Milton J. Cormier

Primary structure of the Aequorea victoria green-fluorescent protein.

Many cnidarians utilize green-fluorescent proteins (GFPs) as energy-transfer acceptors in bioluminescence. GFPs fluoresce in vivo upon receiving energy from either a luciferase-oxyluciferin excited-state complex or a Ca(2+)-activated phosphoprotein. These highly fluorescent proteins are unique due to the chemical nature of their chromophore, which is comprised of modified amino acid (aa) residues within the polypeptide. This report describes the cloning and sequencing of both cDNA and genomic clones of GFP from the cnidarian, Aequorea victoria. The gfp10 cDNA encodes a 238-aa-residue polypeptide with a calculated Mr of 26,888. Comparison of A. victoria GFP genomic clones shows three different restriction enzyme patterns which suggests that at least three different genes are present in the A. victoria population at Friday Harbor, Washington. The gfp gene encoded by the lambda GFP2 genomic clone is comprised of at least three exons spread over 2.6 kb. The nucleotide sequences of the cDNA and the gene will aid in the elucidation of structure-function relationships in this unique class of proteins.

Purification of plant calmodulin by fluphenazine-Sepharose affinity chromatography

The calcium-dependent binding of phenothiazine drugs to calmodulin (Levin, R. M. and Weiss, B. (1977) Mol. Pharmacol. 13, 690–697) has been utilized to develop a rapid purification procedure for calmodulin based on fluphenazine-Sepharose affinity chromatography. Calmodulin from plant, a fungus, porcine brain and the coelenterate, Renillareniformis, were easily purified by the calcium-dependent binding of calmodulin to fluphenazine-Sepharose.

Cloning and expression of the cDNA coding for aequorin, a bioluminescent calcium-binding protein

Aequorin is a bioluminescent protein which consists of a polypeptide chain (apoaequorin), coelenterate luciferin, and bound oxygen. Aequorin produces blue light upon binding Ca2+. We have isolated six recombinant pBR322 plasmids which contain apoaequorin cDNA sequences. A mixed synthetic pBR322 plasmids which contain apoaequorin cDNA sequences. A mixed synthetic oligonucleotide probe was used to identify these cDNAs. An extract of an E. coli strain possessing the largest cDNA contained apoaequorin. This apoaequorin can be converted to aequorin in the presence of coelenterate luciferin, 2-mercaptoethanol, and O2. This cDNA is therefore apparently full-length.

SPECTROPHOTOMETRIC IDENTITY OF THE ENERGY TRANSFER CHROMOPHORES IN RENILLA AND AEQUOREA GREEN‐FLUORESCENT PROTEINS

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 GFPs 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.

High levels of a calcium-dependent modulator protein in spermatozoa and its similarity to brain modulator protein.

Abstract Sperm from hamster, human, rooster, rabbit and sea urchin were found to contain relatively high levels of calcium-dependent modulator protein. Using rabbit sperm the modulator protein was found to be exclusively located in the sperm head fraction (nuclei + acrosomes) with no activity present in the midpiece or tail regions. The modulator protein represents approximately 12% of the total soluble protein found in the sperm head fraction and is similar to porcine and brain modulator proteins in its ability to activate brain cyclic nucleotide phosphodiesterase, its heat stability and electrophoretic migration. We have also observed modulator protein to be present in high levels in sea urchin eggs.

ENERGY TRANSFER VIA PROTEIN-PROTEIN INTERACTION IN RENILLA BIOLUMINESCENCE

Abstract—Radiationless energy transfer is known to play biologically important roles in both photosynthesis and bioluminescence. In photosynthesis, accessory pigments serve as “antennae”, transferring excitation energy into the “reaction centers”. In the bioluminescent coelenterates, energy is transferred from the site of reaction via an accessory protein known as the green‐fluorescent protein (GFP). Coelenterate bioluminescence systems such as that of the sea pansy, Renilla, are well characterized biochemically, and their energy transfer process can be duplicated in vitro using isolated and purified components. We have measured efficient in vitro energy transfer from the electronic excited state of the enzyme‐bound oxyluciferin to the green‐fluorescent protein at protein concentrations of 0.1 μM. We have also demonstrated a 1:l complex between these proteins, under conditions of energy transfer, by the chromato‐graphic technique of Hummel and Dreyer. These observations indicate that bioluminescent energy transfer is mediated via protein‐protein interaction. Furthermore, with inter‐species cross‐reaction studies and protein modification techniques we have shown that the interaction between luciferase and GFP is highly specific. These features make the Renilla system an attractive alternative to the photosynthetic systems as a tool for studying radiationless energy transfer.

An enzymatic assay for calmodulins based on plant NAD kinase activity.

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5s ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5s for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.

[3] Purification of calmodulin by Ca2+-dependent affinity chromatography

Publisher Summary This chapter discusses the purification of calmodulin by Ca 2+ -dependent affinity chromatography. The chapter describes the use of phenothiazine conjugates in calmodulin purification. There are four proven purification steps that can be used effectively for preparing extracts for affinity chromatography: heat treatment, ammonium sulfate fractionation, diethylaminoethyl (DEAE) batch step, and trichloroacetic acid precipitation. Calmodulin have been purified from many different sources representing vertebrates, invertebrates, plants, and fungi. There is no unique combination of these steps that can function universally for the preparation of extracts for affinity chromatography. Each tissue or organism tends to have unique purification problems and for these reasons, this chapter presents an outline of the protocols that are usually applicable for use with phenothiazine affinity chromatography. Frequently, many calmodulin samples eluted from phenothiazine conjugates have other contaminants. Therefore, it is necessary to use a final purification step to remove these contaminants. Ion-exchange steps usually work well for achieving final purification.

Plant and fungal calmodulin: Ca2+-dependent regulation of plant NAD kinase

Although little is known about the role(s) of second messengers, including free Ca2+, in plant cells there has been increasing evidence for a role for Ca2+ in metabolic regulation in plants. The recent demonstration that the Ca2+-binding protein, calmodulin exists in extracts of higher plants and basidiomycete fungi provides a basis for understanding Ca2+-dependent metabolic regulation in plant cells. In this review we summarize the similarities and differences of plant, fungal and mammalian calmodulin. We also discuss the known in vitro functions of calmodulin in higher plants. A Ca2+-calmodulin-dependent NAD kinase has been purified to homogeneity from extracts of pea seedlings and shown to be absolutely dependent upon calmodulin and microM levels of free Ca2+ for activity. The available evidence suggest that this Ca2+-calmodulin-dependent NAD kinase is the major form of plant NAD kinase and that this regulatory enzyme is localized in the chloroplast. A model is presented which predicts that the rate of photosynthesis is regulated by a receptor-mediated change in the level of chloroplastic free Ca2+ upon illumination. Free Ca2+, acting as a second messenger, forms a Ca2+-calmodulin complex thus converting calmodulin to its active conformation. This Ca2+-calmodulin complex then activates chloroplastic NAD kinase resulting in an increased NADP/NAD ratio.

Studies on the bioluminescence of Renilla reniformis III. Some biochemical comparisons of the system to other Renilla species and determination of the spectral energy distributions

Abstract The biochemical requirements for bioluminescence in extracts of Renilla reniformis have been compared to two other Renilla species and have been found to be the same. In addition, the efficiency of cross-reactions between the luciferins and luciferases of the various Renilla species tested indicated that there is little, if any, difference in the chemical structure of the luciferins as well as in the active-site area of the luciferases. Furthermore, the function of 3′,5′-diphosphoadenosine in the light reaction, namely that of activating luciferin, has been found to be the same for all species investigated. In addition to the biochemical comparisons mentioned above, the spectral energy distribution for Renilla reniformis has been determined and compared to the bacterial and firefly emissions and to a newly discovered chemiluminescent reaction. The maximum emission is at 485 mμ with limits between 370 and 650 mμ. Thus, all colors of the spectrum are represented including some near ultraviolet.