Robert H. Cagan

Purification of monellin, the sweet principle of Dioscoreophyllum cumminsii

1. 1. The intensely sweet princile from the fruit of the tropical plant Dioscoreo-pyllum cumminsii Diels has been isolated. The sweet principle, herein named monellin, is purified from aqueous extracts by (NH4)2SO4 fractionation and ion-exchange chromatography, first on DEAE-cellulose, and then on CM-cellulose. 2. 2. The yield is over 50% and the material is homogeneous by disc gel electrophoresis. 3. 3. Monellin is non-dialyzable, shows a strong absorption in the ultraviolet (λmax = 277 nm), and reacts with common protein reagents. Purified monellin is free of carbohydrate (< 5 βg/mg protein). This evidence indicates that monellin is a protein.

Biochemical Studies of Olfaction: Binding Specificity of Odorants to a Cilia Preparation from Rainbow Trout Olfactory Rosettes

Abstract: Cilia isolated from the olfactory epithelium (olfactory rosettes) of rainbow trout (Salmo gairdneri) bind amino acids, which are odor stimuli to this species. We demonstrate that L‐threonine, l‐serine, and l‐alanine bind to a common site, TSA, in the cilia preparation. All possible mixtures of two of the amino acids as competitors, with the third as the 3H‐labeled ligand, were studied. The effect of two combined (unlabeled) competitors was always substantially less than additive compared with their actions singly. Along with additional inhibition studies using mixtures of inhibitors, the data show that the three odorants must interact with at least one common binding site, TSA. Binding of l‐[3H]lysine to site L was unaffected by addition of l‐threonine, l‐serine, or l‐alanine, establishing its independence from site TSA. l‐Arginine inhibited binding of l‐[3H]lysine, showing that both of these basic amino acids interact with site L. The data establish the presence, in trout olfactory cilia, of at least two separate and noninteracting populations of odorant binding sites, TSA and L.

Biochemical studies of taste sensation I. Binding of 14C-labeled sugars to bovine taste papillae

Abstract 1. 1. Binding of 14 C-labeled sugars to preparations of bovine taste papillae has been measured. Suspensioned of circumvallate and fungiform papillae, containing taste buds, bind more 14 C-labeled sucrose than do t hose derived from control (filiform) papillae, which are dovoid of taste buds. 2. 2. Additional studoes used only circumvallate and control papillae. The binding of sucrose to circumvallate papillae can be selectively abolished by heating. 3. 3. Sucrose, fructose, and glucos are known to elecit a behavioral preference response in bovine; these sugars bind to circumvallate papillae. Lactose, which does not elicit a similar behavioral response, shows little binding. 4. 4. It is conducted that specificity for taste stimuli exists in the peripheral taste receptors.

Biochemical studies of taste sensation—XII. Specificity of binding of taste ligands to a sedimentable fraction from catfish taste tissue

The specificity of amino acid binding sites in a sedimentable fraction prepared from catfish taste epithelium was examined. Using seven 3H-labeled amino acids as ligands and the unlabeled amino acids in binding competition assays, the presence of possibly three classes of amino acid binding sites was deduced. Site 1 binds L-THR, L-SER, L-ALA and possibly D-ALA and beta-ALA, Site 2 binds L-SER, L-ALA, GLY, D-ALA, and beta-ALA and Site 3 binds L-ARG and L-LYS. Additional evidence supporting the specificity of Site 2 was obtained from the specificity of enhancement of L-ALA binding. The results demonstrate the presence of some major classes of taste receptor sites, and provide a basis for understanding taste receptor specificity at the biochemical level.

Subcellular Distribution of Glutamate Decarboxylase in Rat Olfactory Bulb: High Content in Dendrodendritic Synaptosomes

: The olfactory bulbs in the CNS contain reciprocal dendrodendritic synapses between the granule cells and the secondary dendrites of mitral cells. Based on pharmacologic and electrophysiologic evidence, these synapses are believed to utilize GABA as an inhibitory neurotransmitter. A dendrodendritic synaptosomal fraction has been isolated from rat olfactory bulbs. The upper portion (PB) of the crude nuclear pellet contains 30–40% of the GAD (glutamate decarboxylase) activity of the olfactory bulb homogenate. When PB is purified on a discontinuous sucrose density gradient, 78–85% of the GAD activity is localized to the region containing the dendrodendritic synaptosomes, which were identified by transmission electron microscopy. The presence of a substantial proportion of GAD, the enzyme that catalyzes synthesis of GABA, in the DDS provides neurochemical support for the hypothesis that GABA functions at the reciprocal dendrodendritic synapses in the olfactory bulb.

Fluorescence characteristics of native and denatured monellin.

Abstract Fluorescence emmission spectral characteristics of the sweet-tasting protein monellin were determined. The spectra of the native (i.e. sweet-tasting) protein revealed that the single trytophan of monellin is in a moderately hydrophobic environment and that some fluorescence from tyrosine is observable. Urea or guanidine hydrochloride caused marked changes in the fluorescence emission, evidenced particularly by appearance of an emission peak near 304 nm due to tyrosine, a bathochromic shift of the tryptophan emission peak, and a marked increase in the total spectral band width of the protein from 56 to 93 nm. Denaturation by guanidine hydrochloride (6M) was considerably more rapid than denaturation by urea (8M). The extent of denaturation of monellin depended upon the concentration of denaturant. Guanidine hydrochloride caused its maximal effects by 3M. The changes with urea occured over a broad concentration range, and may not have been complete even at 8 M urea. The progress of denaturation can be quantified using the halfwidth of the total protein emission band as measured from the trytophan fluorescence maximum. It can also be followed using other fluorescence parameters: the emission polarization, the relative quantum yield, the wavelength maxima of the emission peak(s), or a parameter, r , defined as the ratio of the maximum peak height of the tryptophan emission to that from tyrosine. Although each of these parameters could serve as an index of denaturation for monellin, the most accurate and convenient measure is the halfwidth. It is suggested that the fluorescence emission spectrum of monellin is sensitive to the conformation because the protein has a residue ratio of tyrosine to tryptophan of seven and is composed of two polypeptide chains. The single trytophan of monellin is present in only one of the two chains (of necessity), while the seven tyrosines are distributed between the two chains. Thus denaturation may force not only the randomization of the conformation but also some physical separation of the two chains. It is concluded that the emission spectrum halfwidth, which is sensitive to the increasing contribution from tyrosine emission, is a measure of the overall conformation of the protein and may serve as an indicator of the biological activity, sweetness, of monellin in solution.

Methyltion of the Lysine Residues of Monellin

Summary The €-amino groups of the lysyl residues of monellin were reductively methylated with formaldehyde and sodium borohydride; 20-40% of the lysines could be methylated with essentially complete retention of the sweetness of the protein. The methylated protein yielded dimethyllysine and monomethyllysine upon acid hydrolysis. 3H-Labeled methylated monellin was also prepared with [3H]formaldehyde as the methyl donor; this derivative could be useful in binding studies to taste receptors. The methylated monellin was studied by ion-exchange chromatography, gel filtration, fluorescence spectroscopy, polyacryl-amide gel electrophoresis, and amino acid analysis. Although sweetness was maintained after limited methylation, some change in conformation of the protein did occur.

Formation of Oligomeric Monellin in Protein Denaturants

Abstract Monellin is a protein with an intense sweet taste. It is known to consist of two dissimilar polypeptide chains that are tightly but noncovalently bound. Monellin is also known to contain a single sulfhydryl group; in native monellin it appears to be buried within the interior of the protein. An oligomer of monellin is demonstrated to form in the presence of protein denaturants. It appears to involve dimerization through formation of a disulfide linkage. Formation of larger aggregates, which occurs during removal of denaturant, is postulated to involve the molecular species [monellin]2 as an intermediate.

Biochemical Studies of Taste Sensation V. Binding of Quinine to Bovine Taste Papillae and Taste Bud Cells

Quinine tastes bitter to humans and is rejected by cows in taste tests. In this study quinine was used as a ligand in binding experiments with various types of preparation of taste and non-taste tissues from bovine tongue using fluorescence to quantitate quinine binding. Studies were carried out attempting to demonstrate binding specificity of the ligand using taste papillae in situ, those excised from the tongue, homogenates of entire papillae, homogenates of epidermis of circumvallate papillae, and isolated taste cell suspensions; in each experiment, comparable control tongue tissue devoid of taste buds was included.Binding of small quantities of quinine to tissue can be reliably determined. In general, however, there is as much binding to non-taste preparations as occurs to those which contain taste buds. In contrast to earlier experiments involving taste stimulus molecules for which specific binding could be demonstrated, the present experiments using quinine show that the hypothesis of peripheral spe...

The sulfhydryl group of monellin: its chemical reactivity and importance to the sweet taste.

Summary The presence of a single cysteine in the sweet-tasting protein monellin was confirmed by titrations with p-hydroxy-mercuribenzoate (PHMB) and 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB). The sulfhydryl group in native monellin reacts very slowly with each of these reagents, indicating that the sulfhydryl is relatively inaccessible. In the presence of either 6 M guanidine-HCl, 8 M urea, or 1% sodium dodecyl sulfate, the rate of reaction of the sulfhydryl group with titrant is dramatically increased. Under a variety of conditions, the presence of 1 mole of sulfhydryl per mole of protein (of molecular weight 10,700) was found. Reaction of the sulfhydryl by titration with PHMB or DTNB leads to loss of sweetness. The free sulfhydryl is also lost by carboxymethylation of monellin in the presence of guanidine-HCl, yielding a protein that is not sweet. Exposure to air in the presence of denaturant leads to a decrease in the sweetness of monellin. Sweetness of the PHMB-reacted monellin can be recovered upon treatment of the protein with mercaptoethanol, and the partial loss of sweetness that occurs with air exposure is lessened in the presence of mercaptoethanol. It is postulated that alteration of the single sulfhydryl group of monellin leads to a change in the tertiary structure of the protein and hence its sweet taste. This work was supported in part by Research Contract No. NIH-NIDR-72-2413 from the National Institute of Dental Research, USPHS (to R.H.C.). We thank Mr. H. L. O. Holloway of the Crops Research Institute, Bunso, Ghana, for his continuing cooperation in supplying us with fresh fruits of D. cumminsii. We thank Miss Linda Graham for technical assistance in the purification of monellin.