Photoreactivating-enzyme activity in metazoa.

Photoreactivation of ultraviolet (UV) radiation damage to organisms has been demonstrated in many plant and animal phyla.1 In a few cases, this repair process has been shown to involve a light-dependent photoreactivating enzyme which monomerizes U1-induced pyrimidine dimers in DNA.2 3 However, the biological phenomenon of photoreactivation does not necessarily indicate the involvement of a photoreactivating enzyme, since "indirect photoreactivation," not mediated by such an enzyme, has also been described.4 In most cases where biological photoreactivation has been observed, a photoreactivating enzyme has not yet been demonstrated and the mechansim of reactivation is not known. In the sea urchin Arbacia punctulata, the eggs and zygotes are photoreactivable whereas the sperm are not.5 In this same species,6 we have found that photoreactivating-enzyme activity is demonstrable both in egg and in testis. Such a distribution of photoreactivating activity between sperm and testis is unexpected, since the shed sperm are exposed to sunlight in the shallow waters of the open sea, whereas the testis is always well shielded from both UV and visible radiations of the sun by a virtually opaque covering. The demonstration of a light-dependent enzyme in an internal tissue (i.e., one that is always in the dark) led us to inquire whether such a finding would be unique to this one species or this one tissue. A more general distribution of the enzyme would suggest that it might serve some function other than photoreactivation. Accordingly, we have conducted a survey on the distribution of photoreactivating-enzyme activity in various organs of several species representing the higher phyla of metazoa. In addition, we have fractionated frog liver, an internal organ of a vertebrate with a reasonably high level of photoreactivating activity, to determine the subcellular organelles with which the activity is associated. Materials and Methods.-Tissues from the various metazoan species were homogenized in solutions of approximately isotonic potassium chloride (0.1 molar for amphibians, 0.15 molar for mammals, 0.5 molar for arthropods and echinoderms) containing 0.005 molar glutathione, 0.002 molar ethylenediaminetetraacetate (EDTA), 0.005 molar potassium phosphate buffer, pH 7.0, and 20% (v/v) glycerol. Substitution of sodium chloride for potassium chloride or of mercaptoethanol for glutathione did not affect the activity. The tissues were placed in four volumes of appropriate medium and homogenized either with 8 to 10 passes in a Potter-Elvehjem homogenizer or (for muscle) with a Virtis homogenizer or (for skin) by grinding with sand. The enzyme was then more completely solubilized by two 30sec treatments of the homogenate with a Bronwill "Biosonik" sonicator at a power setting of 60. Sonication for up to 5 min did not diminish the activity of the extracts. The crude homogenates were centrifuged at 2000 X g for 15 min, and the supernatant fractions were assayed for photoreactivating activity. All preparative procedures were performed at 0-40C. Activity was measuired by the assay which Mfuhammed7 developed from the experiments of Goodgal, Rupert, and Hefriott,8 i.e., by the ability of cell extracts to increase, in the light, the biological activity of UV-irradiated transforming DNA. A tissue extract was incubated at 37°C under "blacklight" illumination (320-420 mMu; 7000 ergs mm-2 min-') with transforming DNA from a streptomycin-resistant strain of Hemophilus influenzae. The DNA had been irradiated to 1% survival of the streptomycin marker. A 6000-fold excess of calf thymus DNA, which does