Search PubMed⌕ Search

Biomedical subjects

M R Sussman

Publications and source records attributed to M R Sussman.

At least 37 records · Page 2Linked to original sources
PubMed1995

Immunocytological localization of an epitope-tagged plasma membrane proton pump (H(+)-ATPase) in phloem companion cells.

In higher plants, the plasma membrane proton pump (H(+)-ATPase) is encoded by a surprisingly large multigene family whose members are expressed in different tissues. Using an 18-amino acid epitope tag derived from the animal oncogene c-Myc, we have performed immunocytolocalization measurements of the protein expressed by one member of this family, AHA3 (Arabidopsis H(+)-ATPase isoform 3). Immunofluorescence studies with tissue sections of transgenic plants have revealed that c-Myc-tagged AHA3 is restricted to the plasma membrane of phloem companion cells, whereas other AHA isoproteins are more widely distributed in the plasma membrane of other cell types. Electron microscopy with immunogold-labeled tissue sections suggests that there is a high concentration of proton pumps in the plasma membrane of companion cells but a much lower concentration in the plasma membrane of sieve elements. Due to plasmodesmata connecting the plasma membrane of these two adjacent cell types, it is likely that the proton motive force generated by the proton pump in companion cells can serve to power the uptake of sugar by proton-coupled symporters in either the companion cell or sieve element cell. The abundance of the proton pump in the plasma membrane of companion cells supports an apoplastic model for phloem loading in which the metabolic energy that drives sugar uptake is consumed by AHA3 at the companion cell plasma membrane. These experiments with a genetically altered integral plasma membrane protein demonstrate the utility of using a short c-Myc sequence as an epitope tag in Arabidopsis. Furthermore, our results demonstrate that, using genes encoding individual members of a gene family, it is possible to label plasma membrane proteins immunologically in specific, differentiated cell types of higher plants.

Amino Acid Sequence↗
PubMed1994

The plasma membrane H(+)-ATPase gene family in Arabidopsis: genomic sequence of AHA10 which is expressed primarily in developing seeds.

The plasma membrane H(+)-ATPases in Arabidopsis thaliana represent the largest family of cation translocating P-type ATPases identified in plants or animals. We report here seven new isoforms, which were identified by polymerase chain reaction (PCR) amplification of genomic DNA. Amplifications were performed with degenerate primers corresponding to two short conserved sequence motifs ("CSDK" and "GDGV") found in most P-type ATPases. A comparison was made of three CSDK-side primers, which were used either as totally degenerate mixtures or rendered less degenerate by substitution with deoxyinosine or fluorodeoxyuridine. Amplified genomic fragments were cloned, partially sequenced and shown to correspond to Arabidopsis genes by Southern blot analysis with gene-specific probes. One newly identified isoform, AHA10, was isolated as a cosmid clone and sequenced. The 5' and 3' ends of the gene were determined by comparison with the AHA10 cDNA sequence. AHA10 is the most divergent isoform characterized in the Arabidopsis family. AHA10 appears to be expressed primarily in developing seeds, as indicated by Northern blot analysis of AHA10 mRNA and by the analysis of transgenic plants expressing a beta-glucuronidase (GUS) reporter gene fused to an AHA10 promoter. Our results indicate that one function of this unusually large H(+)-ATPase gene family is to allow for expression of different isoforms in different cell types.

Amino Acid Sequence↗
PubMed1994

Characterization of an Arabidopsis calmodulin-like domain protein kinase purified from Escherichia coli using an affinity sandwich technique.

A full-length cDNA encoding a calcium-dependent protein kinase with a calmodulin-like domain from Arabidopsis thaliana (AK-1 for Arabidopsis kinase-1) has been expressed as a fusion protein (called AK-1-6H) in Escherichia coli and purified to near homogeneity with high specific activity (typically 2000 nmol min-1 mg-1) using an "affinity sandwich" technique. AK-1-6H protein phosphorylation activity using histone as substrate was stimulated up to 50-fold by the addition of calcium alone or up to 5-fold by the addition of specific phospholipids alone; together calcium and these lipids acted synergistically to give up to 100-fold stimulation. We earlier reported that, of a wide array of lipids tested, only phosphatidylinositol and lysophosphatidylcholine stimulated histone phosphorylation by AK-1 [Harper, J. F., Binder, B. M., & Sussman M. R. (1993) Biochemistry 32, 3282-3290]. The properties of lipid stimulation were further explored by testing the effects of lipids on autophosphorylation and on other catalytic properties of the kinase. Although phosphatidylinositol stimulated autophosphorylation up to 11-fold, lysophosphatidylcholine was inactive. Basic peptides such as polylysine (average M(r) approximately 37,100) were potent, mixed-type inhibitors of AK-1-6H with an IC50 of 2 nM. In the presence of phosphatidylinositol, the inhibition was reduced and the IC50 for polylysine was increased to 341 nM. As with autophosphorylation, lysophosphatidylcholine was inactive in alleviating the basic peptide inhibition, which suggests that this lipid's stimulatory effects using exogenous substrate are distinct from those of phosphatidylinositol. These results are consistent with a model in which phosphoinositides directly interact with the kinase protein and alleviate a catalytic block caused by basic charges.

Amino Acid Sequence↗
PubMed1993

Calcium and lipid regulation of an Arabidopsis protein kinase expressed in Escherichia coli.

Calcium-dependent protein kinases (CDPKs) represent a new family of protein kinases which are proposed to contain, in a single polypeptide, both a kinase domain and an adjoining calmodulin-like domain with four calcium-binding EF-hand motifs [Harper, J.F., Sussman, M.R., Schaller, G.E., Putnam-Evans, C., Charbonneau, H., & Harmon, A.C. (1991) Science 252, 951-954]. DNA cloning and Western blot analysis indicate that multiple CDPK isoforms are present in the model plant system Arabidopsis thaliana. One CDPK gene called AK1 was isolated from Arabidopsis as a full-length cDNA. The predicted AK1 protein has a M(r) of 72,645 and is 116 amino acid residues longer at the amino terminus than the prototype CDPK alpha gene previously identified in soybean. The most highly conserved region between these two CDPKs is a region of 31 amino acids that joins the kinase and calmodulin-like domains. To verify the kinase activity of the enzyme encoded by AK1, a fusion of an amino-terminally truncated AK1 to the C-terminus of glutathione S-transferase was expressed in Escherichia coli. The fusion protein was purified and displayed a maximum kinase activity of 40 nmol of phosphate/(min.mg), using histone IIIs as a substrate. The enzyme activity was stimulated 3-6-fold by calcium and 2-5-fold by crude lipid. However, a synergistic stimulation of 16-30-fold was observed by the addition of both calcium and crude lipid. Lipid stimulation was specific for lysophosphatidylcholine and phosphatidylinositol and did not occur with the addition of phosphatidylserine or phosphatidylcholine.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1992

Cell cycle-dependent localization of casein kinase I to mitotic spindles.

Casein kinase I (CKI) is a class of protein kinases ubiquitous to all eukaryotic cells. Recently, cDNA clones encoding several bovine CKI isoforms have been sequenced that show high sequence identity to the HRR25 gene product of the budding yeast Saccharomyces cerevisiae; HRR25 is required for normal cellular growth, nuclear segregation, DNA repair, and meiosis. We have raised polyclonal antibodies to a human erythroid 34-kDa CKI and have sequenced a portion of this kinase. The amino acid sequence identifies the CKI as the alpha-CKI isoform, which is 62% identical to the HRR25 protein kinase. By use of immunofluorescence, the alpha-CKI has been localized to vesicular cytosolic structures and to the centrosome in interphase cells. As cells progress into mitosis, centrospheric staining increases and, in mitosis, alpha-CKI associates with kinetochore fibers. This localization suggests that alpha-CKI, like HRR25, plays a role in the segregation of chromosomes during mitosis and may be cell cycle-regulated both in humans and in yeast.

Amino Acid Sequence↗
PubMed1992

Characterization of a calcium- and lipid-dependent protein kinase associated with the plasma membrane of oat.

A protein kinase that is activated by calcium and lipid has been partially purified from the plasma membrane of oat roots. This protein kinase cross-reacts with four monoclonal antibodies directed against a soluble calcium-dependent protein kinase from soybean described previously [Putman-Evans, C. L., Harmon, A. C., & Cormier, M. J. (1990) Biochemistry 29, 2488-2495; Harper, J. F., Sussman, M. R., Schaller, G. E., Putnam-Evans, C., Charbonneau, H., & Harmon, A. C. (1991) Science 252, 951-954], indicating that the oat enzyme is a member of this calcium-dependent protein kinase family. Immunoblots demonstrate that the membrane-derived protein kinase is slightly larger than that observed in the cytosolic fraction of oat. Limited digestion of the membrane-derived kinase with trypsin generates a smaller water-soluble kinase that is still activated by calcium but is no longer activated by lipid. When posthomogenization proteolysis is minimized, the bulk of the immunoreactive kinase material is localized in the membrane. These results suggest that a calcium-dependent protein kinase observed in the supernatant fraction of oat extracts may originate in situ from a calcium- and lipid-dependent protein kinase which is associated with the oat plasma membrane. They further indicate that, in contrast to animal cells, the predominant calcium- and lipid-dependent protein kinase associated with the plasma membrane of plant cells has biochemical properties and amino acid sequence unlike protein kinase C.

Antibodies, Monoclonal↗
PubMed1991

A calcium-dependent protein kinase with a regulatory domain similar to calmodulin.

Calcium can function as a second messenger through stimulation of calcium-dependent protein kinases. A protein kinase that requires calcium but not calmodulin or phospholipids for activity has been purified from soybean. The kinase itself binds calcium with high affinity. A complementary DNA clone for this kinase has been identified; it encodes a protein with a predicted molecular mass of 57,175 daltons. This protein contains a catalytic domain similar to that of calmodulin-dependent kinases and a calmodulin-like region with four calcium binding domains (EF hands). The predicted structure of this kinase explains its direct regulation via calcium binding and establishes it as a prototype for a new family of calcium-regulated protein kinases.

Amino Acid Sequence↗
PubMed1991

Evidence for a plasma membrane proton pump in phloem cells of higher plants.

Metabolic energy is required for the loading of sucrose into the phloem and translocation of sugars throughout the plant. The proton electrochemical gradient generated by a plasma membrane proton pump (H(+)-ATPase) is thought to provide energy for these processes. The plasma membrane H(+)-ATPase is encoded by a multigene family in Arabidopsis thaliana. Here we characterize the expression of isoform AHA3 (Arabidopsis H(+)-ATPase isoform 3). The AHA3 mRNA start site was mapped and 464 bp of the putative upstream regulatory region sequenced. A translational fusion of AHA3 to the beta-glucuronidase (GUS) reporter gene was constructed and used to generate transgenic Nicotiana and Arabidopsis plants. Using a histochemical stain, expression of the AHA3/GUS fusion was found predominantly in phloem cells of leaves, stems, roots, and flowers. Biochemical measurements of GUS activity in pith and vascular explants confirmed the histochemical localization. Our results support the hypothesis that a proton pump is present in phloem cells, possibly providing energy to drive plasma membrane cotransport systems required for phloem loading and translocation of photosynthates. In addition to AHA3/GUS expression in phloem, expression was observed in pollen and regions of the ovule, tissues whose physiological functions correlate with a requirement for high levels of solute transport.

Arabidopsis↗
PubMed1990

The Arabidopsis thaliana plasma membrane H(+)-ATPase multigene family. Genomic sequence and expression of a third isoform.

The plasma membrane of higher plants contains a H(+)-ATPase as its major ion pump. This enzyme belongs to the P-type family of cation-translocating enzymes and generates the proton-motive force that drives solute uptake across the plasma membrane. In Arabidopsis thaliana the plasma membrane H(+)-ATPase is encoded by a multigene family (Harper, J. F., Surowy, T. K., and Sussman, M. R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 1234-1238). The complete genomic sequence of a third Arabidopsis H(+)-ATPase isoform (referred to as AHA2) is presented here, and the predicted protein sequence is compared with previously published AHA1, AHA3, and tobacco Nicotiana plumbaginifolia NP1 isoforms. The AHA2 gene is most similar to AHA1, with predicted proteins containing 95% amino acid identity. The mRNA start site and 5'-untranslated sequence for AHA2 were determined from cDNA amplified by the polymerase chain reaction. The 5' region contains a 23-base pair (bp) polypyrimidine sequence and a short upstream reading frame. In comparison with the 16 introns reported in AHA3, AHA2 is missing one intron in the 5'-untranslated region and a second intron in the C-terminal coding region. An unusually large intron for Arabidopsis (greater than 1000 bp) is present at the beginning of the coding sequence of both AHA2 and AHA3. In the 3'-untranslated sequence of AHA1 and AHA2 but not AHA3, there is a 65-bp region of 85% identity and a second shorter region of 16-bp identity harboring an unusual putative poly(A) addition signal (dTTTGAAGAAACAAGGC). Northern blot analysis indicates that AHA2 mRNA relative to total cellular RNA is expressed at significantly higher levels in root tissue as compared with shoot tissue.

Amino Acid Sequence↗
PubMed1989

Molecular cloning and sequence of cDNA encoding the plasma membrane proton pump (H+-ATPase) of Arabidopsis thaliana.

In plants, the transport of solutes across the plasma membrane is driven by a proton pump (H+-ATPase) that produces an electric potential and pH gradient. We have isolated and sequenced a full-length cDNA clone that encodes this enzyme in Arabidopsis thaliana. The protein predicted from its nucleotide sequence encodes 959 amino acids and has a molecular mass of 104,207 Da. The plant protein shows structural features common to a family of cation-translocating ATPases found in the plasma membrane of prokaryotic and eukaryotic cells, with the greatest overall identity in amino acid sequence (36%) to the H+-ATPase observed in the plasma membrane of fungi. The structure predicted from a hydropathy plot contains at least eight transmembrane segments, with most of the protein (73%) extending into the cytoplasm and only 5% of the residues exposed on the external surface. Unique features of the plant enzyme include diverged sequences at the amino and carboxyl termini as well as greater hydrophilic character in three extracellular loops.

Amino Acid Sequence↗
PubMed1989

Characterization of a Polyphosphoinositide Phospholipase C from the Plasma Membrane of Avena sativa.

A phosphoinositide-specific phospholipase C activity was identified in oat root (Avena sativa, cv Victory) plasma membranes purified by separation in an aqueous two-phase polymer system. The enzyme is highly active toward inositol phospholipids but only minimally active toward phosphatidylethanolamine and phosphatidylcholine. Activity approaches maximal levels at 200 micromolar phosphatidylinositol 4-phosphate (PIP) and is highly dependent on calcium; it is inhibited by 1 millimolar EGTA and is activated by calcium with an apparent activation constant of 2 micromolar. At 10 micromolar calcium and 200 micromolar inositol phospholipid, the enzyme is specific for phosphatidylinositol 4,5-bisphosphate (PIP(2)) and PIP, which are hydrolyzed at 10 and 4 times, respectively, the rate of phosphatidylinositol (PI) hydrolysis. The principle water soluble products of hydrolysis, as determined by high performance liquid chromatography, are inositol 1,4,5-trisphosphate from PIP(2), inositol 1,4-bisphosphate from PIP, and inositol phosphate from PI.

Journal Article↗
PubMed1988

Purification of integral plasma membrane proteins by reverse-phase high performance liquid chromatography.

Following dissolution in anhydrous trifluoroacetic acid, plasma membrane isolated from two eukaryotic species was directly injected onto a reverse-phase high performance liquid chromatograph column. Upon development with a 60 to 100% (v/v) linear gradient of ethanol containing 0.1% trifluoroacetic acid, most of the polypeptides eluted without retention. Only the lipids and very hydrophobic proteins were retained and resolved. Most noticeable among retained proteins was the Mr 100,000 catalytic polypeptide of each species' primary plasma membrane cation pump, the Na+,K+-ATPase of pig kidney and the H+-ATPase of Neurospora crassa hyphae. This simple 60-min procedure yielded nearly pure ATPase starting from crude membranes and in a completely volatile solvent, without detergent. When fungal plasma membranes were phosphorylated in vitro with [gamma-32P]ATP prior to injection, protein kinase activity was observed and this resulted in the phosphorylation of the H+-ATPase as well as of several other less-abundant hydrophobic membrane proteins. This procedure is useful as an alternative method for the rapid characterization of those membrane-associated polypeptides that contain several hydrophobic, transmembrane sequences.

Adenosine Triphosphatases↗
PubMed1988

Isolation and sequence of tryptic peptides from the proton-pumping ATPase of the oat plasma membrane.

In crude extracts of plant tissue, the M(r) = 100,000 proton-pumping ATPase constitutes less than 0.01% of the total cell protein. A large-scale purification procedure is described that has been used to obtain extensive protein sequence information from this enzyme. Plasma membrane vesicles enriched in ATPase activity were obtained from extracts of oat roots by routine differential and density gradient centrifugation. Following a detergent wash, the ATPase was resolved from other integral membrane proteins by size fractionation at 4 degrees C in the presence of lithium dodecyl sulfate. After carboxymethylation of cysteine residues and removal of detergent, the ATPase was digested with trypsin and resultant peptide fragments separated by reverse phase high performance liquid chromatography. Peptides were recovered with high yield and were readily sequenced by automated Edman degradation on a gas-phase sequencer. Of the eight peptides sequenced, six showed strong homology with known amino acid sequences of the fungal proton-pumping and other cation-transporting ATPases.

Journal Article↗
PubMed1988

Cytochemical localization of ATPase activity in oat roots localizes a plasma membrane-associated soluble phosphatase, not the proton pump.

Cytochemical techniques employing lead-precipitation of enzymically released inorganic phosphate have been widely used in attempts to localize the plasma membrane proton pump (H(+)-ATPase) in electron micrographs. Using Avena sativa root tissue we have performed a side-by-side comparison of ATPase activity observed in electron micrographs with that observed in in vitro assays using ATPases found in the soluble and plasma membrane fractions of homogenates. Cytochemical analysis of oat roots, which had been fixed in glutaraldehyde in order to preserve subcellular structures, identifies an ATPase located at or near the plasma membrane. However, the substrate specificity and inhibitor sensitivity of the in situ localized ATPase appear identical to those of an in vitro ATPase activity found in the soluble fraction, and are completely unlike those of the plasma membrane proton pump. Further studies demonstrated that the plasma membrane H(+)-ATPase is particularly sensitive to inactivation by the fixatives glutaraldehyde and formaldehyde and by lead. In contrast, the predominant soluble ATPase activity in oat root homogenates is less sensitive to fixation and is completely insensitive to lead. Based on these results, we propose a set of criteria for evaluating whether a cytochemically localized ATPase activity is, in fact, due to the plasma membrane proton pump.

Journal Article↗
PubMed1987

Location of a dicyclohexylcarbodiimide-reactive glutamate residue in the Neurospora crassa plasma membrane H+-ATPase.

The proton pump (H+-ATPase) found in the plasma membrane of the fungus Neurospora crassa is inactivated by dicyclohexylcarbodiimide (DCCD). Kinetic and labeling experiments have suggested that inactivation at 0 degrees C results from the covalent attachment of DCCD to a single site in the Mr = 100,000 catalytic subunit (Sussman, M. R., and Slayman, C. W. (1983) J. Biol. Chem. 258, 1839-1843). In the present study, when [14C]DCCD-labeled enzyme was treated with the cleavage reagent, N-bromosuccinimide, a single major radioactive peptide fragment migrating at about Mr = 5,300 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis was produced. The fragment was coupled to glass beads and partially sequenced by automated solid-phase Edman degradation at the amino terminus and at an internal tryptic cleavage site. By comparison to the DNA-derived amino acid sequence for the entire Mr = 100,000 polypeptide (Hager, K., and Slayman, C. W. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 7693-7697), the fragment has been identified as arising by cleavage at tyrosine 100 and tryptophan 141. Covalently incorporated [14C]DCCD was released at a position corresponding to glutamate 129. The DCCD-reactive glutamate is located in the middle of the first of eight predicted transmembrane sequences. When the sequence surrounding the DCCD site is compared to that surrounding the DCCD-reactive residue of two other proton pumps, the F0F1-ATPase and cytochrome c oxidase, no homology is apparent apart from an abundance of hydrophobic amino acids.

Binding Sites↗
PubMed1987

Inhibition and Labeling of the Plant Plasma Membrane H-ATPase with N-Ethylmaleimide.

H(+)-ATPase activity in plasma membranes isolated from Avena sativa root cells is inhibited by N-ethylmaleimide, a covalent modifier of protein sulfhydryl groups. The rate of inhibition is reduced by ADP, MgADP, and MgATP, but even at 40 millimolar ADP the enzyme is only partially protected against inactivation. When plasma membranes are treated wth N-[2-(3)H]ethylmaleimide and analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis, prominent radioactive bands appear at M(r)=100,000 and several other positions. However, only radioactivity in the M(r)=100,000 protein is reduced by the presence of MgADP. These results provide independent evidence that the M(r)=100,000 polypeptide which is observed in purified preparations of the enzyme is the catalytic subunit of the H(+)-ATPase. When tryptic peptides are produced from N-[2-(3)H]ethylmaleimide labeled M(r)=100,000 protein and separated by reverse phase high performance liquid chromatography, two radioactive peaks are observed for which N-[2-(3)H]ethylmaleimide incorporation is reduced in the presence of MgADP.

Journal Article↗