Specific binding of victorin to a 100-kDa protein from oats

Susceptibility of oats to victoria blight, caused by the fungus Cochliobolus victoriae, and sensitivity to the host-specific toxin victorin, produced by the fungus, are controlled by the dominant allele at the Vb locus. It has been postulated that the Vb locus encodes a toxin receptor, although direct evidence for such a receptor is not available. Our recent studies on structure—activity relationships of the toxin established a methodology for producing ¹²⁵I-labeled victorin. Electrophoretic analysis of proteins from isogenic susceptible and resistant oat genotypes following treatment of leaves with radiolabeled victorin showed that victorin binds in a covalent and a genotype-specific manner to a 100-kDa protein only in susceptible oat leaf slices. This in vivo binding was competitively displaced by reduced victorin, a nontoxic protective compound, and appeared to be correlated with biological activity. In vitro binding to the 100-kDa protein in leaf extracts showed several differences from in vivo binding. Binding was not genotype specific and required a reducing agent that was not required for in vivo binding. Differential centrifugation showed that the 100-kDa victorin binding protein was not a cytosolic protein but was enriched in a high-speed particulate fraction. The data support the hypothesis that the 100-kDa protein is the victorin receptor.     

Acyl-acyl carrier protein as a source of fatty acids for bacterial bioluminescence

Pulse-chase experiments with [³H]tetradecanoic acid and ATP showed that the bioluminescence-related 32-kDa acyltransferase from Vibrio harveyi can specifically catalyze the deacylation of a ³H-labeled 18-kDa protein observed in extracts of this bacterium. The 18-kDa protein has been partially purified and its physical and chemical properties strongly indicate that it is fatty acyl-acyl carrier protein (acyl-ACP). Both this V. harveyi [³H]acylprotein and [³H]palmitoyl-ACP from Escherichia coli were substrates in vitro for either the V. harveyi 32-kDa acyltransferase or the analogous enzyme (“34K”) from Photobacterium phosphoreum. TLC analysis indicated that the hexane-soluble product of the reaction is fatty acid. Phosphate ions and, to a lesser extent, organic alcohols stimulated the rate of acyl-protein cleavage. No significant cleavage of either E. coli or V. harveyi tetradecanoyl-ACP was observed in extracts of these bacteria unless the 32-kDa or 34K acyltransferase was present. Since these enzymes are believed to be responsible for the supply of fatty acids for reduction to form the aldehyde substrate of luciferase, the above results suggest that long-chain acyl-ACP is the source of fatty acids for bioluminescence.     

Herbicide resistance in Chlamydomonas reinhardtii results from a mutation in the chloroplast gene for the 32-kilodalton protein of photosystem II

We report the isolation and characterization of a uniparental mutant of Chlamydomonas reinhardtii that is resistant to 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) and 2-chloro-4-ethylamino-6-isopropylamino-s-triazine (atrazine). Such herbicides inhibit photosynthesis by preventing transfer of electrons in photosystem II from the primary stable electron acceptor Q to the secondary stable electron acceptor complex B, which is thought to contain a protein of 32 kDa and a bound quinone. It has been proposed that herbicide binding to the 32-kDa protein alters the B complex so that electron transfer from Q is prohibited. Both whole and broken-cell preparations of the mutant alga show a resistance to the effects of herbicide on electron transfer from Q to B, as measured by fluorescence-induction kinetics. In the absence of herbicide, mutant cells exhibit a slower rate of Q to B electron transfer than do wild-type cells. The 32-kDa protein from wild-type cells, but not mutant cells, binds azido[¹⁴C]atrazine at 0.1 μM. We have isolated psbA, the chloroplast gene for the 32-kDa protein, from both wild-type and herbicide-resistant algae and sequenced the coding regions of the gene that are contained in five exons. The only difference between the exon nucleotide sequences of the wild-type and mutant psbA is a single T-A to G-C transversion. This mutation results in a predicted amino acid change of serine in the wild-type protein to alanine in the mutant. We suggest that this alteration in the 32-kDa protein is the molecular basis for herbicide resistance in the C. reinhardtii mutant.     

Specific binding of a fungal glucan phytoalexin elicitor to membrane fractions from soybean Glycine max

Treatment of soybean tissues with elicitors results in the production of phytoalexins, one of a number of inducible plant defense reactions against microbial infections. The present study uses a β-1,3-[³H]glucan elicitor fraction from Phytophthora megasperma f. sp. glycinea, a fungal pathogen of soybean, to identify putative elicitor targets in soybean tissues. Use of the radiolabeled elicitor disclosed saturable high-affinity elicitor binding site(s) in membrane fractions of soybean roots. Highest binding activity is associated with a plasma membrane-enriched fraction. The apparent K_d value for β-glucan elicitor binding is ≈0.2 × 10^-6 M and the maximum number of binding sites is 0.5 pmol per mg of protein. Competition studies with the [³H]glucan elicitor and a number of polysaccharides demonstrate that only polysaccharides of a branched β-glucan type effectively displace the radiolabeled ligand from membrane binding. Differential displacing activity of the glucans on P. megasperma elicitor binding corresponds closely to their respective ability to elicit phytoalexin production in a cotyledon bioassay.     

High affinity cross-reacting mAb generated by minimal mimicry: Implications for the pathogenesis of anti-nuclear autoantibodies and immunosuppression

The antigen recognition of a profoundly immunosuppressive mAb, mAb 2E1, in vivo was investigated. In addition to the 62-kDa effector cell protease receptor 1, mAb 2E1 bound the 32-kDa T cell adhesion receptor E2 (CD99) and the 86-kDa p80 subunit of the nuclear antigen complex Ku. These molecules share no overall sequence similarity. Peptide mapping experiments identified the mAb 2E1 cross-reacting epitopes as the sequences ⁶⁶GSFSDADLAD⁷⁵ in E2 and ⁵⁷¹GGAHFSVSSLAEG⁵⁸³ in p80 of Ku, sharing a minimal homology motif FSXXXLA, in which X is a nonconserved amino acid. Each of these peptides separately inhibited the binding of mAb 2E1 to E2, effector cell protease receptor 1, and p80 of Ku in a dose-dependent manner. Scatchard plot analysis of ¹²⁵I-labeled mAb 2E1 binding to peripheral blood mononuclear cells revealed a high-affinity interaction with a dissociation constant of 7 × 10⁻¹⁰ M. An anti-E2 mAb bound the same epitope ⁶⁶GSFSDADLAD⁷⁵ recognized by mAb 2E1 but failed to react with p80 of Ku and was not immunosuppressive. These findings demonstrate that high-affinity cross-reacting mAbs can be generated by mimicry of a minimal surface on unrelated molecules. This model of minimal mimicry may determine the nuclear reactivity of certain autoantibodies to Ku and contribute to aberrant immunosuppression in vivo.     

Identification of the triazine receptor protein as a chloroplast gene product

The triazine herbicides inhibit photosynthesis by blocking electron transport at the second stable electron acceptor of photosystem II. This electron transport component of chloroplast thylakoid membranes is a protein-plastoquinone complex termed “B.” The polypeptide that is believed to be a component of the B complex has recently been identified as a 32- to 34-kilo-dalton polypeptide by using a photoaffinity labeling probe, azido-[¹⁴C]atrazine. A 34-kilodalton polypeptide of pea chloroplasts rapidly incorporates [³⁵S]methionine in vivo and is also a rapidly labeled product of chloroplast-directed protein synthesis. Trypsin treatment of membranes tagged with azido-[¹⁴C]atrazine, [³⁵S]methionine in vivo, or [³⁵S]methionine in isolated intact chloroplasts results in identical, sequential alterations of the 34-kilo-dalton polypeptide to species of 32, then 18 and 16 kilodaltons. From the identical pattern of susceptibility to trypsin we conclude that the rapidly synthesized 34-kilodalton polypeptide that is a product of chloroplast-directed protein synthesis is identical to the triazine herbicide-binding protein of photosystem II. Chloroplasts of both triazine-susceptible and triazine-resistant biotypes of Amaranthus hybridus synthesize the 34-kilodalton polypeptide, but that of the resistant biotype does not bind the herbicide.     

Evidence for two nonidentical drug-interaction sites in the human P-glycoprotein

Human P-glycoprotein (Pgp) confers multidrug resistance to cancer cells by ATP-dependent extrusion of a great many structurally dissimilar hydrophobic compounds. The manner in which Pgp recognizes these different substrates is unknown. The protein shows internal homology between its N- and C-terminal halves, each comprised of six putative transmembrane helices and a consensus ATP binding/utilization site. Photoactive derivatives of certain Pgp substrates specifically label two regions, one on each half of the protein. In this study, using [¹²⁵I]iodoarylazidoprazosin ([¹²⁵I]IAAP), a photoactive analog of prazosin, we have demonstrated the presence of two nonidentical drug-interaction sites within Pgp. Taking advantage of a highly susceptible trypsin cleavage site in the linker region of Pgp, we characterized the [¹²⁵I]IAAP binding to the N- and C-terminal halves. cis(Z)-Flupentixol, a modulator of Pgp function, preferentially increased the affinity of [¹²⁵I]IAAP for the C-terminal half of the protein (C-site) by reducing the K_d from 20 to 6 nM without changing the labeling or affinity (K_d = 42–46 nM) of the N-terminal half (N-site). Also, the concentration of vinblastine (Pgp substrate) and cyclosporin A (Pgp modulator) required for 50% inhibition of [¹²⁵I]IAAP binding to the C-site was increased 5- to 6-fold by cis(Z)-flupentixol without any effect on the N-site. In addition, [¹²⁵I]IAAP binding to the N-site was less susceptible than to C-site to inhibition by vanadate which blocks ATP hydrolysis and drug transport. These data demonstrate the presence of at least two nonidentical substrate interaction sites in Pgp.     

Extremely large and slowly processed precursors to the Euglena light-harvesting chlorophyll a/b binding proteins of photosystem II

Antibody to the Euglena light-harvesting chlorophyll a/b binding protein of photosystem II (LHCPII) immunoprecipitated 207-, 161-, 122-, and 110-kDa proteins from total Euglena proteins pulse-labeled for 10 min with [³⁵S]sulfate. The 25.6- and 27.2-kDa LHCPII were barely detectable in the immunoprecipitate. During a 40-min chase with unlabeled sulfate, the amount of radioactivity in the high molecular mass proteins decreased, and the amount of radioactivity in the 25.6- and 27.2-kDa LHCPII increased with kinetics consistent with a precursor-product relationship. The half-life of the high molecular mass proteins was ≈20 min. The major proteins immunoprecipitated from a nuclease-treated rabbit reticulocyte cell-free translation system programmed with Euglena whole cell or poly(A)⁺ RNA had molecular masses corresponding to the molecular masses of the proteins immunoprecipitated from the pulse-labeled in vivo translation products. RNAs of 6.6 and 8.3 kilobases were the only Euglena whole cell and poly(A)⁺ RNAs that hybridized to a 0.7-kilobase EcoRI-BamHI fragment of plasmid pAB165, which contains a portion of the coding sequence for Arabidopsis LHCPII. RNAs of this size are more than sufficient to code for proteins of 207 kDa. Taken together, these findings demonstrate that the LHCPIIs of Euglena are initially synthesized as slowly processed precursors with molecular masses of 207, 161, 122, and 110 kDa.     

Cloned avirulence genes from the tomato pathogen Pseudomonas syringae pv. tomato confer cultivar specificity on soybean

Three different cosmid clones were isolated from a genomic library of the tomato pathogen Pseudomonas syringae pv. tomato, which, when introduced into the soybean pathogen P. syringae pv. glycinea, caused a defensive hypersensitive response (HR) in certain soybean cultivars. Each clone was distinguished by the specific cultivars that reacted hypersensitively and by the intensity of the HR elicited. Unlike wild-type P. syringae pv. tomato isolates, which elicit the HR on all soybean cultivars, all three clones exhibited cultivar specificities analogous to avirulence genes previously cloned from P. syringae pv. glycinea. However, the collective phenotypes of the three clones accounted for HRs on all tested soybean cultivars. One of the three P. syringae pv. tomato clones contained an avirulence gene homologous to avrA, which was previously cloned from P. syringae pv. glycinea race 6. The other two P. syringae pv. tomato clones expressed unique HR patterns on various soybean cultivars, which were unlike those caused by any known P. syringae pv. glycinea race or previously cloned P. syringae pv. glycinea avr gene. Further characterization of the second P. syringae pv. tomato clone indicated that the avirulence phenotype resided on a 5.6-kilobase HindIII fragment that, in Southern blot analyses, hybridized to an identical-size fragment in various P. syringae pathovars, including all tested glycinea races. These results demonstrate that avirulence genes may be distributed among several P. syringae pathovars but may be modified so that the HR is not elicited in a particular host plant. Furthermore, the data raise the possibility that avirulence genes may function in host-range determination at levels above race—cultivar specificity.     

Production and characterisation of gilthead sea bream (Sparus auratus) recombinant parathyroid hormone related protein

The production and purification of gilthead sea bream recombinant parathyroid hormone related protein [sbPTHrP(1-125)] using an Escherichia coli system and one step purification process with continuous elution gel electrophoresis is reported. The cDNA encoding sbPTHrP(1-125) was cloned into a prokaryotic expression vector pET-11a. The recombinant plasmid was used to transfect E. coli BL21(DE3) pLysS and sbPTHrP(1-125) synthesis was induced by addition of 1 mM isopropyl-β-d-thiogalactopyranoside. The rapid one step isolation method gave pure sbPTHrP(1-125) as judged by SDS-PAGE and yielded up to 40 mg/L of culture medium (3.3 mg protein/g of bacteria). The bioactivity of recombinant sbPTHrP(1-125) assessed using an in vitro scale bioassay was found to be equipotent to PTHrP(1-34) in stimulating cAMP accumulation. Assessment of the immunological reactivity of the isolated protein by Western blot revealed it cross-reacts with antisera specific for the N-terminal and C-terminal region of PTHrP. In a radioimmunoassay specific for piscine N-terminal (1-34 aa) PTHrP, the recombinant sbPTHrP(1-125) was equipotent with PTHrP(1-34) in displacing labelled ¹²⁵I-PTHrP(1-36) PTHrP from the antisera. The availability of recombinant sbPTHrP will allow the development of region specific assays and studies aimed at defining post-secretory processing of this protein and its biological activity in fish.     

Purified omega-conotoxin GVIA receptor of rat brain resembles a dihydropyridine-sensitive L-type calcium channel.

The omega-conotoxin GVIA (CTX) receptor has been purified 1900-fold to apparent homogeneity by monitoring both reversible binding of 125I-labeled CTX (125I-CTX) and photoincorporation of N-hydroxysuccinimidyl-4-azidobenzoate-125I-CTX (HSA-125I-CTX). Photoincorporation of HSA-125I-CTX into a 230-kDa protein exhibits a pharmacologic and chromatographic profile indicating that the 230-kDa protein is the CTX-binding subunit of the receptor. The pharmacologic specificity of 125I-CTX binding to the purified CTX receptor closely resembles that of the native membrane-bound form with respect to sensitivity towards CTX (Kd = 32 pM) and other peptide toxin antagonists. The purified CTX receptor comprises the 230-kDa protein (alpha 1) and four additional proteins with apparent molecular masses of 140 (alpha 2), 110, 70 (beta 2), and 60 (beta 1) kDa. This subunit structure closely resembles that of the 1,4-dihydropyridine-sensitive L-type calcium channel.     

Identification and characterization of alpha 1 adrenergic receptors in rat myocardium with a new iodinated radioligand, [125I]IBE 2254

The cardiac alpha adrenergic receptor has been identified in previous studies with tritiated radioligands. However, tritiated radioligands which are relatively low in specific activity, may pose distinct disadvantages in the detection of alpha receptors including the need for large quantitities of tissue, substantial non-receptor, nonspecific binding, and insensitivity at subsaturating ligand concentrations. To overcome these problems we have utilized 2-[β-(4-hydroxyphenyl)ethylamino-methyl] tetralone (BE 2254) a compound shown to be a potent pharmacological alpha₁ adrenergic inhibitor. BE 2254 was iodinated and purified to theoretically high specific activity (2200 Ci/mmol). A membrane fraction that retained stable binding properties and adenylate cyclase activity was prepared from Sprague-Dawley rat hearts. Specific binding of [¹²⁵I]IBE 2254 to cardiac membranes was demonstrable to as little as 10 μg of protein. Nonspecific binding, defined as that component of total binding inhibited by excess, unlabeled BE 2254 (4 μm), ranged from 0 to 20%. At 37°C binding was rapid, saturable, stable, and completely reversible. The association rate constant, k₁, was 1.88 × 10⁸ min^?1m^?1. The dissociation rate constant, k_?1, was 0.09 min^?1. An estimate of the equilibrium dissociation constant (K_D) based upon these kinetic data (480 pm) was in close agreement with the K_D determined by Scatchard analysis (363 pm). The binding capacity of a single class of high affinity binding sites was 280 fmol/mg protein. A series of adrenergic agonists and antagonists competed successfully with [¹²⁵I]IBE 2254 for binding to myocardial membranes with an order of potency indicating alpha site specificity. Dopamine and serotonin were extremely weak competitors at concentrations < 0.1 mm. A series of alpha adrenergic antagonists inhibited [¹²⁵I]IBE 2254 binding with an order of potency consistent with alpha₁ subselectivity: prazosin (alpha₁) > phentolamine (alpha₁/alpha₂) > yohimbine (alpha₂). Moreover, binding was stereoselective for both agonists and antagonists. These observations indicate that the alpha adrenergic antagonist BE 2254 can be iodinated, purified and used with sensitivity and specificity to identify alpha₁ adrenergic receptors in cardiac tissue.     

Neurabin is a synaptic protein linking p70 S6 kinase and the neuronal cytoskeleton

p70 S6 kinase (p70^S6k) is a mitogen-activated protein kinase that plays a central role in the control of mRNA translation. It physiologically phosphorylates the S6 protein of the 40s ribosomal subunit in response to mitogenic stimuli and is a downstream component of the rapamycin-sensitive pathway, which includes the 12-kDa FK506 binding protein and includes rapamycin and the 12-kDa FK506 binding protein target 1. Here, we report the identification of neurabin (neural tissue-specific F-actin binding protein), a neuronally enriched protein of 1,095 amino acids that contains a PDZ domain and binds p70^S6k. We demonstrate the neurabin-p70^S6k interaction by yeast two-hybrid analysis and biochemical techniques. p70^S6k and neurabin coimmunoprecipitate from transfected HEK293 cells. Site-directed mutagenesis of neurabin implicates its PDZ domain in the interaction with p70^S6k, and deletion of the carboxyl-terminal five amino acids of p70^S6k abrogates the interaction. Cotransfection of neurabin in HEK293 cells activates p70^S6k kinase activity. The mRNA of neurabin and p70^S6k show striking colocalization in brain sections by in situ hybridization. Subcellular fractionation of rat brain demonstrates that neurabin and p70^S6k both localize to the soluble fraction of synaptosomes. By way of its PDZ domain, the neuronal-specific neurabin may target p70^S6k to nerve terminals.     

A radioreceptor assay for purified teleost growth hormone.

Highly purified ¹²⁵I-labelled tilapia (Sarotherodon mossambicus) growth hormone (GH) binds to membrane fractions prepared from tilapia liver. Specific (displaceable) binding occurred with the 600, 15,000, and 90,000g fractions with the greatest binding observed with the 90,000g microsomal membrane fraction. Binding was dependent on time and membrane concentration. Specificity studies showed that up to 60% of the ¹²⁵I-labelled tilapia GH bound to the liver microsomal membrane fraction could be displaced by 500 ng of unlabelled hormone. Scatchard analysis of the binding of ¹²⁵I-labelled tilapia GH revealed a single class of binding site with a binding capacity of 125 ± 7.7 fmol/mg protein and a binding affinity of 1.5 × 10¹⁰ ± 0.4 × 10¹⁰ (SEM) l/mol. GH preparations from several vertebrate classes and tilapia prolactin and ovine prolactin at high concentrations displayed competition for the tilapia GH-binding site.¹²⁵I-Labelled tilapia GH demonstrated specific binding to liver microsomal membrane fractions of the teleosts Gillichthys mirabilis, Salmo gairdneri, and Oncorhynchus tschawytscha but not to those of an amphibian Necturus maculosus or a bird Coturnix japonica. Slight, but significant, specific binding was observed with the microsomal membrane fraction of tilapia kidney and gill and of rat liver. These data suggest that this tilapia GH radioreceptor assay may have an application in the detection of teleost GH-like activity.     

CGM1a antigen of neutrophils, a receptor of gonococcal opacity proteins

Neisseria gonorrhoeae (GC) or Escherichia coli expressing phase-variable opacity (Opa) protein (Opa⁺ GC or Opa⁺ E. coli) adhere to human neutrophils and stimulate phagocytosis, whereas their counterparts not expressing Opa protein (Opa⁻ GC or Opa⁻ E. coli) do not. Opa⁺ GC or E. coli do not adhere to human lymphocytes and promyelocytic cell lines such as HL-60 cells. The adherence of Opa⁺ GC to the neutrophils can be enhanced dramatically if the neutrophils are preactivated. These data suggest that the components binding the Opa⁺ bacteria might exist in the granules. CGM1a antigen, a transmembrane protein of the carcinoembryonic antigen family, is exclusively expressed in the granulocytic lineage. The predicted molecular weight of CGM1a is ≈30 kDa. We observed specific binding of OpaI⁺ E. coli to a 30-kDa band of polymorphonuclear leukocytes lysates. To prove the hypothesis that the 30-kDa CGM1a antigen from neutrophils was the receptor of Opa⁺ bacteria, we showed that a HeLa cell line expressing human CGM1a antigen (HeLa-CGM1a) bound Opa⁺ E. coli and subsequently engulfed the bacteria. Monoclonal antibodies (COL-1) against CGM1 blocked the interaction between Opa⁺ E. coli and HeLa-CGM1a. These results demonstrate that HeLa cells when expressing the CGM1a antigens bind and internalize OpaI⁺ bacteria.     

Biological effects of low-dose-rate irradiation of pancreatic carcinoma cells in vitro using ~(125)I seeds

AIM:To determine the mechanism of the radiationinduced biological effects of 125I seeds on pancreatic carcinoma cells in vitro.METHODS:SW1990 and PANC-1 pancreatic cancer cell lines were cultured in DMEM in a suitable environment.Gray’s model of iodine-125(125I)seed irradiation was used.In vitro,exponential phase SW1990,and PANC-1cells were exposed to 0,2,4,6,and 8 Gy using 125I radioactive seeds,with an initial dose rate of 12.13c Gy/h.A clonogenic survival experiment was performed to observe the ability of the cells to maintain their clonogenic capacity and to form colonies.Cell-cycle and apoptosis analyses were conducted to detect the apoptosis percentage in the SW1990 and PANC-1 cells.DNA synthesis was measured via a tritiated thymidine(3H-Td R)incorporation experiment.After continuous low-dose-rate irradiation with 125I radioactive seeds,the survival fractions at 2 Gy(SF2),percentage apoptosis,and cell cycle phases of the SW1990 and PANC-1 pancreatic cancer cell lines were calculated and compared.RESULTS:The survival fractions of the PANC-1 andSW1990 cells irradiated with 125I seeds decreased exponentially as the dose increased.No significant difference in SF2 was observed between SW1990 and PANC-1 cells(0.766±0.063 vs 0.729±0.045,P0.05).The 125I seeds induced a higher percentage of apoptosis than that observed in the control in both the SW1990and PANC-1 cells.The rate of apoptosis increased with increasing radiation dosage.The percentage of apoptosis was slightly higher in the SW1990 cells than in the PANC-1 cells.Dose-dependent G2/M cellcycle arrest was observed after 125I seed irradiation,with a peak value at 6 Gy.As the dose increased,the percentage of G2/M cell cycle arrest increased in both cell lines,whereas the rate of DNA incorporation decreased.In the 3H-Td R incorporation experiment,the dosimetry results of both the SW1990 and PANC-1cells decreased as the radiation dose increased,with a minimum at 6 Gy.There were no significant differences in the dosimetry results of the two cell lines when they were exposed to the same dose of radiation.CONCLUSION:The pancreatic cancer cell-killing effects induced by 125I radioactive seeds mainly occurred via apoptosis and G2/M cell cycle arrest.     

Purification and partial characterization of a receptor protein for mouse interferon gamma.

A receptor protein for mouse interferon gamma has been purified from solubilized plasma membranes of the mouse monomyelocytic cell line WEHI-3. Sequential wheat germ agglutinin and ligand affinity chromatography of membranes extracted with octyl beta-D-glucopyranoside resulted in at least a 680-fold purification of the receptor, as measured by precipitating it in association with liposomes composed of phosphatidylcholine. The purified receptor bound 125I-labeled recombinant mouse interferon gamma (rMuIFN-gamma) with a Kd of 10 nM, a value comparable to that obtained with isolated membranes (3.5 nM). PAGE analysis of radiolabeled (with either 35S or 125I) receptor preparations consistently revealed a major band of 95 kDa. This species was degraded with time to smaller fragments, principally one of 60 +/- 5 kDa. Treatment with peptide:N-glycosidase F reduced the apparent molecular masses of the proteins in the 95- and 60-kDa regions by 15-20 kDa each. GR-20, a monoclonal antibody against the receptor, completely inhibited specific binding of 125I-labeled rMuIFN-gamma to WEHI-3 cells, blocked the induction of priming by rMuIFN-gamma of macrophage-mediated tumor cell killing, removed binding activity for 125I-labeled rMuIFN-gamma from solubilized membranes, and immunoprecipitated a single 95-kDa protein from the extract of surface labeled (125I) WEHI-3 cells. Cross-linking of 125I-labeled rMuIFN-gamma to its receptor yielded a complex of 125 +/- 5 kDa, consistent with the binding of the dimeric form of mouse interferon gamma (32 kDa) to a membrane protein of 95 kDa. These data suggest that the receptor for mouse interferon gamma (or a ligand-binding subunit thereof) is a glycoprotein of 95 kDa.     

Visualization of the Ca-transport system of the mitotic apparatus of sea urchin eggs with a monoclonal antibody

Monoclonal antibodies have been obtained to components of Ca²⁺-sequestering vesicles from the endoplasmic reticulum of HeLa cells by isolating hybridomas that were generated by the in vitro immunization of lymphocytes followed by fusion with plasmocytoma cells. One of these monoclonal antibodies specifically labels punctate structures which appear in the mitotic apparatus of sea urchin eggs at the beginning of prophase and disappear upon the completion of cytokinesis. The antibody inhibits the Ca²⁺ uptake of the membrane system in vitro. It reacts with one 46-kDa protein out of the complex protein mixture from the membrane fraction. We take all this as evidence that in fact a specific Ca²⁺-transport system is part of the mitotic apparatus, that such a system is very conserved, and that it is most probably derived from the endoplasmic reticulum.     

Association of the circadian rhythmic expression of GmCRY1a with a latitudinal cline in photoperiodic flowering of soybean

Photoperiodic control of flowering time is believed to affect latitudinal distribution of plants. The blue light receptor CRY2 regulates photoperiodic flowering in the experimental model plant Arabidopsis thaliana. However, it is unclear whether genetic variations affecting cryptochrome activity or expression is broadly associated with latitudinal distribution of plants. We report here an investigation of the function and expression of two cryptochromes in soybean, GmCRY1a and GmCRY2a. Soybean is a short-day (SD) crop commonly cultivated according to the photoperiodic sensitivity of cultivars. Both cultivated soybean (Glycine max) and its wild relative (G. soja) exhibit a strong latitudinal cline in photoperiodic flowering. Similar to their Arabidopsis counterparts, both GmCRY1a and GmCRY2a affected blue light inhibition of cell elongation, but only GmCRY2a underwent blue light- and 26S proteasome-dependent degradation. However, in contrast to Arabidopsis cryptochromes, soybean GmCRY1a, but not GmCRY2a, exhibited a strong activity promoting floral initiation, and the level of protein expression of GmCRY1a, but not GmCRY2a, oscillated with a circadian rhythm that has different phase characteristics in different photoperiods. Consistent with the hypothesis that GmCRY1a is a major regulator of photoperiodic flowering in soybean, the photoperiod-dependent circadian rhythmic expression of the GmCRY1a protein correlates with photoperiodic flowering and latitudinal distribution of soybean cultivars. We propose that genes affecting protein expression of the GmCRY1a protein play an important role in determining latitudinal distribution of soybeans.     

Dephosphorylation of a major sperm membrane protein is induced by egg jelly during sea urchin fertilization

A 160-kilodalton (kDa) protein of Arbacia punctulata sperm is constitutively phosphorylated on serine residues, as shown by in vivo³²PO₄^3- labeling. Exposure of sperm to solubilized egg jelly results in the immediate dephosphorylation (within 5 sec) of this protein and a simultaneous increase in its electrophoretic mobility (to an apparent molecular mass of 150 kDa). In its dephosphorylated form (150 kDa), the protein is a major component of the flagellar plasma membrane. In fact, silver-stained polyacrylamide gels show the 160/150-kDa protein to be the third most abundant protein in the whole flagellum. The spermatozoan must pass through the egg jelly layer on its way to the egg surface. The jelly-induced dephosphorylation of such an abundant sperm membrane protein may be an important event in the successful interaction of sperm and egg during fertilization.