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M S Springer

Publications and source records attributed to M S Springer.

At least 73 records · Page 4Linked to original sources
PubMed1990

Interaction between the C5a receptor and Gi in both the membrane-bound and detergent-solubilized states.

C5a elicits a variety of responses from the polymorphonuclear leukocyte all of which utilize G proteins as transducing elements. In the present study, we report the consequences of the interaction between the C5a receptor and the G proteins and describe a system which may allow identification of the transducing proteins. C5a binding to polymorphonuclear leukocyte membranes is inhibited by pertussis, but not cholera, toxin and by a variety of guanine nucleotides. In the absence of nucleotide, we observed a single class of sites with a Kd of 17 pM. The presence of guanosine 5'-3-O-(thio)triphosphate (GTP gamma S) did not alter this affinity but did result in a concentration-dependent decrease in the number of binding sites. Surprisingly, we did not observe the concomitant appearance of a low affinity state implying that, if such a state exists, its affinity is below our limit of detection (5 nM). The receptor and G protein retained their functional interaction following solubilization of the membrane in digitonin. In the absence of nucleotide, we observed a single class of sites with a Kd of 28 pM. Addition of GTP gamma S suppressed binding, and, as was found in membranes, this inhibition is due almost entirely to a decrease in the number of sites. Again we failed to detect the appearance of a lower affinity state. Gel filtration studies of the detergent-solubilized receptor and receptor-C5a complexes indicate that the receptor is precoupled to G protein in the absence of ligand (C5a).

Binding Sites↗
PubMed1990

DNA hybridization, cladistics, and the phylogeny of phalangerid marsupials.

Single-copy DNA/DNA hybridization experiments and numerical cladistic analyses of anatomical characters were used to investigate relationships among nine phalangerid (Marsupialia) species from four different genera. Both rate-dependent and rate-independent analyses of molecular data indicate that species of Trichosurus from one clade and that Strigocuscus, Phalanger, and Spilocuscus form a second. Within the latter group, Spilocuscus is excluded from a Strigocuscus-Phalanger clade, which, in turn, is not fully resolved on a jackknife strict consensus tree. Minimum-length Dollo, Wagner, and Camin-Sokal parisomy trees based on 35 anatomical characters, in contrast, suggest placement of Strigocuscus with Trichosurus rather than with Spilocuscus and Phalanger. However, there are two derived characters that support the alternative arrange of Strigocuscus with Spilocuscus and Phalanger and one character that further unites Strigocuscus and Phalanger. Thus, DNA hybridization results are not inconsistent with the distribution of derived character states among anatomical characters, only with minimum-length trees based on character data.

Animals↗
PubMed1990

DNA/DNA hybridization studies of the carnivorous marsupials. I: The intergeneric relationships of bandicoots (Marsupialia: Perameloidea).

A complete suite of comparisons among six bandicoot species and one outgroup marsupial was generated using the hydroxyapatite chromatography method of DNA/DNA hybridization; heterologous comparisons were also made with three other bandicoot taxa. Matrices of delta Tm's, delta modes, and delta T50Hs were generated and corrected for nonreciprocity, homoplasy, and, in the case of delta Tm's, normalized percent hybridization; these matrices were analyzed using the FITCH algorithm in Felsenstein's PHYLIP (version 3.1). Uncorrected and nonreciprocity-corrected matrices were also jackknifed and analyzed with FITCH to test for consistency. Finally, sample scores for delta Tm, delta mode, and delta T50H matrices were bootstrapped and then subjected to phylogenetic analysis. These manipulations were carried out, in part, to address criticisms of the statistics used to summarize DNA/DNA hybridization (especially T50H) and the method itself. However, with the exception of an unresolved trichotomy among the two Echymipera species and Peroryctes longicauda, all trees showed the same branchpoints. Except in the case of the tree generated from reciprocal-corrected delta Tm data, nodes were stable under jackknifing; and, again excepting the above-mentioned trichotomy, all nodes were supported by 95% or more of the bootstrapped trees. These results suggest that, despite arguments to the contrary, all three summary statistics can be valid for DNA/DNA hybridization data. Of taxonomic interest is the placement of Echymipera spp. and Peroryctes longicauda together and separate from the more distant Peroryctes raffrayanus; the genus Peroryctes is thus at least paraphyletic. The trees further grouped Echymipera-plus-Peroryctes as the sister group of Isoodon-plus-Perameles. Limited hybridizations with Macrotis lagotis suggest that its current position as representative of an entirely distinct family of perameloids is correct.

Animals↗
PubMed1989

Rates of single-copy DNA evolution in phalangeriform marsupials.

DNA/DNA hybridization was used to investigate the relationships of taxa representing the phalangeriform marsupial families Acrobatidae, Burramyidae, Macropodidae, Petauridae, Phalangeridae, and Pseudocheiridae and (as an outgroup) the bandicoot family Peramelidae. In the course of this, a marked rate slowdown was noted in the burramyid lineage represented by Cercartetus caudatus; ANOVA (with Tukey's test) and F-ratio tests of both corrected and uncorrected data matrices confirmed this rate disparity. As burramyids are small, short-generation-time phalangeriforms, these data present a striking counterexample to the common view that rates of change in DNA sequences are inversely correlated with generation time.

Analysis of Variance↗
PubMed1988

Solubilization of the functional C5a receptor from human polymorphonuclear leukocytes.

The C5a receptor has been extracted in an active state from the membranes of human polymorphonuclear leukocytes with the detergents digitonin and beta-dodecyl maltoside. The solubilized receptor exhibits a single class of high affinity binding sites with a Kd = 90 pM, a value similar to that found with intact membranes. Physical studies with the soluble receptor demonstrate that it exists in two forms which differ in molecular mass. Gel filtration experiments with receptor to which C5a has been bound give an apparent molecular mass for the complex of 150-200 kDa. When the experiments were repeated with nonliganded receptor, most of the C5a binding activity eluted with an apparent mass of 150-200 kDa. However, the peak had a pronounced trailing shoulder indicating that, in the nonliganded state, a portion of the receptor population exists in a smaller form, which may be converted to the larger form on binding C5a. The molecular mass of the smaller form, estimated to be 30-70 kDa, is consistent with that of the binding subunit of the receptor. These data imply that the larger form, and therefore the bulk of the solubilized receptor, is oligomeric, a conclusion which is supported by cross-linking studies. When C5a was cross-linked to the soluble receptor two specific complexes with molecular masses of 52 and 95 kDa were formed. The former is the covalent adduct of C5a and the binding subunit of the receptor and the latter appears to be a complex between the 52-kDa species and an additional polypeptide.

Cell Membrane↗
PubMed1985

Identification of the polymorphonuclear leukocyte C5a receptor.

The peptide C5a is thought to play an important role in the inflammatory response primarily through its action on the polymorphonuclear leukocyte (PMN). The receptor for C5a on human PMN has now been identified by affinity labeling. Cross-linking 125I-C5a to intact PMN with disuccinimidyl suberate produced a species that had a molecular mass on sodium dodecyl sulfate gels of 5.2 X 10(4) daltons. We believe this species represents a complex between C5a and its receptor for the following reasons. The band is eliminated if the cross-linking experiment is performed in the presence of a large excess of unlabeled C5a, but is unaffected by the presence of nonspecific protein or the chemotactic factors N-formyl-Met-Leu-Phe and leukotriene B4. The 5.2 X 10(4)-dalton species is not observed if the cross-linker is omitted. Finally, the dose-response curves for the inhibition of binding of 125I-C5a by unlabeled C5a and the inhibition of cross-linking are similar. Subtraction of the molecular mass of C5a from that of the complex gives a molecular mass for the binding moiety of the C5a receptor of 4.0 X 10(4) daltons.

Binding, Competitive↗
PubMed1984

Sensory transduction in Escherichia coli: regulation of the demethylation rate by the CheA protein.

The reversible methylation of three membrane proteins plays an essential role in bacterial chemotaxis. Chemotactic stimuli bring about changes in the levels of methylation of these proteins, at least in part, by regulation of the demethylation reaction. Addition of attractants causes an increase in the methylation level and a transient, but essentially complete, inhibition in the rate of the demethylation reaction, while addition of repellents results in a decrease in level and a transient increase (of at least 25- to 30-fold) in rate. We have now found that the increase, but not the decrease, in rate requires the presence of the cheA gene product, a protein that is distinct from the demethylase. The demethylation reaction is therefore regulated by two distinct mechanisms--one, which involves the CheA protein, that mediates the increase in rate and a second, which does not involve the CheA protein, that mediates the decrease in rate. Several pieces of evidence already in the literature imply that the CheA protein functions downstream of the methylation system at the flagellar end of the chemotactic machinery. These data, in conjunction with the newer results, suggest that the CheA protein helps to regulate the demethylation reaction through a feedback mechanism.

Bacterial Proteins↗
PubMed1983

Synthetic leukotriene B4 is a potent chemotaxin but a weak secretagogue for human PMN.

We have examined the effects of very pure (greater than 99.8%) chemically synthesized leukotriene B4 of verified structure on the chemotactic and secretory behavior of human polymorphonuclear leukocytes (PMN). The synthetic material is highly chemotactic and shows the same concentration dependence of this activity as does natural LTB4. Synthetic LTB4 is also a weak degranulating agent in cytochalasin B treated PMN. Maximally it released 11%, 17% and 26% as much N-acetyl-beta-D-glucosaminidase, myeloperoxidase and lysozyme as did N-formyl-methionine-leucine-phenylalanine (fMLP). Thus LTB4 differs significantly from other chemotaxins, such as C5a and fMLP, in that it is a poor secretagogue for enzymes of the specific and azurophilic granules of human PMN.

Acetylglucosaminidase↗
PubMed1982

Ordered methylation of the methyl-accepting chemotaxis proteins of Escherichia coli.

The methylation of glutamic acid residues on 3 membrane proteins, MCPI, MCPII, and MCPIII, plays an essential role in the chemotactic response of Escherichia coli. In the absence of a chemotactic stimulus the methyl-accepting chemotaxis proteins (MCPs) are methylated to a basal level. This level rises to a new plateau when an attractant is added and then falls to its original basal value when the attractant is removed. We have now been able to show that the properties of the glutamic acid residues which become methylated after stimulation with an attractant differ from the properties of the residues methylated on the basal level. Our data imply that there is a preferred order in which those residues are methylated and demethylated.

Bacterial Proteins↗
PubMed1979

Attractants and repellents control demethylation of methylated chemotaxis proteins in Escherichia coli.

A group of methylated proteins, the methyl-accepting chemotaxis proteins (MCP), has been shown to play a central role in bacterial chemotaxis. Both methylation and demethylation of MCP occur continuously in the absence of added stimuli; these two processes are in balance such that a basal level of methylation is maintained. Attractants cause the methylation level to increase to a new value, whereas repellents bring about a decrease in level. Therefore, attractants and repellents must somehow perturb the balance between methylation and demethylation of MCP. In this report the effect of attractants on demethylation of MCP was monitored in two ways: (i) by following the disappearance of [methyl-3H]MCP and (ii) by measuring formation of [3H]methanol, the product of MCP demethylation. Both methods showed that addition of attractants causes a transient inhibition of MCP demethylation. Repellent addition has previously been shown to stimulate MCP demethylation. It is therefore concluded that control of demethylation plays a crucial role in changing the level of methylation of MCP in response to attractants and repellents.

Bacterial Proteins↗
PubMed1979

Pleiotropic aspartate taxis and serine taxis mutants of Escherichia coli.

Mutants that at one time were thought to be specifically defective in taxis toward aspartate and related amino acids (tar mutants) or specifically defective in taxis toward serine and related amino acids (tar mutants) are now shown to be pleiotropic in their defects. The tar mutants also lack taxis toward maltose and away from Co2+ and Ni2+. The tsr mutants are altered in their response to a variety of repellents. Double mutants (tar tsr) fail in nearly all chemotactic responses. The tar and tsr mutants provide evidence for two complementary, converging pathways of information flow: certain chemoreceptors feed information into the tar pathway and others into the tsr pathway. The tar and tsr products have been shown to be two different sets of methylated proteins.

Aspartic Acid↗
PubMed1979

On the mechanism of sensory transduction in bacterial chemotaxis.

Sensory transduction in bacterial chemotaxis is beginning to be understood at the molecular level. At the receptor end, we have some considerable knowledge about the molecular properties of chemoreceptors. At the effector end, we know that flagella rotate and that the direction of rotation is determined by attractants and repellents, although we do not yet know the molecular features of the motor and the gear shift. Between the receptors and the effectors is a system for integrating the sensory transduction, which somewhow involves methylation of membrane proteins and possibly a change in membrane potential, but further details of how the mechanism works remain to be elucidated. It seems to us likely that the facts and concepts learned from a study of sensory transduction in bacteria can be applied to answering questions about transduction mechanisms in eukaryotic cells. Examples include the following: How do sensory stimuli produce their effects in sensory receptor cells? How do neurotransmitters act at receptors of postsynaptic cells to produce the variety of effects possible (changes in membrane potential, in secretion, in contraction, etc.)? How do hormones interact with their receptors to bring about various responses?

Carrier Proteins↗
PubMed1978

Attraction by repellents: an error in sensory information processing by bacterial mutants.

Normal Escherichia coli bacteria are repelled by acetate, benzoate, and indole and attracted by alpha-aminoisobutyrate. We have isolated mutants that are attracted to acetate, benzoate, and indole and may be repelled by alpha-aminoisobutyrate. These reversed-taxis mutants are defective in a central processing component: a set of methylated proteins known as MCP 1. The mechanism of reversal of taxis is discussed.

Acetates↗
PubMed1978

Failure of sensory adaptation in bacterial mutants that are defective in a protein methylation reaction.

Chemotactic bacteria, such as E. coli, detect changes in the chemical composition of the environment. Addition of an attractant or repellent leads to an immediate response, characterized by a change in the swimming behavior of the cells--a process known as sensory excitation. However, the response gradually disappears with time, despite the continued presence of the chemical--a process known as sensory adaptation. We report here the behavior of a class of nonchemotactic mutants (cheX) that can carry out sensory excitation but are defective in the process of sensory adaptation. These mutants are also defective in the ability to carry out a protein methylation reaction which has previously been implicated in the adaptation process (Goy, Springer and Adler, 1977). The results presented here establish a firm relationship between the methylation reaction and sensory adaptation.

Adaptation, Physiological↗
PubMed1977

Sensory transduction in Escherichia coli: a requirement for methionine in sensory adaptation.

Chemotaxis of E. coli is a behavioral response to a change in the concentration of a stimulatory compound. The response is transient; thus, E. coli undergoes sensory adaptation. In this communication, we show that L-methionine is required by E. coli for adaptation to increases in the concentration of chemical attractants, but is not required for the maintenance of the adapted state. When the concentration of the attractant is lowered to its initial level, cells regain their sensitivity to the attractant. This process of deadaptation does not require methionine. We suggest that the methylation of a membrane protein, a reaction previously shown to be involved in chemotaxis [Kort, E.N., Goy, M.F., Larsen, S.H. & Adler J. (1975) Proc. Natl. Acad. Sci. USA 72, 3939-3943] underlies these phenomena.

Chemotaxis↗
PubMed1977

Sensory transduction in Escherichia coli: role of a protein methylation reaction in sensory adaptation.

The behavioral response of Escherichia coli to the addition of a stimulatory compound is transient; thus the organism undergoes sensory adaptation. When the compound is removed, E. coli undergoes the inverse process, called deadaptation, and very rapidly regains its sensitivity to the stimulus. In this communication we demonstrate that the previously reported methylation of several cytoplasmic membrane proteins is correlated with, and very likely controls, the state of adaptation of the cell. In the absence of an added stimulus these proteins are methylated to a basal level. When the bacteria are stimulated by the addition of an attractant, the extent of methylation increases over a period of several minutes to a new level, which is maintained as long as the attractant is present. The magnitude of the increase in methylation is a function of the size of the stimulus and is directly proportional to the duration of the behavioral response. Upon removal of the attractant the level of methylation very rapidly falls to the basal value. Previously we have shown that adaptation requires methionine, but maintenance of the adapted state and de-adaptation do not [Springer, M. S., Goy, M. F. & Adler, J. (1975) Proc. Natl. Acad. Sci. USA 74, 183-187]; here we demonstrate that methylation requires methionine but maintenance of an attractant-induced level of methylation and the demethylation that occurs following removal of the attractant do not. These results strongly indicate a role for protein methylation in sensory adaptation.

Adaptation, Physiological↗