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R MacColl

Publications and source records attributed to R MacColl.

At least 37 records · Page 2Linked to original sources
PubMed1995

Studies on the dissociation of cryptomonad biliproteins.

The spectroscopic properties of two biliproteins, phycocyanin 645 and phycoerythrin 566, have been studied by treating the proteins with two different agents, NaSCN at pH 6.0, or pH 4.0 without NaSCN. For phycoerythrin 566, treatment with NaSCN revealed that the visible CD spectrum of its chromophores was separated into a pair of different spectra, and each of these spectra was observed as a negative and one or more positive bands. For phycocyanin 645, two negative CD bands have been observed previously, together with two or more positive bands, in the dimer (alpha 2 beta 2) state, and NaSCN treatment caused elimination of one of these negative bands. The dimer was stable at pH 6.0, but at pH 4.0 the spectra of phycocyanin 645 had one less negative band than those at pH 6.0. Chromatography demonstrated that phycocyanin 645 was a monomer (alpha beta) at pH 4.0. Monomers of cryptomonad biliproteins have never been observed before. Excitation at 514 nm, in picosecond time-resolved fluorescence studies, produced lifetimes of 11.0 and 45.2 ps for dimers and monomers, respectively. Excitation at 566 nm yielded times of 1.38 and 1.24 ps, for dimers and monomers, respectively. CD in the far UV showed that monomers and dimers had very similar secondary structures. These results have been used to test an hypothesis that proposed two types of exciton splitting among the chromophores of phycocyanin 645, and perhaps phycoerythrin 566 could also have this chromophore organization.

Animals↗
PubMed1995

Spectroscopic study of the interaction of aluminum ions with human transferrin.

Transferrin is the plasma protein responsible for transporting Fe3+ from the absorption to the utilization site. Interactions of apo- and holo-transferrin with Al3+ were studied by circular dichroism (CD), UV-visible, and fluorescence spectrometry. Binding of Al3+ to both metal-ion binding sites of apo-transferrin was confirmed by fluorescence studies. No interaction of Al3+ with holo-transferrin was observed, indicating that Al3+ cannot displace Fe3+ under the experimental conditions employed. An increase in tryptophan fluorescence (lambda max at 330 nm) by excitation at either 280 or 295 nm was observed after Al3+ interaction with apo-transferrin. There was no shift in wavelength of the fluorescence band of apo-transferrin after interaction with Al3+, but the intensity did increase. Since excitation at 295 nm is specific for tryptophan residues, tryptophan but not tyrosine must be responsible for the change in fluorescence intensity. Decreased fluorescence is the result of Fe3+ binding to apo-transferrin. The CD spectrum of apo-transferrin was slightly affected in the far UV by Al3+ binding, but a salient change was noted in the near UV at approximately 288 nm where tyrosine and tryptophan absorb. It is concluded that a small conformational change in the protein was induced by Al3+ binding to apo-transferrin.

Aluminum↗
PubMed1994

Exciton splitting in phycoerythrin 545.

Phycoerythrin 545 is a biliprotein having a polypeptide structure of alpha 2 beta 2, and each alpha and beta polypeptide has chromophores. Circular dichroism (CD) and absorption spectroscopy in the visible region together with various biochemical protocols have been used to study these chromophores. The CD spectrum exhibits overlapping positive and negative bands. Exciton splitting between closely-spaced pairs of chromophores produces a CD spectrum that has positive and negative bands of equal rotational strengths, a conservative spectrum. Alternatively, any positive or negative band could arise from a single chromophore. The results of this study demonstrate that exciton splitting is the likely cause of the negative and corresponding positive bands. The CD spectra of the separated alpha and beta polypeptides, under conditions where the polypeptide structure is denatured, have no negative bands. When the polypeptides are allowed to refold individually, the chromophores on the beta polypeptide regain a combination of negative and positive CD bands. The spectrum of the alpha polypeptide shows no evidence of exciton splitting under these refolding conditions. In another approach, urea is added to the protein in low concentrations, which result in changes in the conformation and perhaps association of the protein. A difference CD spectrum of native protein minus protein in 0.8 M urea shows a spectrum characteristic of exciton splitting. Moreover, the remaining CD spectra in 0.8, 1.6, or 2.4 M urea still show the possibility of further exciton splitting, but a slightly different wavelengths from the spectrum that is deleted by 0.8 M urea. This finding may suggest that there are two types of exciton splitting.

Circular Dichroism↗
PubMed1994

Chromophore topography and exciton splitting in phycocyanin 645.

The biliprotein phycocyanin 645 has been purified from a photosynthetic cryptomonad Chroomonas species. It is composed of two copies each of two polypeptides (alpha and beta); each alpha polypeptide has one chromophore, and each beta polypeptide has three. There are one cryptoviolin and two phycocyanobilins on each beta polypeptide and one 697-nm bilin on each alpha polypeptide for a total of eight chromophores on the protein. Circular dichroism (CD) spectroscopy has been used to investigate the arrangement of these chromophores. Comparisons among the intact protein (alpha 2 beta 2) and various urea-treated products have yielded a tentative model for chromophore topography. The six chromophores on the two beta polypeptides are segregated into three pairs. The chromophores of each pair are close enough to experience electronic interactions. One pair, consisting of the two cryptoviolins, produces exciton splitting on the blue edge of the visible CD spectrum, and the two pairs of phycocyanobilins cause exciton splitting on the red edge of this spectrum. Deconvolution shows that the CD spectrum of each pair has a positive and a negative band, which are nearly conservative as expected for exciton coupling. The two chromophores on the alpha polypeptides are more isolated. The pairing of cryptoviolin chromophores occurs across two beta polypeptides, but the more likely position of each of the two pairs of phycocyanobilins is probably not across a beta-beta interface but within a single beta polypeptide. The exciton splitting events both increase the range of visible light absorption for the protein and establish the routes of exciton migration through the protein.

Circular Dichroism↗
PubMed1993

The binding domain structure of retinoblastoma-binding proteins.

The retinoblastoma gene product (Rb), a cellular growth suppressor, complexes with viral and cellular proteins that contain a specific binding domain incorporating three invariant residues: Leu-X-Cys-X-Glu, where X denotes a nonconserved residue. Hydrophobic and electrostatic properties are strongly conserved in this segment even though the nonconserved amino acids vary considerably from one Rb-binding protein to another. In this report, we present a diagnostic computer pattern for a high-affinity Rb-binding domain featuring the three conserved residues as well as the conserved physico-chemical properties. Although the pattern encompasses only 10 residues (with only 4 of these explicitly defined), it exhibits 100% sensitivity and 99.95% specificity in database searches. This implies that a certain pattern of structural and physico-chemical properties encoded by this short sequence is sufficient to govern specific Rb binding. We also present evidence that the secondary structural conformation through this region is important for effective Rb binding.

Amino Acid Sequence↗
PubMed1990

Effect of oligosaccharides and chloride on the oligomeric structures of external, internal, and deglycosylated invertase.

External invertase exists in an oligomeric equilibrium of dimer, tetramer, hexamer, and octamer, the concentrations of which vary with pH, time, and concentration of enzyme [Chu, F.K., Watorek, W., & Maley, F. (1983) Arch. Biochem. Biophys. 223, 543-555; Tammi, M., Ballou, L., Taylor, A., & Ballou, C.E. (1987) J. Biol. Chem. 262, 4395-4401]. To assess the influence of carbohydrate on this equilibrium, we investigated the self-association of external invertase (10 oligosaccharides per subunit), deglycosylated external invertase (2 oligosaccharides per subunit), and internal invertase (no carbohydrate) under various conditions. In addition, the effect of carbohydrate on the interaction of the subunits of these various invertases to form heterooligomers was studied. Chloride ion was found to promote subunit association in the various invertases irrespective of their glycosylation status. However, external invertase was less responsive to chloride ion relative to the internal and deglycosylated invertases. The higher oligomers of deglycosylated invertase were stable at 47 degrees C whereas those of external invertase dissociated rapidly into dimers, suggesting that the additional oligosaccharides in external invertase destabilize subunit interaction. Hybridization experiments with the various invertases showed that the dimers of internal invertase formed heterooligomers with either external or deglycosylated invertase. By contrast, the monomers of external and internal invertases formed their respective homodimers, but not heterodimers. These results suggest that the oligosaccharide content of invertase not only influences the extent of self-association but also affects heterooligomer formation.

Chlorides↗
PubMed1990

Biliprotein light-harvesting strategies, phycoerythrin 566.

A series of experiments on the light-harvesting properties of the cryptomonad biliprotein phycoerythrin 566 has been carried out on purified protein isolated from Cryptomonas ovata. Although this pigment has an absorption maximum at 566 nm, a property very close to that of other phycoerythrins, it was found to have a totally unique set of chromophores. The chromophores (bilins) responsible for its absorption spectrum were analyzed by a number of approaches. Chromophore-containing peptides were produced by trypsin treatment and purified in order to isolate the individual peptide-bound bilins free of overlapping absorption. These chromopeptides, after comparison with appropriate controls, showed that three spectrally distinct bilins occurred on the purified oligomeric protein. Two of the bilins were the well-known phycoerythrobilin and cryptoviolin, but the third was previously undiscovered and had an absorption spectrum between that of cryptoviolin and phycocyanobilin. Since the spectral diversity of the three bilins was fully maintained in solvents that minimize the effects of apoprotein on the spectra of the bilins, it is likely that the three bilins are also structurally dissimilar. The alpha and beta subunits, which constitute the protein, were separated by ion-exchange chromatography, and the new bilin was found to be the sole chromophore on the alpha subunit. It was also found that at least two alpha subunits could be separated and they both had this unusual bilin (cryptobilin 596). The beta subunit, therefore, contained both phycoerythrobilin and cryptoviolin. On the basis of the spectra of the three chromopeptides, the absorption spectrum of the protein was modeled using the known absorptivities of cryptoviolin and phycoerythrobilin.

Animals↗
PubMed1987

Kinetics of allophycocyanin's trimer-monomer equilibrium.

Kinetic studies of the dissociation of allophycocyanin trimers to monomers have been performed by using stopped-flow techniques. The dissociation was monitored by two techniques: by light scattering to observe the molecular weight changes directly and by 650-nm absorbance to observe the linkage of quaternary structure to spectra. The light-scattering experiments showed a simple exponential decay of trimers to monomers with a dissociation constant of 0.23 s-1. The absorption changes were complex, with two processes occurring. The faster absorption change appeared to be almost simultaneous with the molecular weight change (about 0.27 s-1) and was perhaps totally coordinated with it. The slower absorption change (0.071 s-1) was possibly a result of a conformational change in the chromophore arising during the conversion from newly dissociated monomers to equilibrium monomers.

Cyanobacteria↗
PubMed1987

Inhibition of fibrinolytic system by liquoid (polyanetholesulfonate). A contributing factor in the liquoid-induced renal cortical necrosis.

Liquoid induces microvascular thrombosis and renal cortical necrosis in experimental animals. We hypothesized that thrombosis and renal cortical necrosis may, at least in part, result from the inhibition of the fibrinolytic system by liquoid. Effects of liquoid on plasminogen activation by rat kidney, purified human tissue plasminogen activator (TPA), urokinase, streptokinase, and on the amidolytic activities of TPA, urokinase, and plasmin were studied using chromogenic substrates and clot lysis. Liquoid had a strong inhibitory effect on the fibrinolytic system in vivo and in vitro. The inhibition was most effective at the plasminogen activation level, with activation by streptokinase being most susceptible. The demonstrated stoichiometric binding between liquoid and plasminogen, and to a lesser degree the direct inactivation of plasminogen activators and plasmin, is probably responsible for the reduction of plasminogen activation in circulation and in the kidneys.

Animals↗
PubMed1985

Comparative properties of amplified external and internal invertase from the yeast SUC2 gene.

Saccharomyces cerevisiae external and internal invertases have been amplified by introducing the normal and modified SUC2 genes into yeast multicopy plasmids, which were then used to transform a yeast strain resistant to repression by glucose. Amino acid compositional analysis of these enzymes, in addition to end group sequencing, confirmed the DNA sequence data of Taussig and Carlson (Taussig, R., and Carlson, M. (1983) Nucleic Acids Res. 11, 1943-1954), indicating that both enzymes were encoded in the same gene. Comparison of the properties of carbohydrate-containing external invertase and its nonglycosylated internal form revealed that although the carbohydrate did not appear to influence the conformation of the peptide backbone, as determined by circular dichroism analyses, its presence considerably enhanced the ability of guanidine HCl-denatured external invertase to be renatured relative to internal invertase. The Mr of the internal enzymes was found to be greatly dependent on pH with the enzyme being a monomer at pH 9.4, a dimer at pH 8.3, and an apparent octamer at pH 4.9.

Amino Acid Sequence↗
PubMed1985

Picosecond fluorescence of cryptomonad biliproteins. Effects of excitation intensity and the fluorescence decay times of phycocyanin 612, phycocyanin 645, and phycoerythrin 545.

The fluorescence of purified biliproteins (phycocyanin 645, phycocyanin 612, and phycoerythrin 545) from three cryptomonads, Chroomonas species, Hemiselmis virescens, and Rhodomonas lens, and C-phycocyanin from Anacystis nidulans has been time resolved in the picosecond region with a streak camera system having less than or equal to 2-ps jitter. The fluorescence lifetimes of phycocyanins from Chroomonas species and Hemiselmis virescens are 1.5 +/- 0.2 ns and 2.3 +/- 0.2 ns, respectively, regardless of the fluence of the 30 ps, 532-nm excitation pulse. (Fluence [or photons/cm2] = f intensity [photons/cm2s]dt.). In contrast, that of C-phycocyanin is 2.3 +/- 0.2 ns when the excitation fluence is 8.2 X 10(11) photons/cm2 and decreases to a decay approximated by an exponential decay time of 0.65 +/- 0.1 ns at 7.2 X 10(16) photons/cm2. The cryptomonad phycoerythrin fluorescence decay lifetime is also dependent on intensity, having a decay time of 1.5 +/- 0.1 ns at low fluences and becoming clearly biphasic at higher fluences (greater than 10(15) photons/cm2). We interpret the shortening of decay times for C-phycocyanin and phycoerythrin 545 in terms of exciton annihilation, and have discussed the applicability of exciton annihilation theories to the high fluence effects.

Cyanobacteria↗
PubMed1984

Exciton interaction in allophycocyanin.

The absorption and circular dichroism (CD) spectra of allophycocyanin II in the trimer and monomer (dissociated) forms were resolved into four and two components, respectively. The short-wavelength region of the visible spectra was approximated by a chimera of Lorentzian- and Gaussian-shaped bands having a bandwidth of ca. 65 nm. The rest of the bands have a pure Gaussian form. The characteristic 652-nm band in the absorption spectrum (656 nm in the CD spectrum) is shown to arise from exciton interaction between two fluorescent phycocyanobilin chromophores.

Circular Dichroism↗
PubMed1984

Spectroscopic properties of tetrapyrroles on denatured biliproteins.

Four biliproteins (phycoerythrin 545, phycocyanin 612, phycocyanin 645, and C-phycocyanin) were denatured by a high concentration of urea and then studied by absorption spectroscopy. Low pH and high protein concentrations conserved the tetrapyrroles' color, and mercaptoethanol and dithiothreitol promoted bleaching. One of these tetrapyrroles, cryptoviolin, appeared not to be hypochromic in the presence of depleting phycocyanobilin, but its absorbance did decay when phycocyanobilin is absent. The product from the treatment of phycocyanobilin with mercaptoethanol or dithiothreitol overlapped spectrally with cryptoviolin and gave the false appearance of maintaining a constant cryptoviolin concentration. Failure to note this effect could result in erroneous cryptoviolin/phycocyanibilin ratios.

Chemical Phenomena↗
PubMed1983

Phycocyanin 645. The chromophore assay of phycocyanin 645 from the cryptomonad protozoa Chroomonas species.

Phycocyanin 645 was isolated and purified from the cryptomonad Chroomonas species. Its chromophore content was obtained from absorption spectra in acidic 8.0 M urea for both whole protein and the separated subunits. The principal method used to separate the alpha and beta subunits was gel filtration through a Sephacryl S-200 column in acidic urea. The subunits were shown to be completely separated during this procedure by sodium dodecyl sulfate-gel electrophoresis. Spectra were analyzed by three component Beer's law equations. The whole protein was found to consist of four phycocyanobilins (Amax at 662 nm), two cryptoviolins (Amax at 590 nm), and two unnamed bilins with an Amax at 697 nm. The separated subunits were analyzed, and the beta subunit was shown to have two phycocyanobilins for each cryptoviolin and alpha was composed of the 697-nm bilin exclusively. A comparison of the total amounts of alpha and beta from the Sephacryl columns showed that the molar ratios of phycocyanobilin on beta to the 697-nm bilin on alpha was 2:1, and the ratio of cryptoviolin on beta to 697-nm bilin on alpha was 1:1. We therefore propose that, assuming a symmetrical distribution, each beta subunit on the alpha 2 beta 2 protein has two phycocyanobilins and one cryptoviolin and each alpha subunit has one 697-nm bilin. This chromophore distribution differs from one previously reported in which the subunits were separated on a BioRex 70 cation exchange resin in 12% formic acid via a 4-10 M urea gradient.

Chromatography, Gel↗
PubMed1983

Stability of allophycocyanin's quaternary structure.

The dissociation of allophycocyanin trimers to monomers was examined under a variety of conditions. For alkyl ureas and alcohols the dissociation increased as the straight-chain alkyls increased in length. The effect of branching chains was smaller. Tetrapropylammonium chloride was found to be a very effective agent for trimer dissociation when compared to ureas and alcohols with similar or longer alkyl chains. An explanation for these observations is that the hydrocarbons have an affinity for nonpolar regions in the contact areas between monomers in a trimeric structure. A comparison among several inorganic salts demonstrated that the chaotropic salts (NaSCN greater than NaClO4 much greater than NaNO3 greater than NaBr) fostered increased trimer dissociation, while nonchaotropes (KF, (NH4)2SO4, K phosphate, and NaCl) produced no measurable amounts of monomer. Allophycocyanin dissolved in D2O was much more stable against dissociation than when dissolved in H2O. All the above observations were consistent with hydrophobic forces being the dominant source of trimer stabilization. The equilibrium constant for the dissociation of trimers to monomers was calculated to be about 6 X 10(-16) mol2 liter-2. Calculations were made of the apparent total number of amino acids (40) in the two contact regions on each monomer. An absorption change analogous but not necessarily identical to a conversion of allophycocyanin II to III was noted when (NH4)2SO4 was present. When allophycocyanin's nonexchangeable hydrogens were replaced by deuteriums, it was more readily dissociated to monomers.

Alcohols↗