Formation of hybrid phycobilisomes by association of phycobiliproteins from Nostoc and Fremyella

Formation of phycobilisomes has been accomplished in vitro from isolated phycobiliprotein fractions obtained from the same blue-green alga (intrageneric) and from different blue-green algae (intergeneric). Phycobilisomes, which are supra-molecular complexes of phycobiliproteins, serve as major light-harvesting antennae for photosynthesis in blue-green and red algae. Intrageneric association into energetically functional phycobilisomes, previously reported to occur with Nostoc sp. allophycocyanin and phycoerythrin-phycocyanin complexes [Canaani, O., Lipschultz, C. A. & Gantt, E. (1980) FEBS Lett. 115, 225-229], has been obtained with Fremyella diplosiphon. By their spectral properties (absorption, fluorescence excitation, and emission) and electron microscopic images, the native and in vitro-associated phycobilisomes were virtually indistinguishable. Intergeneric phycobilisomes have been produced from allophycocyanin of Nostoc sp. strain Mac. and phycoerythrin-phycocyanin of F. diplosiphon, as well as from the reverse mixtures. The yield of intergeneric phycobilisomes, favored by higher phycobiliprotein content in 0.75 M phosphate, pH 7.0/2.0 M sucrose, was 40-60%. Energy transfer to the terminal long-wavelength-emitting allophycocyanin in the phycobilisomes was evident from the 670-675 nm fluorescence emission peaks. Furthermore, excitation spectra showed the contribution of the respective phycoerythrins (Fremyella, λ_max 570; Nostoc, λ_max 573 and 553 nm), as well as that of phycocyanin and short-wavelength-absorbing allophycocyanin. Phycobilisomes of Nostoc and Fremyella, analyzed by NaDodSO₄/polyacrylamide gel electrophoresis, possessed a number of polypeptides having similar molecular weights: the usual α- and β-phycobilin-containing polypeptides of M_r 15,000-22,000, a faint band at M_rca. 95,000, and a prominent band at M_rca. 31,000. The M_r 31,000 polypeptide is assumed to provide the recognition site for attachment of the phycoerythrin-phycocyanin complexes with the allophycocyanin core. In vitro association was not obtained between allophycocyanin from Nostoc and phycoerythrin-phycocyanin complexes from Phormidium persicinum or Porphyridium sordidum.     

Molecular composition of cyanobacterial phycobilisomes.

Phycobilisomes isolated from eight different species of cyanobacteria contain in addition to the light-harvesting phycobiliproteins, a small number of colorless polypeptides with molecular weights higher than those of the chromopolypeptide subunits of the phycobiliproteins. In the phycobilisomes of the species examined, from four to nine colorless polypeptides were resolved by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Those of highest molecular weight (70,000-120,000) also occurred in the washed membrane fraction of the cell and may therefore be derived from the thylakoids, to which the phycobilisomes are attached in vivo. Colorless polypeptides of lesser molecular weight (30,000-70,000) appeared to be specific constituents of the phycobilisome. In strains of cyanobacteria that adapt chromatically, their synthesis, like that of the major phycobiliproteins, is regulated by light quality.     

Nuclear and cytoplasmic genes controlling synthesis of variant mitochondrial polypeptides in male-sterile maize

The polypeptides synthesized in vitro by mitochondria isolated from etiolated maize shoots of a number of different nuclear and cytoplasmic genotypes have been compared by using polyacrylamide gel electrophoresis. We have previously shown that mitochondria from maize plants carrying the T or C forms of cytoplasmically inherited male sterility (cms-T and cms-C mitochondria) can be distinguished from each other and from the mitochondria of normal (N) plants by the synthesis of a single additional or variant polypeptide species. Using lines that carry the T cytoplasm, and that differ principally in the presence or absence of nuclear “restorer” alleles that suppress the male-sterile phenotype, we find that these nuclear genes specifically suppress synthesis of the 13,000 M_r variant polypeptide. A 21,000 M_r polypeptide that is synthesized by N mitochondria is not detectable among the translation products of cms-T mitochondria from either restored or nonrestored lines. Results obtained with a number of lines possessing dominant restorer alleles from different sources indicate that it is the restorer gene at the Rf₁ locus that is primarily responsible for regulating synthesis of the 13,000 M_r polypeptide. Mitochondria from lines with the S form of cytoplasmic male sterility (cms-S) were found to synthesize a group of minor polypeptides, ranging in molecular weight from 42,000 to 88,000, which were not detected in N, cms-T, or cms-C mitochondria. In the case of the S and C forms of male sterility no differences were found between the translation products of mitochondria from restored and nonrestored lines.     

Characterization of cyanobacterial phycobilisomes in zwitterionic detergents

Properties of cyanobacterial phycobilisomes (from Synechococcus spp. 6301 and 6312 and Synechocystis sp. 6701) prepared in the presence of two different zwitterionic detergents were compared to those of phycobilisomes detached from membranes with the nonionic detergent Triton X-100 and then freed from Triton by sedimentation through high-salt sucrose density gradients. The absorption spectra, polypeptide composition, and ultrastructure of phycobilisomes were independent of the detergent used during the preparation. Phycobilisomes from certain cyanobacteria aggregated in the absence of detergent. Such aggregation was not seen in preparations containing zwitterionic detergents. Aggregation of phycobilisomes led to a partial quenching of their fluorescence. Electron microscopy showed the three types of phycobilisomes to have a hemi-disc appearance, although their detailed structures were quite different.     

Cyanobacterial phycobilisomes: Selective dissociation monitored by fluorescence and circular dichroism

Phycobilisomes are supramolecular assemblies of phycobiliproteins responsible for photosynthetic light collection in red algae and cyanobacteria. They can be selectively dissociated by reduction of temperature and buffer concentration. Phycobilisomes isolated from Fremyella diplosiphon transfer energy collected by C-phycoerythrin and C-phycocyanin to allophycocyanin. The energy transfer to allophycocyanin is nearly abolished at 2°C, as indicated by a blue shift in fluorescence emission, and is accompanied by a decrease in the circular dichroism in the region of allophycocyanin absorbance. Further dissociation of the phycobilisomes can be attained by reduction of buffer concentration and holding at 2°C. Energy transfer to C-phycocyanin is nearly abolished, and decreases occur in the circular dichroism in the region of C-phycocyanin and C-phycoerythrin absorbance. Complete dissociation of the phycobilisomes at low buffer concentration and 2°C requires extended time. Energy transfer to C-phycocyanin is further reduced and the circular dichroism maximum of C-phycoerythrin at 575 nm is lost. Circular dichroism provides information on the hexamer-monomer transitions of the phycobiliproteins, whereas fluorescence is indicative of hexamer-hexamer interactions. We consider that hydrophobic interactions are fundamental to the maintenance of the structure and function of phycobilisomes.     

Two tumor antigens and their polypeptides in adenovirus type 12-infected and transformed cells

A tumor (T) antigen, designated T antigen g, was visualized as fine fluorescent granules in nuclei of adenovirus type 12 (Ad12)-infected cells by immunofluorescence with sera from rats bearing HY cell tumors (H sera). HY cells are rat cells incompletely transformed by the Acc I-H endonuclease fragment (0-4.7 map units) of Ad12 DNA. The antigen is different from the usually described T antigen, designated T antigen f, which is visualized as fluorescent flecks or filaments in both nucleus and cytoplasm of Ad12-infected cells when tested with narrowly reacting T sera. Extracts of [³⁵S]methioninelabeled infected cells were immunoprecipitated with H sera, and the resultant precipitate was analyzed by the two-dimensional gel electrophoresis technique of O'Farrell. The autoradiogram showed the presence of a cluster of several polypeptides (M_r 35,000-40,000, pI 5.0-5.5) that was absent in extracts of mock-infected cells. A similar autoradiogram of infected cells analyzed with narrowly reacting T sera showed the presence of a small polypeptide (M_r 10,000, pI 6.4), that was absent in extracts of mock-infected cells. The results show that M_r 35,000-40,000 polypeptides are components of T antigen g and a M_r 10,000 polypeptide is a component of T antigen f. Ad12-transformed cells showed a similar result. T antigen g was present and T antigen f was absent in HY cells. Both T antigen g and T antigen f were present in CY cells, which are rat cells completely transformed by the EcoRI-C endonuclease fragment (0-16 map units) of Ad12 DNA. The possible functions of these proteins are discussed.     

Isolation of the in vivo emitter in bacterial bioluminescence

A blue fluorescence protein has been isolated and purified from extracts of the luminous bacterium Photobacterium phosphoreum. It is a single polypeptide of molecular weight 22,000 with absorption maxima at 274 and 418 nm. It is efficiently fluorescent (ϕ_F 0.45), with a fully corrected spectral maximum (476 nm) and distribution identical to the in vivo bioluminescence from this same type of bacterium. At low concentration this fluorescence shifts towards the red and becomes identical to the in vitro bioluminescence emission. This spectral shift apparently results from a change in the protein pulled by dissociation of the chromophore (K_d [unk] 10^-7 M). If the blue fluorescence protein is included in the in vitro bioluminescence reaction with reduced FMN, oxygen, aldehyde, and luciferase (P. phosphoreum), the bioluminescence spectrum is shifted towards the blue from its maximum at 490 nm to one at 476 nm, where it is again identical in all respects to the in vivo bioluminescence spectrum. This is accompanied by an increase in the initial light intensity by an order of magnitude at saturating levels of blue fluorescence protein, and the specific light yield of the luciferase is increased 4-fold. It is suggested that the blue fluorescence protein acts as a sensitizer of the bacterial bioluminescence reaction.     

Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of thylakoid membranes at 4 degrees C: Characterizations of two additional chlorophyll a-protein complexes

Lithium dodecyl sulfate/polyacrylamide gel electrophoresis of Chlamydomonas reinhardtii thylakoid membranes at room temperature gave two chlorophyll-protein complexes, CP I and CP II, as had been reported previously. However, when the electrophoresis was performed at 4°C, there was an increase in the amount of chlorophyll associated with CP I and CP II, and in addition, three other chlorophyll-protein complexes appeared. Two of these complexes, designated CP III and CP IV, were characterized and found to be similar in their compositions. Each complex contains four to five molecules of chlorophyll a, one molecule of β-carotene, and one polypeptide chain. The apoprotein of CP III is polypeptide 5 (M_r 50,000) and that of CP IV is polypeptide 6 (M_r 47,000); the two polypeptides are structurally unrelated. Chlorophyll-protein complexes similar to C. reinhardtii CP III and CP IV were also detected in higher plants (e.g., Pisum sativum). The apoproteins of the higher plant complexes are immunochemically related to those of the C. reinhardtii complexes, as shown by crossed immunoelectrophoresis. Absorption spectra of CP III and CP IV at -196°C revealed a component at 682 nm. This observation, together with the previous results on photosystem II mutants [Chua, N.-H. & Bennoun, P. (1975) Proc. Natl. Acad. Sci. USA 72, 2175-2179], provides indirect evidence that CP III and CP IV may be involved in the primary photochemistry of photosystem II.     

Cytoplasmic and chloroplast synthesis of phycobilisome polypeptides

In vivo labeling of eukaryotic phycobilisomes in the presence of inhibitors of translation on 70S and 80S ribosomes demonstrates that some of the polypeptides of this light-harvesting complex are synthesized in the cytoplasm while others are synthesized in the chloroplast. The major pigmented polypeptides, the α and β subunits of the biliproteins (molecular weights between 15,000 and 20,000) and the anchor protein (molecular weight about 90,000) are translated on 70S ribosomes. This suggests that these polypeptides are made within the algal chloroplast. Because the α and β subunits comprise a group of closely related polypeptides, the genes encoding these polypeptides may reside in the plastid genome as a multigene family. Other prominent phycobilisome polypeptides, including a nonpigmented polypeptide that may be involved in maintaining the structural integrity of the complex, are synthesized on cytoplasmic ribosomes. Because the synthesis of phycobilisomes appears to require the expression of genes in two subcellular compartments, this system may be an excellent model for: (i) examining interaction between nuclear and plastid genomes: (ii) elucidating the molecular processes involved in the evolution of plastid genes: (iii) clarifying the events in the synthesis and assembly of macromolecular complexes in the chloroplast.     

Characterization of L-CAM, a major cell adhesion molecule from embryonic liver cells

We have developed a method for purifying L-CAM, the cell adhesion molecule from embryonic chicken liver cells, and have compared its properties with those of N-CAM, the neural cell adhesion molecule. L-CAM was released from membranes with trypsin, purified by a series of chemical techniques, and used to generate monoclonal antibodies which allowed the identification of the intact L-CAM molecule from membranes. The monoclonal antibodies were used to isolate trypsin-released L-CAM in a single step by affinity chromatography. Material purified by either technique was predominantly a component of M_r 81,000 on NaDodSO₄/polyacrylamide gel electrophoresis with a pI of 4.0-4.5. Rabbit antibodies to this component and to the M_r 81,000 species that had been further purified on NaDodSO₄/polyacrylamide gel electrophoresis displayed all of the activities of anti-L-CAM. Some of the trypsin-released L-CAM bound specifically to lentil lectin, suggesting that L-CAM is a glycoprotein. The apparent molecular weight of material having L-CAM antigenic determinants depended upon the procedures used to extract membranes; this appears to account for the various values reported previously in the literature. Both the rabbit serum antibodies and the monoclonal antibodies detected the M_r 81,000 species on immunoblots of unfractionated trypsin-released material. Immunoblots of whole liver cell membranes with the same antibodies revealed a major M_r 124,000 component, with minor components of M_r 94,000 and 81,000. Active L-CAM derivatives released by trypsin in the presence of EGTA were detected as a species of M_r 40,000. L-CAM derivatives obtained by extraction of membranes with EDTA alone appeared as species of M_r 53,000, 62,000, and 81,000. The combined results suggest that L-CAM on the cell surface is an acidic glycoprotein of M_r 124,000. In the presence of calcium, the molecule can be released from membranes by trypsin as a soluble M_r 81,000 fragment; in the absence of calcium, it is released by either endogenous proteases or by trypsin as a variety of smaller fragments.     

Higher plant chloroplasts: Evidence that all the chlorophyll exists as chlorophyll-protein complexes

By using the polyacrylamide gel electrophoresis system described in this report, it was possible to fractionate all the photosynthetic pigments of maize (Zea mays L.) thylakoids into chlorophyll—protein complexes with negligible formation of free or detergent-complexed chlorophyll. Identical sodium dodecyl sulfate extracts of thylakoids have previously resulted in up to 50% of the chlorophyll migrating as free chlorophyll after electrophoresis. The major difference from previous gel electrophoresis systems is the replacement of sodium dodecyl sulfate in the electrophoresis buffer by Deriphat 160 (disodium N-lauryl-β-iminodipropionate), a zwitterionic detergent. The results suggest that: (i) no significant amount of free chlorophyll exists in the chloroplast thylakoid membranes in vivo, and (ii) most of the free pigment seen previously on gels was generated during the electrophoresis and was not a result of the solubilization technique. Additionally, the new chlorophyll-protein complexes resolved appear to have different characteristics (pigment content and size) that those observed in former systems.     

Native phytochrome: Inhibition of proteolysis yields a homogeneous monomer of 124 kilodaltons from Avena

Phytochrome purified from Avena as the red-absorbing form, Pr, by an established immunoaffinity column procedure is heterogeneous. Two major polypeptides and one minor polypeptide with apparent molecular masses of 118, 114, and 112 kilodaltons (kDal), respectively, are observed on NaDodSO₄/polyacrylamide gel electrophoresis. In contrast, only a single band of 124 kDal is obtained when phytochrome is rapidly immunoprecipitated after extraction either (i) as the far-red absorbing form, Pfr, in detergent-free buffer or (ii) in either spectral form in a 100°C NaDodSO₄-containing buffer. On two-dimensional gel electrophoresis the three column-purified species have pIs of 5.8, 6.0, and 6.0, whereas 124-kDal phytochrome is a single spot with a pI of 5.9. Incubation as Pr in extracts causes progressive conversion of the 124-kDal polypeptide to the 118- and 114-kDal species. This process is inhibited by phenylmethylsulfonyl fluoride, suggesting that Pr is susceptible and Pfr resistant to limited proteolysis during extraction. These data, and the fact that the cell-free translation product of phytochrome mRNA is also 124 kDal [Bolton, G. W. & Quail, P. H. (1982) Planta, in press], indicate that the native monomer from Avena is a single species of 124 kDal. Thus the heterogeneous preparations of slightly lower molecular weight (“large” or “120-kilodalton” phytochrome) previously extensively characterized appear to have consisted of a mixture of partially degraded molecules that have undergone limited proteolysis during purification as Pr, as is established practice. A reexamination of the molecular properties of phytochrome appears necessary.     

Comparison of the exoproducts of gram-negative bacteria by SDS-page

The protein exoproducts released during exponential growth of Gram-negative bacteria were analysed and compared by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-Page). The following bacterial strains were tested: Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, Enterobacter cloacae, Serratia liquefaciens, Serratia rubidaea, Proteus mirabilis, Proteus vulgaris, Salmonella minnesota, Pseudomonas aeruginosa and Pseudomonas fluorescens. It is demonstrated by SDS-Page that members of one species show identical protein pattern, whereas different species show besides comparable protein bands a species characteristic pattern. All members of Enterobacteriaceae were shown to release proteins whose molecular weights fell into the following size regions: Each strain was shown to synthesize a polypeptide of molecular weight 34,000 and one or more polypeptides within the molecular weight range 25,000–29,000. This profile was shown to be clearly different from that of Pseudomonas strains where 20 or more distinct polypeptides ranging from 12,500 to 160,000 Mr were detectable.     

Characterization of immunologically significant unique B16 melanoma proteins produced in vivo and in vitro

Proteins unique to B16 melanoma cells grown in culture and as solid tumors have been independently described recently. As both proteins have apparent M_r of 65,000-70,000, we have more fully compared their biochemical characteristics. The M_r 65,000 protein, which is isolated from B16 cells in culture, has an aggregate molecular weight of >200,000 under nonreducing conditions. This M_r 65,000 form is shed into the culture medium exclusively and is the predominant molecular species of serum-free culture supernatants. It was found that B700, a major membrane protein of B16 cells in solid tumors, and the M_r 65,000 preparation from cultured cells were electrophoretically identical when compared under four separate sets of conditions. These two proteins also share immunologic determinants. The results suggest the identity of these two proteins isolated by completely different approaches.     

Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the Ti plasmid of Agrobacterium tumefaciens

We have shown that the structural gene for octopine synthase (a crown gall-specific enzyme) is located in a central portion of the T-DNA that came from the Ti plasmid of agrobacterium tumefaciens and is expressed after it has been transferred to the plant cells. Polyadenylylated RNA was prepared from polysomes isolated from an octopine-producing crown gall callus and purified by selective hybridization to one of five recombinant plasmids. Each such plasmid contained a different fragment of T-DNA of pTi-15955 (octopine-type Ti plasmid). Purified mRNA was translated in vitro in rabbit reticulocyte lysates, and the translation products were immunoprecipitated with antibody against octopine synthase. Total and immunoprecipitated products were characterized by their molecular weights. A polypeptide of M_r 40,000 (the same as authentic octopine synthase) was synthesized in vitro by crown gall mRNA selectively hybridized to three of the five fragments of T-DNA and precipitated with antibody against octopine synthase. This polypeptide was not immunoprecipitated with normal rabbit antibody nor was it synthesized when mRNA from the habituated callus was substituted. A mRNA 1500 bases long was detected when total mRNA was fractionated on an agarose gel, transferred to nitrocellulose, and used for hybridization to three of the five ³²P-labeled T-DNA fragments. This apparent mRNA for octopine synthase hybridized to the same three fragments of T-DNA as the mRNA for the M_r 40,000 polypeptide and was not detected in the habituated callus.     

Nodulin-24 gene of soybean codes for a peptide of the peribacteroid membrane and was generated by tandem duplication of a sequence resembling an insertion element

A nodulin gene coding for a polypeptide with an apparent M_r of 24,000 (nodulin-24) was isolated from soybean (Glycine max). DNA sequence analysis of this gene revealed that its coding capacity is for a polypeptide of only M_r 15,100 and is interrupted by four introns. The three middle exons and their flanking segments appear to have been generated by duplications of a unit resembling an insertion sequence. This unit is bounded by a 12-base-pair inverted repeat and encompasses the 54-base-pair exon corresponding to each of three central hydrophobic domains of the protein, nodulin-24. The resulting repeated hydrophobic structure of this protein may be responsible for an apparent increase in M_r from 15,100 to 24,000. In vitro translation and immunological studies suggest that nodulin-24 is a precursor and is processed cotranslationally into a M_r 20,000 polypeptide. This polypeptide is a component of the membrane envelope enclosing the bacteroids (peribacteroid membrane) synthesized during symbiosis with Rhizobium. The low degree (<6%) of sequence divergence among the repeated units suggests that this gene has been generated recently during the evolution of symbiotic nitrogen fixation in soybean.     

Giant circular dichroism of high molecular weight chlorophyllide-apomyoglobin complexes

Chlorophyllide a and the apoprotein of myoglobin (Mb) spontaneously form three types of complex. The M (M_r ≈ 3 × 10⁵) and H (M_r ≥ 4 × 10⁶) complexes, but not the L (M_r ≈ 1.7 × 10⁴), display a circular dichroism (CD) spectrum that is highly red-shifted, nonconservative, and very intense—characteristics shared by the CD spectra of reaction center complexes from purple photosynthetic bacteria. At its 710-nm peak, the H complex CD spectrum has a larger magnitude, 0.06 differential absorbance per unit total absorbance, than has been reported for chlorophyll in any medium.     

Molecular characterization of the terminal energy acceptor of cyanobacterial phycobilisomes.

Cyanobacteria harvest light energy through multimolecular structures, the phycobilisomes, regularly arrayed at the surface of the photosynthetic membranes. Phycobilisomes consist of a central core from which rods radiate. A large polypeptide (LCM, 75-120 kDa) is postulated to act both as terminal energy acceptor and as a linker polypeptide that stabilizes the phycobilisome architecture. We report here the characterization of the gene (apcE) that encodes this LCM polypeptide in Calothrix sp. PCC 7601. It is located upstream from the genes encoding the major components of the phycobilisome core (allophycocyanin) and is part of the same operon. The deduced amino acid sequence shows that the N-terminal region of LCM shares homology with the other phycobiliprotein subunits and thus constitutes the chromoprotein domain. The other part of the molecule is made up of four repeated domains that are highly homologous to the N-terminal regions of the phycocyanin rod linker polypeptides. The predicted secondary structure of the different domains of the LCM is discussed in relation to the different roles and properties of this large molecule.     

Stoichiometric model of the photosynthetic unit of Ectothiorhodospira halochloris

A stoichiometric model of the photosynthetic unit of Ectothiorhodospira halochloris has been obtained by means of scanning transmission electron microscope mass determination and mass mapping in conjunction with polyacrylamide gel electrophoresis. One reaction center, consisting of four single polypeptides, including one cytochrome, is surrounded by six identical light-harvesting complexes, each containing three polypeptides with 2:2:2 stoichiometry. This stoichiometric model was incorporated into the three-dimensional structure of the photosynthetic unit as derived from surface relief reconstructions of the two surfaces of shadowed membranes. The reaction center protrudes substantially from both membrane surfaces and has the cytochrome attached to the periplasmic face in a noncentrosymmetric fashion. The reaction center may assume various orientations within the photosynthetic complexes.     

The role of cell surface proteins in platelet stimulation by monosodium urate crystals

To test whether urate crystal-membrane protein interactions mediate platelet stimulation, platelet membrane proteins were radiolabeled by lactoperoxidase, extracted with 1% Triton X–100, and incubated with urate crystals. The crystal-associated and supernate fractions were isolated and analyzed by sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and by 2-dimensional isoelectric focusing followed by SDS-PAGE. Four of the lactoperoxidaseradiolabeled polypeptides that associated with urate crystals had reduced molecular weights and pIs of M_r = 105,000, pI 4.9; M_r = 123,000, pI = 4.9; M_r = 123,000, pI = 5.3; and M_r = 132,000, pI = 4.8–6.3, respectively. These proteins were characterized with regard to their labeling intensities, apparent isoelectric points, apparent molecular weights (reduced and nonreduced), lectin-binding properties, carbohydrate- and protein-staining characteristics, and presence or absence in Glanzmann's thrombasthenia. They have been identified as derived from glycoproteins Ib, IIb, and III (Phillips-Agin nomenclature) and an unidentified membrane protein. To test whether removal of these proteins would result in a diminution of platelet response to urate, intact platelets were digested with chymotrypsin, resulting in cleavage of more than 80% of these proteins and a 5-fold reduction in secretory responsiveness to urate crystals. Response to a second platelet stimulus, collagen, was unaffected, indicating an intact secretory mechanism. In addition, when platelets were preincubated with F(ab′)₂ fragments of an antibody directed against these proteins, platelet secretory response to urate was inhibited by 50%, whereas the responses to collagen and thrombin were unaffected. Thus, membrane proteins appear to mediate platelet stimulation by urate crystals.