Physiological and biochemical characterization of aggregation-deficient mutants of Dictyostelium discoideum: detection and response to exogenous cyclic AMP

1978 ◽  
Vol 24 (4) ◽  
pp. 455-465 ◽  
Author(s):  
E. K.-L. Lo ◽  
M. B. Coukell ◽  
A. S. Tsang ◽  
J. L. Pickering
Keyword(s):  
Dictyostelium Discoideum ◽  
Biochemical Characterization ◽  
Biochemical Properties ◽  
Chemotactic Response ◽  
Bacterial Suspension ◽  
Camp Phosphodiesterase ◽  
Contact Sites ◽  
Solid Substratum ◽  
Camp Receptor ◽  
Physiological And Biochemical

Forty independently isolated aggregation-deficient (Agg−) mutants of Dictyostelium discoideum, which have been partially characterized genetically, were examined for the ability to respond chemotactically to exogenous cAMP and for certain cellular and extracellular activities thought to be involved in this process. Only five of the mutants failed to produce a statistically significant chemotactic response to cAMP. When shaken in buffer, most of the Agg− mutants produced reduced levels of the cAMP-binding sites and the cell-bound cAMP phosphodiesterase reported to be components of a cell-surface cAMP receptor involved in the chemotactic response. In addition, most of the mutants failed to form contact sites A and were unable to develop on water agar plates. When the mutants were pulsed with 100 nM cAMP, 36 of 40 mutants produced detectable contact sites A and three-quarters of these strains underwent further development when placed on a solid substratum. Treatment with cAMP pulses also stimulated certain mutants to form higher levels of cAMP receptors. These observations suggest that the impaired differentiation of the plasma membrane of many Agg− mutants is due, at least in part, to the abnormal synthesis and (or) secretion of cAMP by these strains. When grown in bacterial suspension, all 40 of the mutants produced extracellular phosphodiesterase (ePD) activity, and all but 8 of the mutants secreted detectable amounts of the ePD inhibitor. In general, Agg− strains carrying mutations at the same aggregation locus (aggF–J) exhibited very similar physiological and biochemical properties.

scholarly journals Biochemical characterization of specific Alanine Decarboxylase (AlaDC) and its ancestral enzyme Serine Decarboxylase (SDC) in tea plants (Camellia sinensis)

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Peixian Bai ◽  
Liyuan Wang ◽  
Kang Wei ◽  
Li Ruan ◽  
Liyun Wu ◽  
...  
Keyword(s):  
Gene Duplication ◽  
Camellia Sinensis ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Protein Sequences ◽  
Biochemical Properties ◽  
High Similarity ◽  
New Gene ◽  
Tea Plants

Abstract Background Alanine decarboxylase (AlaDC), specifically present in tea plants, is crucial for theanine biosynthesis. Serine decarboxylase (SDC), found in many plants, is a protein most closely related to AlaDC. To investigate whether the new gene AlaDC originate from gene SDC and to determine the biochemical properties of the two proteins from Camellia sinensis, the sequences of CsAlaDC and CsSDC were analyzed and the two proteins were over-expressed, purified, and characterized. Results The results showed that exon-intron structures of AlaDC and SDC were quite similar and the protein sequences, encoded by the two genes, shared a high similarity of 85.1%, revealing that new gene AlaDC originated from SDC by gene duplication. CsAlaDC and CsSDC catalyzed the decarboxylation of alanine and serine, respectively. CsAlaDC and CsSDC exhibited the optimal activities at 45 °C (pH 8.0) and 40 °C (pH 7.0), respectively. CsAlaDC was stable under 30 °C (pH 7.0) and CsSDC was stable under 40 °C (pH 6.0–8.0). The activities of the two enzymes were greatly enhanced by the presence of pyridoxal-5′-phosphate. The specific activity of CsSDC (30,488 IU/mg) was 8.8-fold higher than that of CsAlaDC (3467 IU/mg). Conclusions Comparing to CsAlaDC, its ancestral enzyme CsSDC exhibited a higher specific activity and a better thermal and pH stability, indicating that CsSDC acquired the optimized function after a longer evolutionary period. The biochemical properties of CsAlaDC might offer reference for theanine industrial production.

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

Monoclonal antibody recognizing gp80, a membrane glycoprotein implicated in intercellular adhesion of Dictyostelium discoideum

1983 ◽  
Vol 3 (5) ◽  
pp. 863-870
Author(s):  
B A Murray ◽  
H L Niman ◽  
W F Loomis
Keyword(s):  
Monoclonal Antibody ◽  
Dictyostelium Discoideum ◽  
Posttranslational Modification ◽  
Rabbit Antiserum ◽  
Intercellular Adhesion ◽  
Blocking Effect ◽  
Antigenic Determinants ◽  
Membrane Glycoprotein ◽  
Developmentally Regulated ◽  
Contact Sites

WE have raised a monoclonal antibody, designated E28D8, which reacts with an 80,000-dalton membrane glycoprotein (gp80) of Dictyostelium discoideum. gp80 has been implicated in the formation of the EDTA-resistant adhesions ("contact sites A") which appear during development. The monoclonal antibody reacted with other developmentally regulated proteins of D. discoideum, confirming previous results indicating the presence of common antigenic determinants recognized by polyclonal rabbit antibodies directed to gp80. Periodate sensitivity of the determinants suggests that carbohydrate may be necessary for reactivity. Thus, the determinant recognized by E28D8 may result from a posttranslational modification common to a number of proteins. Some of the proteins that carry the determinant were preferentially localized to posterior cells in slugs. Monoclonal antibody E28D8 did not inhibit contact-sites-A-mediated intercellular adhesion. However, gp80 affinity purified on immobilized monoclonal antibody was able to neutralize the adhesion-blocking effect of rabbit antiserum to gp80. Although gp80 itself may not be essential for cell-cell adhesion, it appears to carry the determinants associated with adhesion.

Chemotaxis-associated properties of separated prestalk and prespore cells of Dictyostelium discoideum

1986 ◽  
Vol 64 (8) ◽  
pp. 722-732 ◽  
Author(s):  
J. D. Mee ◽  
D. M. Tortolo ◽  
M. B. Coukell
Keyword(s):  
Light Scattering ◽  
Cell Surface ◽  
Dictyostelium Discoideum ◽  
Binding Sites ◽  
Large Fraction ◽  
Cell Types ◽  
The Other ◽  
Deficient Mutant ◽  
Camp Phosphodiesterase ◽  
Intracellular Cgmp

During development, prestalk and prespore cells of Dictyostelium discoideum become organized in multicellular structures. This physical association makes it difficult to characterize the two cell types biochemically and physiologically. In the present study, we have separated prestalk and prespore cells from 16-h slugs by the method of Tsang and Bradbury and have examined a number of chemotaxis-associated properties of these cells. When assayed on phosphate-buffered agar under both gradient and nongradient conditions, isolated prestalk cells responded chemotactically to cAMP and, unexpectedly, to folate and certain folate derivatives. In contrast, separated prespore cells failed to respond appreciably to any of these compounds. Neither prestalk nor prespore cells of strain HC91 exhibited a cAMP-induced increase in intracellular cGMP. However, a cGMP response was observed in both prestalk and prespore cells of strain NP368, a cGMP phosphodiesterase deficient mutant. Both cell types exhibited comparable cAMP-mediated light-scattering changes and possessed similar levels of surface cAMP- and folate-binding sites. On the other hand, prestalk cells had at least fourfold higher cAMP phosphodiesterase and folate deaminase activities than prespore cells, and a large fraction of both activities was on the cell surface. Therefore, the greater chemotactic response of prestalk cells to cAMP and folate on agar might be due, in part, to their increased capacity to generate a chemoattractant gradient. Results obtained in this study demonstrate that prestalk and prespore cells separated by this procedure can be used in certain physiological as well as biochemical experiments.

scholarly journals cAMP induces a rapid and reversible modification of the chemotactic receptor in Dictyostelium discoideum.

1985 ◽  
Vol 100 (3) ◽  
pp. 715-720 ◽  
Author(s):  
C Klein ◽  
J Lubs-Haukeness ◽  
S Simons
Keyword(s):  
Dictyostelium Discoideum ◽  
Concentration Dependence ◽  
Time Course ◽  
Camp Synthesis ◽  
Sds Page ◽  
Reversible Modification ◽  
Camp Receptor ◽  
Camp Stimulation ◽  
Stimulation Of

Stimulation, within 1 min after cAMP stimulation, of aggregation-competent Dictyostelium discoideum amebae was found to cause a rapid (within 1 min) modification of the cell's surface cAMP receptor. The modified receptor migrated on SDS PAGE as a 47,000-mol-wt protein, as opposed to a 45,000-mol-wt protein labeled on unstimulated cells. The length of time this modified receptor could be detected depended upon the strength of the cAMP stimulus: 3-4 min after treatment with 10(-7) M cAMP, cells no longer possessed the 47,000-mol-wt form of the cAMP receptor. Instead, the 45,000-mol-wt form was present. Stimulation of cells with 10(-5) M cAMP, however, resulted in the persistent (over 15 min) expression of the modified receptor. The time course, concentration dependence, and specificity of stimulus for this cAMP-induced shift in the cAMP receptor were found to parallel the cAMP-stimulated phosphorylation of a 47,000-mol-wt protein. In addition, both phenomena were shown to occur in the absence of endogenous cAMP synthesis. The possibility that the cAMP receptor is phosphorylated in response to cAMP stimulation, and the role of this event in cell desensitization, are discussed.

scholarly journals Rem2, a new member of the Rem/Rad/Gem/Kir family of Ras-related GTPases

2000 ◽  
Vol 347 (1) ◽  
pp. 223-231 ◽  
Author(s):  
Brian S. FINLIN ◽  
Haipeng SHAO ◽  
Keiko KADONO-OKUDA ◽  
Nan GUO ◽  
Douglas A. ANDRES
Keyword(s):  
Cellular Localization ◽  
Biochemical Characterization ◽  
Fluorescent Protein ◽  
Dissociation Constants ◽  
Intrinsic Rate ◽  
Biochemical Properties ◽  
Cell Physiology ◽  
Gtp Binding ◽  
C Terminus ◽  
Green Fluorescent

Here we report the molecular cloning and biochemical characterization of Rem2 (for Rem, ad and G-related 2), a novel GTP-binding protein identified on the basis of its homology with the Rem, Rad, Gem and Kir (RGK) family of Ras-related small GTP-binding proteins. Rem2 mRNA was detected in rat brain and kidney, making it the first member of the RGK family to be expressed at relatively high levels in neuronal tissues. Recombinant Rem2 binds GTP saturably and exhibits a low intrinsic rate of GTP hydrolysis. Surprisingly, the guanine nucleotide dissociation constants for both Rem2 and Rem are significantly different than the majority of the Ras-related GTPases, displaying higher dissociation rates for GTP than GDP. Localization studies with green fluorescent protein (GFP)-tagged recombinant protein fusions indicate that Rem2 has a punctate, plasma membrane localization. Deletion of the C-terminal seven amino acid residues that are conserved in all RGK family members did not affect the cellular distribution of the GFP fusion protein, whereas a larger deletion, including much of the polybasic region of the Rem2 C-terminus, resulted in its redistribution to the cytosol. Thus Rem2 is a GTPase of the RGK family with distinctive biochemical properties and possessing a novel cellular localization signal, consistent with its having a unique role in cell physiology.

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