Showing papers by "Alexander N. Glazer published in 1983"

The structure of a "simple" phycobilisome.

Abstract: This report describes the properties of a relatively simple phycobilisome, Synechococcus 6301 (Anacystis nidulans). Morphology. -- Examination of wild type and mutant phycobilisomes by electron microscopy has shown them to have two morphologically differing substructures when seen in "face-view". There is a core consisting of two contiguous objects, disc-like in face-view projection, 115 A in diameter, and six rods, each composed of several stacked discs 60 A thick and 120 A in diameter, which radiate from the core in a hemidiscoidal arrangement. Each of the core components consists of four discs approximately 30 A thick. Rod substructures. -- Each of the discs in the rod substructure is a phycocyanin hexamer held together by interaction with a specific linker polypeptide, i. e., it has the composition (alpha beta)6 . X, where X is the linker polypeptide and alpha beta a phycocyanin monomer. The disc proximal to the core is an (alpha beta)6 . 27,000 complex. A small portion, Mr approximately 2,000,, of the Mr 27,000 polypeptide is essential to the attachment of this disc to the core. From studies of phycobilisomes from nitrogen-starved cells, and from mutants containing lowered amounts of phycocyanin relative to allophycocyanin, the second disc has been established to be an (alpha beta)6 . 33,000 complex. Either (alpha beta)6 . 33,000 or (alpha beta)6 . 30,000 complexes occupy the positions in the rods distal to the (alpha beta)6 .33,000 discs. Core substructure. -- Structural studies on the core and on core-rod junctions were greatly facilitated by the isolation of a mutant, strain AN112, which produces phycobilisomes with rods only one disc in length but with normal cores. Partial dissociation of these incomplete phycobilisomes under a variety of conditions, and separation and characterization of the resulting sub-complexes, has led to the determination of the composition of four distinct "trimeric" complexes, each of which is present in two copies per phycobilisome. These complexes, which account for the composition of the core, are as follows: (alpha beta)3AP . 10,500 with lambda maxF at 662 nm; (alpha beta)3AP with lambda maxF at 660 nm; (alpha 2AP alpha APB beta 3AP) . 10,500 with lambda maxF at 680 nm; where apha AP and alpha AFB are alpha subunits of allophycocyanin and allophycocyanin B, respectively, and beta AP is a subunit common to these two biliproteins; (alpha beta)2 AP . 18,300 . 40,000* . 11,000* with lambda maxF at 680 nm, where the Mr 40,000* and 11,000* polypeptides are derived from a Mr 75,000 polypeptide by tryptic digestion.(ABSTRACT TRUNCATED AT 400 WORDS)

Core substructure in cyanobacterial phycobilisomes

Abstract: The tricylindrical core of Synechocystis 6701 phycobilisomes is made up of four types of allophycocyanin-containing complexes: A, (αAP βAP)3; B, (αAP βAP)3 .10K; C, (ααβ).10K; D, (αAP βAP)2.18.5K.99K; where AP is allophycocyanin, APB is allophycocyanin B, and 10K, 18.5K, and 99K are polypeptides of 10,000, 18,500, and 99,000 daltons, respectively. The 18.5K polypeptide is a hitherto unrecognized biliprotein subunit with a single phycocyanobilin prosthetic group. The tricylindrical core is made up of 12 subcomplexes in the molar ratio of A:B:C:D: of 4:4:2:2. Complexes C and D act as terminal energy acceptors. From these results and previous analysis of the bicylindrical core of Synechococcus 6301 phycobilisomes [14,15] it is proposed that the two cylinders of the Synechocystis 6701 core, proximal to the thylakoid membrane, each have the composition ABCD, and that the distal cylinder has the composition A2B2.

BRIEF COMMUNICATION Immunofluorescence Measurement in a Flow Cytometer Using Low-Power Helium-Neon Laser Excitation'

Abstract: Helium-neon lasers are economical and efficient light sources; their utility in flow cytometry to date has been limited by the lack of fluorescent probes that can be excited at 633 nm. Allophycocyanin (APC), a highly fluorescent phycobiliprotein, can be used as an antibody label and has spectral characteristics suitable for use with He-Ne lasers; we undertook to resolve whether a low-power (7 mW) He-Ne laser could provide sufficient excitation to permit flow cytometric detection of APC-labeled antibodies on cell surfaces. We made an APC conjugate of monoclonal antibody 4F2, which reacts with an antigen abundant on the surfaces of activated human T-lymphocytes; APC-4F2 was used to stain blood mononuclear cells that had been cultured with and without phytohemagglutinin (PHA). Cells so stained were examined in a flow cytometer with orthogonal illumination at 633 nm from a 7 mW He-Ne laser; antibody-bearing cells were detectable by fluorescence emission above 665 nm. Cells from the same cultures were stained with fluorescein-labeled 4F2 antibody and examined in a flow cytometer with argon ion laser excitation at 488 nm. Percentages of antibody-bearing cells determined from APC fluorescence and from fluorescein fluorescence were in good agreement. It thus appears that He-Ne lasers and APC-antibodies are usable for immunofluorescence measurements; the sensitivity attainable with this technique remains to be determined.

Immunofluorescence measurement in a flow cytometer using low-power helium-neon laser excitation

Abstract: Helium-neon lasers are economical and efficient light sources; their utility in flow cytometry to date has been limited by the lack of fluorescent probes that can be excited at 633 nm. Allophycocyanin (APC), a highly fluorescent phycobiliprotein, can be used as an antibody label and has spectral characteristics suitable for use with He-Ne lasers; we undertook to resolve whether a low-power (7 mW) He-Ne laser could provide sufficient excitation to permit flow cytometric detection of APC-labeled antibodies on cell surfaces. We made an APC conjugate of monoclonal antibody 4F2, which reacts with an antigen abundant on the surfaces of activated human T-lymphocytes; APC-4F2 was used to stain blood mononuclear cells that had been cultured with and without phytohemagglutinin (PHA). Cells so stained were examined in a flow cytometer with orthogonal illumination at 633 nm from a 7 mW He-Ne laser; antibody-bearing cells were detectable by fluorescence emission above 665 nm. Cells from the same cultures were stained with fluorescein-labeled 4F2 antibody and examined in a flow cytometer with argon ion laser excitation at 488 nm. Percentages of antibody-bearing cells determined from APC fluorescence and from fluorescein fluorescence were in good agreement. It thus appears that He-Ne lasers and APC-antibodies are usable for immunofluorescence measurements; the sensitivity attainable with this technique remains to be determined.

Cyanobacterial photosystem I: Morphology and aggregation behavior

Abstract: A simple procedure is described for the preparation of cyanobacterial photosystem I particles with a full complement of antenna chlorophyll a from Triton X-100-solubilized thylakoid membranes. In the presence of ≥0.1% Triton X-100, photosystem I particles from Synechococcus 6301 were largely monodisperse. These particles, when negatively stained, appeared to approximate prolate ellipsoids 18 × 8 nm. A value of 4-5 × 10-19 cm3 was estimated for the volume of the stain-exclusion envelopes of the particles. At low concentrations of Triton X-100, photosystem I particles formed linear aggregates or sheets one-layer thick. The manner of aggregation was strongly dependent on protein concentration. Shadowed preparations of the sheets indicated a thickness of 8.0-8.5 nm.