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Renilka reniformis is an anthozoan coelenterate capable of exhibiting bioluminescence. Bioluminescence in ReniUa results from the oxidation of coelenterate luciferin (coelenterazine) by luciferase [Renilka-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.51. In vivo, the excited state luciferin4uciferase complex undergoes the process of nonradiative energy transfer to an accessory protein, green fluorescent protein, which results in green bioluminescence. In vitro, Renila luciferase emits blue light in the absence ofany green fluorescent protein. A Renifa cDNA library has been constructed in Agtll and screened by plaque hybridization with two oligonucleotide probes. We report here the isolation and characterization of a luciferase cDNA and its gene product. The recombinant luciferase expressed in Escherichia coli is identical to native luciferase as determined by SDS/PAGE, immunoblot analysis, and bioluminescence emission characteristics.Renilla reniformis (class Anthozoa) is a bioluminescent soft coral found in shallow coastal waters ofNorth America, which displays blue-green bioluminescence upon mechanical stimulation (1, 2). The components involved in Renilla bioluminescence have been described in detail (3). Renilla luciferase [Renilla-luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5] catalyzes the oxidative decarboxylation of coelenterazine in the presence of dissolved oxygen to yield oxyluciferin, C02, and blue light (Am. = 480 nm) (4). This reaction has a bioluminescence quantum yield of =7%. The stoichiometry of this reaction and the detailed mechanism leading to excited-state formation have been described (4,5).The color of in vitro-catalyzed bioluminescence changes from blue to green upon addition of submicromolar amounts of an energy-transfer acceptor green fluorescent protein (GFP), which has been purified from Renilla and characterized (6). This green fluorescence (Amax = 509 nm) is identical to the in vivo emission in Renilla. The energy-transfer process is nonradiative; an increase in both the quantum yield (6) and calculated lifetimes has been determined for this process (7). Luciferase and GFP form a specific 1:1 rapid equilibrium complex in solution (8).The elucidation of mechanisms involved in nonradiative energy transfer processes as well as determination of detailed structural information on both luciferase and GFP have been hindered by a lack of material. To overcome this, we have cloned, sequenced, and expressed in Escherichia coli a cDNA encoding Renilla luciferase. § MATERIALS AND METHODS Amino Acid Sequence Determination of ReniUa Luciferase. Native Renilla luciferase was isolated as described (4). Purified luciferase was digested with Staphlococcal protease V-8 (9). The resulting peptides were purified by HPLC and subjected to NH2-terminal Edman sequencing as described (10). Based on these peptide sequences two 17-base oligonucleotide probes were synthesized with an Applied Biosystems DNA synthesizer at the Molecular Genetics Instrumentation Faci...

Luciferase from the anthozoan coelenterate Renilla reniformis (Renilla luciferin:oxygen 2-oxidoreductase (decarboxylating), EC 1.13.12.5.) catalyzes the bioluminescent oxidation of Renilla luciferin producing light (lambdaB 480 nm, QB 5.5%), oxyluciferin, and CO2 (Hori, K., Wampler, J.E., Matthews, J.C., and Cormier, M.J. (1973), Biochemistry 12, 4463). Using a combination of ion-exchange, molecular-sieve, sulfhydryl-exchange, and affinity chromatography, luciferase has been purified, approximately 12 000-fold with 24% recovery, to homogeneity as judged by analysis with disc and sodium dodecyl sulfate-polyacrylamide gel electrophoresis, gel filtration, and ultracentrifugation. Renilla luciferase is active as a nearly spherical single polypeptide chain monomer of 3.5 X 10(4) daltons having a specific activity of 1.8 X 10(15) hp s-1 mg-1 and a turnover number of 111 mumol min-1 mumol-1 of enzyme. This enzyme has a high content of aromatic and hydrophobic amino acids such that it has an epsilon280nm 0.1% of 2.1 and an average hydrophobicity of 1200 cal residue-1. The high average hydrophobicity of luciferase, which places it among the more hydrophobic proteins reported, is believed to account, at least in part, for its tendency to self-associate forming inactive dimers and higher molecular weight species.

ABSTRACIA calcium-dependent protein kinase activity from suspension-cultured soybean cells (Glycine max L. Wayne) was shown to be dependent on calcium but not calmodulin. The concentrations of free calcium required for half-maximal histone H1 phosphorylation and autophosphorylation were similar (-2 micromolar). The protein kinase activity was stimulated 100-fold by >10 micromolar-free calcium. When exogenous soybean or bovine brain calmodulin was added in high concentration (1 micromolar) to the purified kinase, calcium-dependent and -independent activities were weakly stimulated (<2-fold). Bovine serum albumin had a similar effect on both activities. The kinase was separated from a small amount of contaminating calmodulin by sodium dodecyl sulfate polyacrylamide gel electrophoresis. After renaturation the protein kinase autophosphorylated and phosphorylated histone H 1 in a calcium-dependent manner.Following electroblotting onto nitrocellulose, the kinase bound 45Ca2+ in the presence of KCI and MgCl2, which indicates that the kinase itself is a high-affinity calcium-binding protein. Also, the mobility of one of two kinase bands in SDS gels was dependent on the presence of calcium. Autophosphorylation of the calmodulin-free kinase was inhibited by the calmodulin-binding compound N-(6-aminohexyl)-5-chloro-I-naphthalene sulfonamide (W-7), showing that the inhibition of activity by W-7 is independent of calmodulin. These results show that soybean calciumdependent protein kinase represents a new class of protein kinase which requires calcium but not calmodulin for activity.One of the ways in which Ca2" may play a second messenger role in plants is to activate protein kinases in response to stimuli such as growth regulators, light, or stress (5). The receptor and transducer of the calcium signal could be the Ca2"-binding protein calmodulin or the protein kinase itself. There have been several reports ofcalcium/calmodulin-dependent protein kinases (1,20,21,23,25,31) (86-CRCR-1-1969).2Abbreviations: CDPK, calcium-dependent protein kinase; W-7, N-(6-aminohexyl)-5-chloro-l-naphthalene sulfonamide; W-5, N-(6-aminohexyl)--naphthalene sulfonamide; TAPP, 2-trifluoromethyl-1OH-10-(3' aminopropyl) phenothiazine; M,, relative mass; Ko.5, concentration required for 50% activation; IC50, concentration required for 50% inhibition.kinase from suspension-cultured soybean cells (22). This CDPK2 was completely inhibited by 1 mM W-7, a calmodulin-binding compound, and was activated a slight amount (30%) by micromolar concentrations of calmodulin (22)

Abstract— Spectral properties of guanidine‐denaturated and pronase‐digested green‐fluorescent proteins (GFP) from two species of bioluminescent coelenterates have been investigated. Spectrophotometric titrations of Renilla and Aequorea GFP, following denaturation in 6M guanidine HCl at elevated temperature, revealed identical absorption peaks in acid (383–384 nm) and in alkali (447–448 nm) and a single isosbestic point in the visible region at 405 nm. Both proteins exhibited a spectrophotometric pK. of 8.1 in guanidine ‐HCl. Pronase digestion of the heat‐denaturated GFP's generated a methanol‐soluble blue‐fluorescent peptide with identical fluorescence emission spectra (λmax= 430 nm, uncorrected; φf1= 0.003) for both coelenterate species. These data suggest that the large absorption differences between native Renilla and Aequorea GFP molecules result from unique protein environments imported to a common chromophore.