Showing papers by "Alexander N. Glazer published in 1982"

Fluorescent phycobiliprotein conjugates for analyses of cells and molecules.

Abstract: The synthesis of a novel class of reagents for fluorescence analyses of molecules and cells is reported. These compounds consist of a highly fluorescent phycobiliprotein conjugated to a molecule having biological specificity. Phycoerythrin-immunoglobulin, phycoerythrin-protein A, and phycoerythrin-avidin conjugates were prepared. These conjugates bind specifically to beads containing a covalently attached target molecule and render them highly fluorescent. Femtomole (10(-15) mole) quantities of phycoerythrin conjugates can be detected because of the high extinction coefficient (epsilon M = 2.4 x 10(6) cm-1 M-1 for 2.4 x 10(5) daltons) and high fluorescence quantum yield (Q = 0.8) of the phycobiliprotein moiety. An important feature of these conjugates is that they emit in the orange-red spectral region, where background fluorescence is less than at shorter wavelengths. Phycoerythrin conjugates are well-suited for two-color flow cytofluorimetric analyses employing a single excitation line. The distributions of Leu antigens (also called OKT antigens) on the surface of T-lymphocytes were analyzed using fluoresceinated antibody as the green-fluorescent stain and biotinylated antibody counter-stained with phycoerythrin-avidin as the red one. This one-laser two-color analysis showed that cells express Leu-3a and Leu-3b or neither antigen. In contrast, the distributions of Leu-2a (a marker of suppressor and cytotoxic T-cells) and Leu-3a (a marker of helper and inducer T-cells) are mutually exclusive. These studies show that phycobiliprotein conjugates can be applied to fluorescence-activated cell sorting and analysis, fluorescence microscopy, and fluorescence immunoassay.

Rod Substructure in Cyanobacterial Phycobilisomes: Phycoerythrin Assembly in Synechocystis 6701 Phycobilisomes

Abstract: 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.