratios. The integrity was measured by RNA Integrity Number (RIN). Our results demonstrated that the most efficient procedures were GenElute, RNeasy and Aurum, producing a sufficient quantity of RNA for NGS. The Bioanalyzer profiles and RIN values revealed that the most efficient methods guaranteeing for RNA integrity were RNeasy and Aurum combined with an initial preservation in RNAlater. This research represents the first attempt to standardize a method for high-quality RNA extraction from sea urchin embryos at the pluteus stage, providing a new resource for this established model marine organism.

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Occurrence and properties of a thermostable protease secreted by sea-urchin embryos.

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