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PubMed1977

Subunit structure and chromophore composition of rhodophytan phycoerythrins. Porphyridium cruentum B-phycoerythrin and b-phycoerythrin.

A comparative study is presented of the two phycoerythrins of the unicellular red alga Porphyridium cruentum. Native B-phycoerythrin has a molecular weight of 236,000 +/- 18,000 in 0.05 M potassium phosphate at pH 7.0, and an absorption spectrum with maxima at 545 nm (epsilonM = 2.41 X 10(6) M-1 cm-1) and 563 nm, and a shoulder at 498 nm. The protein carries 38 phycoerythrobilin and at least two phycourobilin prosthetic groups per 240,000 daltons. B-Phycoerythrin is composed of three dissimilar subunits, alpha and beta, each of 17,500 daltons, and gamma of 30,200 daltons. Physical, chemical, and spectroscopic data are consistent with a subunit structure (alphabeta)6gamma for B-phycoerythrin. The alpha and beta subunits carry solely phycoerythrobilin chromophores, while the gamma subunit carries both phycoerythrobilin and phycourobilin groups. The NH2-terminal sequences of the alpha and beta subunits determined by sequential Edman degradation, are shown below: alpha subunit: Met-Lys-Ser-Val-Ile-(Gly-Arg-Phe: beta subunit: Met-Leu-Asp-Ala-Phe-(Thr)-Arg-Val-Val-Val-Asn-Ala-Asx-Ala-( )-Ala-Ala-Tyr-Val. The NH2 terminus of the gamma subunit is blocked. b-Phycoerythrin is polydisperse and exhibits native molecular weights ranging from approximately 40,000 to approximately 260,000, depending on pH, ionic strength, and protein concentration. The absorption spectrum is characterized by maxima at 543 nm (epsilonM = 3.41 X 10(5) M-1 cm-1/35,000 daltons) and 563 nm. The protein carries six phycoerythrobilin groups per 35,000 daltons. b-Phycoerythrin is made up of two dissimilar types of subunits, alpha and beta, of 17,500 daltons each. The alpha and beta subunits derived from b-phycoerythrin appeared equivalent to the corresponding subunits of B-phycoerythrin on the basis of the following criteria: (a) identical chromatographic behavior on Bio-Rex 70 in acid urea; (b) similar amino acid compositions; (c) identical mobilities on polyacrylamide gels in the presence of sodium dodecyl sulfate; (d) similar phycoerythrobilin contents; (e) identical NH2-terminal sequences. These data support, but do not establish unambiguously, the conclusion that b-phycoerythrin may be a component of B-phycoerythrin. The absence or presence, and relative height, of an absorption peak (or shoulder) at 498 nm represents the major difference among the absorption spectra of different classes of phycoerythrins. The present study shows that this spectral feature is dependent on the presence and amount of phycourobilin chromophores in the native protein and is correlated with the presence of the gamma subunit.

Amino Acid Sequence↗

PubMed1991

Phycoerythrins of marine unicellular cyanobacteria. III. Sequence of a class II phycoerythrin.

The genes for the alpha and beta subunits of a novel six bilin-bearing (class II) phycoerythrin were cloned from Synechococcus sp. WH8020 and sequenced. The cloned genes (mpeA and mpeB) were detected by homology with the genes for C-phycoerythrin from Pseudanabaena sp. PCC7409. The mpe locus occurs once in the genome and is arranged similarly to that of many other phycobiliproteins, with mpeA shortly 3' of mpeB. Sequence comparison suggests that this phycoerythrin (and perhaps all class II phycoerythrins) occupy a branch of the phycoerythrin family separate from five-chromophore per alpha beta (class I) phycoerythrins, C-phycoerythrin, and B-phycoerythrin. The position of the sixth chromophore of the class II phycoerythrin of WH8020 was determined by comparison of the amino acid sequence of the chromopeptides (Ong, L. J., and Glazer, A. N. (1991) J. Biol Chem. 266, 9515-9527) with the sequence deduced from the gene. This located the chromophore at residue 75 of the alpha subunit, very close to the alpha-83 chromophore in the primary structure and, presumably, in the three-dimensional structure.

Amino Acid Sequence↗

PubMed1980

Biliprotein assembly in the disc-shaped phycobilisomes of Rhodella violacea. Electron microscopical and biochemical analysis of B-phycoerythrin and B-phycoerythrin--C-phycocyanin aggregates.

Hexameric B-phycoerythrin (alpha beta)6 gamma is a double disc of about 10.7 x 4.3 nm; each single disc consists of a six membered periphery (alpha beta)3, the subunits of which are assumed to be associated in alternating positions with little or no staggering. A central subunit, almost certainly the gamma-subunit, penetrates both rings linking them tightly together. Hexameric C-phycocyanin (alpha beta)6 has the same construction but lacks this central subunit. In urea gel electrophoresis B-phycoerythrin I and II separate into three bands alpha, beta, and gamma in a relative molar ratio of 6:6:1. The molecular weights of the alpha-, beta-, gamma-subunits, estimated from SDS gels were 18 700, 18 700 and 29 200 and 18 300, 18 300 and 29 900 for B-phycoerythrin I and II, respectively, resulting in molecular weights of 253 600 and 249 500 for both hexameric aggregates. In density gradient centrifugation a sedimentation coefficient s20,w . 10(13) of 11.3 and a molecular weight of 244 000 were calculated. In sedimentation analyses of partially dissociated phycobilisomes a fragment consisting of two phycoerythrin hexamers with a sedimentation constant of 18 S (dodecamer) and tripartite units with two B-phycoerythrin hexamers associated with one polar C-phycocyanin hexamer with a sedimentation constant of 22 S were demonstrated. The corresponding molecular weight of the tripartite units, about 800 000, coincides well with morphological measurements on the basis of an average protein packing density and with earlier estimates on cross-linked biliprotein aggregates in gradient gel electrophoresis. The spaces of 1.2 to 3.0 nm between the hexamers give rise to a strong 6.0 nm periodicity within the tripartite units, the weak 3.0 nm periodicity originates from the double-rings of the constituent hexamers.

Centrifugation, Density Gradient↗

PubMed1993

Rod structure of a phycoerythrin II-containing phycobilisome. II. Complete sequence and bilin attachment site of a phycoerythrin gamma subunit.

The major phycoerythrins of marine unicellular cyanobacteria Synechococcus spp. and those of red algae are isolated as stable complexes with the composition (alpha beta)6 gamma. The gamma subunits carry bilins and in this respect are unique among phycobilisome rod linker polypeptides. There has been no complete amino acid sequence data on any gamma subunit. Synechococcus sp. WH8020 phycoerythrin II (PE II) gamma subunit was isolated from purified PE II hexamers. Partial amino acid sequence determination showed that the gamma subunit was encoded by the mpeC gene, an open reading frame 275 base pairs 3' of mpeA and mpeB, which encode the alpha and beta subunits of PE II, respectively (Wilbanks, S. M., and Glazer, A. N. (1993) J. Biol. Chem. 268, 1226-1235). A single phycourobilin is attached through a thioether bond to gamma-Cys-49. Derivatization with 4-vinylpyridine showed that the only other cysteinyl residue, gamma-Cys-64, is unsubstituted. MpeC encodes a polypeptide of 293 residues with a predicted molecular weight of 32,100 and a pI of 8.9. These properties are like those of non-bilin-bearing linker polypeptides associated with C-phycoerythrin and hence the gamma subunit is designated gamma (LR32). Alignment of the sequence of the PE II-gamma with those of the latter polypeptides shows that PE II-gamma has a 49-residue extension at the N terminus, that encompasses the phycourobilin attachment site, and is shorter by a similar number of residues at the C terminus. These differences in linker polypeptide structure offer a possible explanation for the observed much higher stability of PE II hexamers relative to those of C-phycoerythrins.

Amino Acid Sequence↗

PubMed1991

Phycoerythrins of marine unicellular cyanobacteria. I. Bilin types and locations and energy transfer pathways in Synechococcus spp. phycoerythrins.

Marine Synechococcus strains WH8103, WH8020, and WH7803 each possess two different phycoerythrins, PE(II) and PE(I), in a weight ratio of 2-4:1. PE(II) and PE(I) differ in amino acid sequence and in bilin composition and content. Studies with strain WH7803 indicated that both PE(II) and PE(I) were present in the same phycobilisome rod substructures and that energy absorbed by PE(II) was transferred to PE(I). Strain WH8103 and WH8020 PE(I)s carried five bilin chromophores thioether-linked to cysteine residues in sequences homologous to those previously characterized in C-, B-, and R-PEs. In contrast, six bilins were attached to strain WH8103 and WH8020 PE(II)s. Five of these were at positions homologous to bilin attachment sites in other phycoerythrins. The additional bilin attachment site was on the alpha subunit. The locations and bilin types in these PE(s) and in the marine Synechocystis strain WH8501 PE(I) (Swanson, R. V., Ong, L. J., Wilbanks, S. M., and Glazer, A. N. (1991) J. Biol. Chem. 266, 9528-9534) are: (table; see text) Since phycourobilin (PUB) (lambda max approximately 495 nm) transfers energy to phycoerythrobilin (PEB) (lambda max approximately 550 nm), inspection of these data shows that the invariant PEB group at beta-82 is the terminal energy acceptor in phycoerythrins. The adaptations to blue-green light, high PUB content and the presence of an additional bilin on the alpha subunit, increase the efficiency of light absorption by PE(II)s at approximately 500 nm.

Amino Acid Sequence↗

PubMed1988

Indirect immunofluorescence labelling with complexes of phycoerythrin and monoclonal anti-phycoerythrin antibodies (PEAPE complexes).

We describe a method of immunofluorescence which is a lateral application of the principles of the APAAP immunohistochemical technique. Immune complexes of R-phycoerythrin and monoclonal anti-R-phycoerythrin (PEAPE complexes) were used in an indirect immunofluorescence technique to detect the binding to cells of monoclonal antibodies directed to IgM, HLA-DR and B cell activation and differentiation antigens. PEAPE complexes were linked to cell surface bound mAbs by unlabelled anti-mouse Ig antibodies to produce high levels of fluorescent staining. The sensitivity of this method of indirect immunofluorescence was enhanced by the sequential application of several cycles of anti-mouse Ig and PEAPE complexes.

Animals↗

PubMed2003

Effect of heat on phycoerythrin fluorescence: Influence of thermal exposure on the fluorescence emission of R-phycoerythrin.

The goal of this work was to measure and model the effect of thermal exposure on the fluorescence emission of R-phycoerythrin (R-PE). The long-term objective of our work is to assess the feasibility of encapsulating R-PE for use as the critical component of a time-temperature integrator (TTI) for ascertaining the degree of inactivation of food pathogens such as Salmonella. In this article we present a study to measure and model the thermally induced fluorescence emission decay of R-PE in several isothermal experiments. We used the isothermal data to determine the kinetic parameters, based on a general n(th) order reaction, and evaluated the utility of the resulting model by using it to predict R-PE fluorescence emission decay for several nonisothermal experiments based on published USDA safe harbor guidelines for cooked beef products. The transient experiments were conducted over the same temperature range used in the isothermal study. Very good agreement was obtained between theory and experiment at temperatures of 62.8 degrees C and above, although the model slightly underpredicted the extent of fluorescence emission decay at 60 degrees C. Our results indicate that R-PE fluorescence emission decay kinetics is well behaved and that the protein is a strong candidate for use as a time-temperature integrator.

Biosensing Techniques↗

PubMed1991

Immunofluorescent labeling using covalently linked anti-phycoerythrin antibodies and phycoerythrin polymers.

We have developed methods for covalently attaching anti-phycoerythrin (PE) MAbs to other antibodies, and for using PE or polymers of PE in conjunction with these conjugates to rapidly produce specific, high intensity fluorescent labeling of antigens. The performance of these systems was examined on the surface of microspheres and on the cell surface. The noncovalent means by which PE is incorporated into the label complex in this method makes it possible to use crude algal homogenate successfully as a source of PE in immunofluorescence assays. The intensity of labeling achieved using this method is comparable to, or in some cases better than, that obtained using a direct PE conjugate. With the aim of amplifying the intensity of fluorescence obtained, we produced two types of covalently linked complexes. These were an anti-PE MAb, designated PE6, linked to itself (i.e., PE6-PE6), and PE linked to itself (i.e., PE-PE). When used in a two-stage procedure in place of monomeric PE alone, these complexes increased the intensity of fluorescence obtained on the surface of microspheres by more than fourfold. On the cell surface, the performance of this system varied from one antigen to the next but in most cases was restricted to, at best, a 60% increase in the intensity of fluorescence and in many cases only about a 30% increase.

Antibodies, Monoclonal↗

PubMed2002

CpeR is an activator required for expression of the phycoerythrin operon (cpeBA) in the cyanobacterium Fremyella diplosiphon and is encoded in the phycoerythrin linker-polypeptide operon (cpeCDESTR).

In the cyanobacteria, phycobilisomes are assembled from (alphabeta)(6) hexamers of the coloured phycobiliproteins, allophycocyanin, phycocyanin and phycoerythrin (PE). The precise architecture of the phycobilisome is determined by the various colourless linker proteins that bind to the biliprotein hexamers. Genes for beta and alpha subunits of PE make up one operon (cpeBA), whereas genes for PE-associated linker polypeptides are in a second operon. In the chromatically adapting cyanobacterium Fremyella diplosiphon green light is required for the transcription of both cpeBA and the operon encoding the PE-associated linkers (cpeCDE). From the genome of F. diplosiphon we have identified an open reading frame, cpeR, which, when expressed from a shuttle plasmid, is capable of suppressing various mutations that cause a decrease in PE synthesis. The introduction of a shuttle plasmid bearing cpeR+ into wild-type F. diplosiphon caused PE expression in red light. Fremyella diplosiphon cpeR-, created by in vitro mutagenesis and in vivo homologous recombination, is fully PE and, in this strain, cpeCDE is transcribed normally whereas the transcript from cpeBA is undetectable. Polymerase chain reaction (PCR) amplification of cDNA showed that cpeR is transcribed as part of the cpeCDE operon on an extended transcript. As CpeR is an activator required for expression of the cpeBA operon, we propose that at the onset of green light the operons cpeCDESTR and cpeBA are expressed in series as a genetic cascade.

Bacterial Proteins↗

PubMed1996

Differential transcription of phycobiliprotein components in Rhodella violacea. Light and nitrogen effects on the 33-kilodalton phycoerythrin rod linker polypeptide, phycocyanin, and phycoerythrin transcripts.

In Rhodella violacea phycoerythrin (PE) has two transcripts, a premessenger and a mature messenger (the gene contains an intron). Phycocyanin, which is plastid-encoded, and the 33-kD PE rod linker polypeptide, which is nuclear-encoded, have only one transcript. The PE premessenger had a rapid turnover; mature transcripts were stable in the light and more stable in the dark. In the presence of rifampicin, cells that shifted from dark to light exhibited an active translation of preexisting transcripts. There are indications of a modulation of the nuclear genome expression by the chloroplast; it may involve an unstable, plastid-encoded translational activator. All transcripts disappeared rapidly during nitrogen starvation. If nitrogen addition was carried out in the dark, active transcription and translation resumed as in light conditions, but ceased after 2 d. Both nitrogen and light were required for a total recovery after nitrogen starvation. Compared with the transcripts of phycobilisome components studied so far in cyanobacteria and Rhodophyceae, the mature transcripts of R. violacea are very stable when nitrogen is not limiting. The unstable PE premessenger is a good indicator of active transcription. This organism is therefore an interesting model to study the regulation of gene expression and the interactions between chloroplastic and nuclear genomes.

Amino Acid Sequence↗

PubMed1982

Rod substructure in cyanobacterial phycobilisomes: phycoerythrin assembly in synechocystis 6701 phycobilisomes.

Synechocystis 6701 phycobilisomes consist of a core of three cylindrical elements in an equilateral array from which extend in a fanlike manner six rods, each made up of three to four stacked disks. Previous studies (see Gingrich, J. C., L. K. Blaha, and A. N. Glazer, 1982. J. Cell Biol. 92:261-268) have shown that the rods consist of four disk-shaped complexes of biliproteins with "linker" polypeptides of 27-, 33.5-, 31.5-, and 30.5-kdaltons, listed in order starting with the disk proximal to the core: phycocyanin (alpha beta)6-27 kdalton, phycocyanin (alpha beta)6-33.5 kdalton, phycoerythrin (alpha beta)6-31.5 kdalton, phycoerythrin (alpha beta)6-30.5 kdalton, where alpha beta is the monomer of the biliprotein. Phycoerythrin complexes of the 31.5- and 30.5-kdalton polypeptides were isolated in low salt. In 0.05 M K-phosphate-1 mM EDTA at pH 7.0, these complexes had the average composition (alpha beta)2-31.5 and (alpha beta)-30.5 kdalton polypeptide, respectively. Peptide mapping of purified 31.5- and 30.5-kdalton polypeptides showed that they differed significantly in primary structure. In 0.65 M Na-K-phosphate at pH 8, these phycoerythrin complexes formed rods of stacked disks of composition (alpha beta)6-31.5 or (alpha beta)6-30.5 kdaltons. For the (alpha beta)-30.5 kdalton complex, the yield of rod assemblies was variable and the self-association of free phycoerythrin to smaller aggregates was an important competing reaction. Complementation experiments were performed with incomplete phycobilisomes from Synechocystis 6701 mutant strain CM25. These phycobilisomes are totally lacking in phycoerythrin and the 31.5- and 30.5-kdalton polypeptides, but have no other apparent structural defects. In high phosphate at pH 8, the phycoerythrin-31.5-kdalton complex formed disk assemblies at the end of the rod substructures of CM25 phycobilisomes whereas no interaction with the phycoerythrin-30.5 kdalton complex was detected. In mixtures of both the phycoerythrin-31.5 and -30.5 kdalton complexes with CM25 phycobilisomes, both complexes were incorporated at the distal ends of the rod substructures. The efficiency of energy transfer from the added phycoerythrin in complemented phycobilisomes was approximately 96%. The results show that the ordered assembly of phycoerythrin complexes seen in phycobilisomes is reproduced in the in vitro assembly process.

Cyanobacteria↗

PubMed1990

Structure and light-regulated expression of phycoerythrin genes in wild-type and phycobilisome assembly mutants of Synechocystis sp. strain PCC 6701.

Phycoerythrin is a major pigmented component of the phycobilisome, a cyanobacterial light-harvesting complex. It contains bilin-type chromophores that absorb and transfer light energy to chlorophyll protein complexes of the photosynthetic membranes. In many cyanobacteria, phycoerythrin expression is regulated by light wavelength in a response known as chromatic adaptation. Green light-grown cells contain higher levels of this biliprotein than do cells grown in red light. The phycoerythrin gene set from the unicellular cyanobacterium Synechocystis sp. strain PCC 6701 was cloned and sequenced, and the 5' end of the phycoerythrin mRNA was localized. The amino acid sequences of the phycoerythrin subunits from Synechocystis strain 6701 and Fremyella diplosiphon were 90% identical. As observed in F. diplosiphon, the Synechocystis strain 6701 phycoerythrin transcript accumulated to high levels in green light-grown cells and low levels in red light-grown cells. Similar nucleotide sequences, which might control gene expression, occurred upstream of the transcription initiation sites of the phycoerythrin genes in both organisms. While the phycoerythrin structure and light-regulated transcript accumulation were similar in Synechocystis strain 6701 and F. diplosiphon, the steady-state levels of phycoerythrin subunits during growth in red light were quite different for the two organisms. This observation suggests that control of phycoerythrin levels in Synechocystis strain 6701 is complex and may involve posttranscriptional processes. We also characterized the phycoerythrin genes and mRNA levels in two phycobilisome assembly mutants, UV16-40 and UV16.

Amino Acid Sequence↗

PubMed1993

Refined crystal structure of phycoerythrin from Porphyridium cruentum at 0.23-nm resolution and localization of the gamma subunit.

The three-dimensional structure of the light-harvesting pigment-protein b-phycoerythrin from the red alga Porphyridium cruentum has been determined at 0.23-nm resolution. The b-phycoerythrin structure is very similar to the structure of B-phycoerythrin from Porphyridium sordidum. Besides three non-identical residues there are only small differences between b-phycoerythrin and B-phycoerythrin alpha and beta subunits, respectively. In the crystals b-phycoerythrin forms an (alpha beta)6 hexamer (molecular mass: 236 kDa), whereas B-phycoerythrin additionally contains a 30-kDa gamma subunit. The comparison of the b-phycoerythrin and B-phycoerythrin electron-density maps clearly reveals, that the gamma subunit is located inside the (alpha beta)6 aggregate.

Crystallography, X-Ray↗

PubMed1989

The complete amino-acid sequence of the alpha and beta subunits of B-phycoerythrin from the rhodophytan alga Porphyridium cruentum.

Determination of the complete amino-acid sequence of the subunits of B-phycoerythrin from Porphyridium cruentum has shown that the alpha subunit contains 164 amino-acid residues and the beta subunit contains 177 residues. When the sequences of B- and C-phycoerythrins are aligned with those of other phycobiliproteins, it is obvious that B-phycoerythrin lacks a deletion at beta-21-22 present in C-phycoerythrin. However, relative to C-phycoerythrin from Fremyella diplosiphon (Calothrix) (Sidler, W., Kumpf, B., Rüdiger, W. and Zuber, H. (1986) Biol. Chem. Hoppe-Seyler 367, 627-642), B-phycoerythrin has deletions at beta-141k-o, beta-142, beta-143, beta-147 and beta-148. The four singly-linked phycoerythrobilins at positions alpha-84, alpha-143a, beta-84 and beta-155, and the doubly-linked phycoerythrobilin at position beta-50/61 are at sites homologous to the attachment sites in C-phycoerythrin. The aspartyl residues (alpha-87, beta-87, and beta-39), that interact with the bilins at alpha-84, beta-84, and beta-155 in C-phycocyanin, are found in the homologous positions in B-phycoerythrin. B-Phycoerythrin, in common with other phycobiliproteins, contains a N gamma-methylasparagine residue at position beta-72.

Amino Acid Sequence↗

PubMed1981

Phycoerythrins of the Red Alga Callithamnion: VARIATION IN PHYCOERYTHROBILIN AND PHYCOUROBILIN CONTENT.

Phycoerythrins of several species of the higher red alga Callithamnion show virtually identical spectra, typical of R-phycoerythrins, with absorption maxima at 565, 539, and 497 nanometers. One species, Callithamnion roseum, produces a phycoerythrin lacking the peak at 539 nanometers. Comparison of a "typical" R-phycoerythrin from Callithamnion byssoides with the "atypical" phycoerythrin of C. roseum shows that both proteins carry 35 bilins per native molecule of 240,000 daltons; however, C. byssoides phycoerythrin carries 27.6 phycoerythrobilin and 7.3 phycourobilin groups, whereas C. roseum phycoerythrin carries 24.1 phycoerythrobilin and 10.9 phycourobilin groups. These differences in the relative amounts of the bilin prosthetic groups account in large measure for the differences between the absorption spectra of the native proteins. The ratio of phycoerythrobilin to phycourobilin in C. roseum phycoerythrin can be modulated by varying the light intensity during growth.Data on the physical, immunological and spectroscopic properties of Callithamnion phycoerythrins indicate that the variation in the relative number of the two bilin prosthetic groups does not affect significantly the conformation of the biliprotein.

Journal Article↗

PubMed2006

Comparative molecular population genetics of phycoerythrin locus in Prochlorococcus.

As the only remainder type of phycobiliproteins in Prochlorococcus, the actual role of phycoerythrin still remains unknown. Previous studies revealed that two different forms of phycoerythrin gene were found in two ecotypes of Prochlorococcus that are specifically adapted to either high light (HL) or low light (LL) conditions. Here we analyze patterns of phycoerythrin nucleotide variation in the HL- and LL-Prochlorococcus populations. Our analyses reveal a significantly greater number of non-synonymous fixed substitutions in peB and peA than expected based on interspecific comparisons. This pattern of excess non-synonymous fixed substitutions is not seen in other five phycoerythrin-related genes (peZ/V/Y/T/S). Several neutrality statistical tests indicate an excess of rare frequency polymorphisms in the LL-Prochlorococcus data, but an excess of intermediate frequency polymorphisms in the HL-Prochlorococcus data. Distributions of the positively selected sites identified using the likelihood ratio test, when mapped onto the phycoerythrin tertiary structure, reveal that HL- and LL-phycoerythrin should be under different selective patterns. These findings may provide insights into the likely role of selection at the phycoerythrin locus and motivate further research to unveil the function of phycoerythrin in Prochlorococcus.

Adaptation, Biological↗

PubMed2000

One-step purification of R-phycoerythrin from the red macroalga Palmaria palmata using preparative polyacrylamide gel electrophoresis.

Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria widely used as a fluorescent probe. In this study, phycoerythrin of the red macroalga Palmaria palmata was extracted by grinding the algal sample in liquid nitrogen, homogenisation in phosphate buffer and centrifugation. Phycoerythrin was then purified from this crude extract using preparative polyacrylamide gel electrophoresis (PAGE) with a continuous elution system and detected by its pink colour and fluorescence. The pigment presented a typical spectrum of R-phycoerythrin, with three absorbance maxima at 499, 545 and 565 nm, and displayed a fluorescence maximum at 578 nm. The absorbance ratio A565/A280, a criterion for purity, was 3.2. A single protein of relative molecular mass 240,000 was detected on native-PAGE with silver staining. Sodium dodecyl sulphate-PAGE demonstrated the presence of two major subunits with Mr 20,000 and 21,000, respectively, and a very minor subunit of Mr 30,000. These observations are consistent with the (alphabeta)6gamma subunit composition characteristic of R-phycoerythrin. Phycoerythrin of Palmaria palmata was determined to be present in larger amounts in autumn and showed a good stability up to 60 degrees C and between pH 3.5 and 9.5. In conclusion, phycoerythrin of Palmaria palmata was purified in a single-step using preparative PAGE. Obtaining pure R-phycoerythrin of Palmaria palmata will allow one to evaluate its fluorescence properties for future applications in biochemical techniques.

Electrophoresis, Polyacrylamide Gel↗

PubMed1982

Rod substructure in cyanobacterial phycobilisomes: analysis of Synechocystis 6701 mutants low in phycoerythrin.

Synechocystis 6701 phycobilisomes contain phycoerythrin, phycocyanin, and allophycocyanin in a molar ratio of approximately 2:2:1, and other polypeptides of 99-, 46-, 33.5-, 31.5-, 30.5-, and 27-kdaltons. Wild-type phycobilisomes consist of a core of three cylindrical elements in an equilateral array surrounded by a fanlike array of six rods each made up of 3-4 stacked disks. Twelve nitrosoguanidine-induced mutants were isolated which produced phycobilisomes containing between 0 and 53% of the wild-type level of phycoerythrin and grossly altered levels of the 30.5- and 31.5-kdalton polypeptides. Assembly defects in these mutant particles were shown to be limited to the phycoerythrin portions of the rod substructures of the phycobilisome. Quantitative analysis of phycobilisomes from wild-type and mutant cells, grown either in white light or chromatically adapted to red light, indicated a molar ratio of the 30.5- and 31.5-kdalton polypeptides to phycoerythrin of 1:6, i.e., one 30.5- or one 31.5-kdaltons polypeptide per (alpha beta)6 phycoerythrin hexamer. Presence of the phycoerythrin-31.5-kdalton complex in phycobilisomes did not require the presence of the 30.5-kdalton polypeptide. The converse situation was not observed. These and earlier studies (R. C. Williams, J. C. Gingrich, and A. N. Glazer. 1980. J. Cell Biol. 85:558-566) show that the average rod in wild type Synechocystis 6701 phycobilisomes consists of four stacked disk-shaped complexes: phycocyanin (alpha beta)6-27 kdalton, phycocyanin (alpha beta)6-33.5 kdalton, phycoerythrin (alpha beta)6-31.5 kdalton, and phycoerythrin-30.5 kdalton, listed in order starting with the disk proximal to the core.

Cyanobacteria↗