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N E Hoffman

Publications and source records attributed to N E Hoffman.

At least 37 records · Page 2Linked to original sources
PubMed1994

The pacL gene of Synechococcus sp. strain PCC 7942 encodes a Ca(2+)-transporting ATPase.

An ATP-dependent Ca2+ uptake activity was identified in plasma membrane vesicles prepared from Synechococcus sp. strain PCC 7942. This activity was insensitive to agents which collapse pH gradients and membrane potentials but sensitive to vanadate, indicating that the activity is catalyzed by a P-type Ca(2+)-ATPase. A gene was cloned from Synechococcus sp. strain PCC 7942 by using a degenerate oligonucleotide based on a sequence conserved among P-type ATPases. This gene (pacL) encodes a product similar in structure to eukaryotic Ca(2+)-ATPases. We have shown that pacL encodes a Ca(2+)-ATPase by demonstrating that a strain in which pacL is disrupted has no Ca(2+)-ATPase activity associated with its plasma membrane. In addition, Ca(2+)-ATPase activity was restored to the delta pacL strain by introducing pacL into a second site in the Synechococcus sp. strain PCC 7942 chromosome.

Base Sequence↗
PubMed1993

Characterization of a gene encoding a Ca(2+)-ATPase-like protein in the plastid envelope.

By screening an Arabidopsis expression library with an antiserum against chloroplast envelope proteins, we have isolated a partial cDNA with an open reading frame that encodes a polypeptide similar to P-type cation-transporting ATPases. The corresponding genomic clone was isolated and the complete coding sequence was deduced after identification and mapping of introns. The gene has been designated PEA1 (plastid envelope ATPase) and the predicted polypeptide PEA1p. PEA1p has 946 amino acids and a molecular mass of 104 kDa. This protein is 40-44% identical to various mammalian plasma membrane Ca(2+)-ATPases but lacks the C-terminal calmodulin binding domain present in the mammalian polypeptides. When aligned with mammalian plasma membrane Ca(2+)-ATPases, PEA1p has a 70- to 80-amino acid N-terminal region that extends beyond the N terminus of these enzymes. This extension has some similarity to the transit peptide of the plastid envelope phosphate translocator and may function to target the protein to the plastid. Antibodies raised against a portion of PEA1p recognize a single 90- to 95-kDa polypeptide in chloroplast inner envelope preparations. Transcript abundance as determined by RNase protection was found to be 7- to 9-fold higher in roots than in leaves. Possible roles for a plastid envelope calcium pump are suggested.

Amino Acid Sequence↗
PubMed1993

Characterization of a chloroplast homologue of the 54-kDa subunit of the signal recognition particle.

Proteins and RNAs homologous to components of the eukaryotic signal recognition particle (SRP) have been identified in a number of prokaryotic organisms. Here we report the isolation of an Arabidopsis thaliana cDNA, FFC (fifty-four chloroplast homologue), that encodes a chloroplast protein (54CP) homologous to the 54-kDa subunit of the signal recognition particle. 54CP shares 27% identity with mammalian SRP54 and 44% identity with the Escherichia coli ffh gene product suggesting a prokaryotic origin for this gene. FFC is a nuclear gene encoding a 62-kDa cytoplasmically synthesized precursor that is capable of being imported into isolated chloroplasts and processed to a 53-kDa stromal polypeptide. Antibodies generated against a portion of 54CP recognize an endogenous 53-kDa chloroplast protein that sediments at 4 S and 70 S, the latter form may be associated with ribosomes. The FFC transcript is most abundant in green shoot tissue whereas etiolated buds and roots have transcript levels about 30 and 10%, respectively, relative to light grown green shoots. By analogy with the eukaryotic signal recognition particle which targets secretory proteins to the endoplasmic reticulum, it is speculated that 54CP may target chloroplast proteins to either the thylakoid or envelope membranes.

Amino Acid Sequence↗
PubMed1993

The gamma subunit of R-phycoerythrin and its possible mode of transport into the plastid of red algae.

R-phycoerythrin is the major light-harvesting pigment protein of most red algal phycobilisomes. It is composed of three pigmented polypeptide subunits, the alpha, beta, and gamma. While alpha and beta phycoerythrin subunits are each unique in the red alga Aglaothamnion neglectum, there are two different gamma subunits with distinct molecular masses. Both gamma subunits are pigmented by virtue of covalently attached linear tetrapyrroles. The amino acid sequence of one of the gamma subunits, as deduced from the nucleotide sequence of a cDNA clone, has no significant similarity to any known sequence in the data bases. This result is surprising, since the gamma subunit of phycoerythrin is thought to have a function that is similar to cyanobacterial linker polypeptides. The A. neglectum gamma subunit is synthesized as a 36-kDa precursor protein that is processed at the amino terminus to yield a 33-kDa mature protein. The amino-terminal extension was able to direct the pea small subunit of Rubisco into isolated pea chloroplasts. This result suggests that red algae transport proteins into the plastid by a mechanism similar to that of higher plants. There are significant changes in levels of mRNA encoding the gamma 33 subunit when A. neglectum is grown under different conditions of illumination and in nitrogen-deficient medium. These changes parallel those previously observed for transcripts encoding the alpha and beta phycoerythrin subunits. Hence, there may be coordinated expression of nuclear and plastid-encoded phycoerythrin subunit genes.

Amino Acid Sequence↗
PubMed1993

Primary structure and characterization of an Arabidopsis thaliana calnexin-like protein.

A cDNA clone (pTE-83) encoding a protein (CNX1p) related to the microsomal Ca(2+)-binding protein, calnexin, was isolated from an Arabidopsis thaliana expression library. Southern and Northern hybridization indicated that CNX1 is a single-copy gene encoding a message of 1900 nucleotides. The open reading frame encodes a polypeptide with 530 amino acids, a molecular mass of 60.5 kDa, and overall 48% identity to dog calnexin. Both animal calnexin and CNX1p contain a large luminal domain followed by a single potential membrane-spanning domain near the C terminus and a small C-terminal domain exposed to the cytoplasm. The in vitro translation product from the cloned cDNA yielded a polypeptide of 67 kDa that was co-translationally imported into dog microsomes and processed to a 64-kDa product. Antibodies generated against the C-terminal half of the protein cross-react with an identically sized protein present in the microsomal fraction from Arabidopsis. Both the imported and native proteins are cleaved by trypsin to a 59-kDa product indicating that the gene product was indeed correctly processed and translocated into dog microsomes and that the membrane topology of CNX1p resembles that of dog calnexin. The presence of a calnexin-like protein within the plant kingdom indicates that this protein is widespread and involved in processes fundamental to all eukaryotes.

Amino Acid Sequence↗
PubMed1993

Biogenesis of a photosystem I light-harvesting complex. Evidence for a membrane intermediate.

CAB-7p is a chlorophyll a/b binding protein of photosystem I (PSI). It is found in light-harvesting complex I 680 (LHCI-680), one of the chlorophyll complexes produced by detergent solubilization of PSI. Two types of evidence are presented to indicate that assembly of CAB-7p into PSI proceeds through a membrane intermediate. First, when CAB-7p is briefly imported into chloroplasts or isolated thylakoids, we initially observe a fast-migrating membrane form of CAB-7p that is subsequently converted into PSI. The conversion of the fast-migrating form into PSI does not require stroma or ATP. Second, trypsin treatment of thylakoids containing radiolabeled CAB-7p indicates that there are at least two membrane forms of the mature 23-kD protein. The predominant form is completely resistant to proteolysis; a second form of the protein is cleaved by trypsin into 12- and 7-kD polypeptides. We interpret this to mean that the intermediate is a cleavable form that becomes protease resistant during assembly. This notion is supported by the observation that CAB-7p in LHCI-680 is largely cleaved by trypsin into 12- and 7-kD polypeptides, whereas CAB-7p in isolated PSI particles is trypsin resistant. In vitro, we generated a mutant form of CAB-7p, CAB-7/BgI2p, that was able to integrate into thylakoid membranes but was unable to assemble into PSI. The membrane form of CAB-7/BgI2p, like LHCI-680, was predominantly cleaved by trypsin into 12- and 7-kD fragments. We suggest that the mutant protein is arrested at an intermediate stage in the assembly pathway of PSI. Based on its mobility in nondenaturing gels and its susceptibility to protease cleavage, we suggest that the intermediate form is LHCI-680. We propose the following distinct stages in the biogenesis of LHCI: (a) apoprotein is integrated into the thylakoid, (b) chlorophyll is rapidly bound to apoprotein forming LHCI-680, and (c) LHCI-680 assembles into the native PSI complex.

Base Sequence↗
PubMed1992

Deletion Mutants of Chlorophyll a/b Binding Proteins Are Efficiently Imported into Chloroplasts but Do Not Integrate into Thylakoid Membranes.

Chlorophyll a/b binding polypeptides (CABp) are integral thylakoid membrane proteins containing three membrane-spanning helices. We have created a series of mutations in tomato CABp to test whether individual membrane helices with hydrophilic flanking sequences, when fused to a transit peptide, can be imported into chloroplasts and correctly targeted to thylakoid membranes. All of the mutated precursors, including those with large C-terminal and internal deletions, were imported successfully, showing that these regions of the mature CABp are not required for import into chloroplasts. All mutants tested, containing either one or two membrane helices, were found primarily in the stroma and not in the thylakoids. The small amount of protein found associated with the thylakoids was largely resistant to alkali extraction but was sensitive to protease, unlike wild-type protein, which is resistant to both treatments. When incubated with thylakoids in the absence of stroma and/or ATP, a significant amount of wild-type protein assumes a form that is resistant to alkali extraction but is protease sensitive, like the imported deletion proteins. This form of the wild-type protein is not chased into a protease-resistant form by adding stroma and/or ATP. These results suggest that CABp can spontaneously associate with membranes as an aberrant species that is not an intermediate in the process of integration. The inability of the deletion forms of CABp to assume a protease-resistant conformation suggests that correct integration is afforded by elements within the entire protein that collectively contribute to the proper conformation of the protein. The ability of deletion mutants to associate with thylakoids in a nonphysiological way suggests that the study of such mutants may not be useful in elucidating thylakoid-targeting signals.

Journal Article↗
PubMed1991

Isolation and characterization of tomato cDNA and genomic clones encoding the ubiquitin gene ubi3.

We report here the isolation and nucleotide sequence of tomato cDNA and genomic clones encoding a ubiquitin extension protein homologous to the yeast gene ubi3. Sites similar to upstream activating sites commonly found in the promoters of yeast ribosomal genes were observed in the tomato promoter. The tomato ubi3 promoter also contained elements found in the rbcS promoter from pea. The transcription initiation site was determined to occur 66 bp upstream of the initiating Met. RFLP mapping revealed that the gene was located on chromosome 1, 23 cM from marker TG301. A ubi3 gene-specific probe hybridized to a single 800 nt transcript. Expression was reduced in heat-shocked plants and plants kept in the dark. Expression was highest in young leaves and immature green fruit and lowest in mature leaves and petals. We isolated the original cDNA clone using an antibody prepared against chloroplast polypeptides. Immunological studies did not detect ubiquitin or ubiquitin extension proteins in the chloroplast. However, higher-molecular-weight chloroplast proteins were detected with ubiquitin antisera suggesting that ubiquitin conjugates are transported into the chloroplast.

Amino Acid Sequence↗
PubMed1990

Evidence that Isolated Developing Chloroplasts Are Capable of Synthesizing Chlorophyll b from 5-Aminolevulinic Acid.

Developing chloroplasts isolated from cucumber (Cucumis sativus L. var Beit Alpha) cotyledons are capable of incorporating [(14)C]5-aminolevulinic acid into chlorophyll (Chl) b and Chl a when incubated under photosynthetic illumination. Thin layer chromatography and high pressure liquid chromatography were employed to analyze the pigments. The specific radioactivity in Chl a was over three times higher than that found in Chl b. Both Chl a and b synthesizing activities in organello decayed rapidly at approximately the same rate. We conclude that concomitant synthesis of Chl a/b-binding apoprotein is not required for Chl b synthesis.

Journal Article↗
PubMed1990

Validation of prognostic models in primary biliary cirrhosis.

Prognostic models in primary biliary cirrhosis have been validated for large population groups but the predictive value for individual patients has not been tested. We used data from ten deceased patients with primary biliary cirrhosis to test three prognostic models: the Shapiro model (bilirubin); the Christensen model (age, bilirubin, albumin, presence of cirrhosis or cholestasis, azathioprine treatment); and the Dickson model (age, bilirubin, albumin, prothrombin time, oedema). The predictive value of each model for individual patients was determined by assessing whether it would have accurately predicted appropriate timing of liver transplantation in patients prior to death. The Dickson model predicted that four of nine cases would have been considered for liver transplantation one year before death and one of seven cases two years before death. The Christensen model predicted that this procedure would have been considered in three of seven cases two years before death. The Shapiro model was demonstrated to be the least predictive of the three tested. Although none of the three models assessed was found to accurately predict survival, no model predicted a worse survival than actually occurred. Liver transplantation is indicated in those cases with a poor predicted survival.

Aged↗
PubMed1989

Determination of ranitidine in plasma by high-performance liquid chromatography.

A high-performance liquid chromatographic method has been developed for the determination of ranitidine in plasma. Ranitidine was extracted with acetonitrile by adding it to the plasma and then salting it out with potassium carbonate. The chromatographic column was 5-microns ODS silica, the mobile phase being acetonitrile-7 mM triethylammonium ion in phosphoric acid (pH 3.00) (30:70, v/v). The ranitidine peak was monitored at a wavelength of 315 nm, the retention time for ranitidine being 4.6 min. A limit of detection of 3 ng ml-1 was obtained for a 100-microliters injection of ranitidine. The method was found to be reproducible with a relative standard deviation (RSD) between 0.8-5.3% (n = 5) over the concentration range 25-80 ng ml-1 in plasma. The ranitidine concentration was determined in 18 different patients' plasmas. Ranitidine and its metabolites ranitidine S-oxide, ranitidine N-oxide and desmethyl-ranitidine, were also studied for chromatographic resolution from each other. It was shown that a group of common drugs did not interfere with ranitidine determination.

Chromatography, High Pressure Liquid↗
PubMed1988

High-performance liquid chromatographic determination of fusidic acid in plasma.

Fusidic acid was determined in plasma by a high-performance liquid chromatographic method. Fusidic acid was extracted from plasma with acetonitrile that was salted out with ammonium sulfate. Prior to salting out cadmium sulfate was mixed with the acetonitrile-plasma mixture to help remove interfering constituents. A 150 mm X 4.6 mm column packed with 5-microns cyanopropyl stationary phase was used for chromatography. The mobile phase was acetonitrile-20 mM sodium dihydrogenphosphate (pH 3.50) (39:61, v/v). An ultraviolet-visible detector was set at 204 nm. The presence of water in the injection solvent had a significant effect on the fusidic acid peak height. A number of clinically important acquired immunodeficiency syndrome drugs did not interfere with the fusidic acid determination. The relative standard deviation varied between 0.99 and 7.8%. A limit of detection of 200 ng/ml was obtained for a 80-microliters injection.

Chromatography, High Pressure Liquid↗
PubMed1988

A light-entrained circadian clock controls transcription of several plant genes.

Diurnal oscillations in steady-state mRNA levels and transcription rates were measured for seven transcripts (five of which encode chloroplast-localized proteins) in tomato seedlings: photosystem I and photosystem II chlorophyll a/b binding proteins (CAB/I and CAB/II), small subunit of RuBisCO (RBCS), actin, subunit II of the photosystem I reaction center (PSAD), subunit I of the photosystem II oxygen-evolving enzyme (OEE1), and a biotin-binding protein of unknown function. CAB/II mRNA levels were found to oscillate greater than 20-fold, showing a peak at noon, while only marginal diurnal oscillations are seen in RBCS transcripts. The oscillations are at least partially controlled at the transcriptional level. Transcription rates of both CAB/II and RBCS, measured by nuclear run-on experiments, were found to oscillate, with a peak around 8 a.m. Transcription rates of the 'biotin' clone also oscillated, with a peak around noon. Transfer of plants to constant darkness or constant light conditions alters the amplitude of the transcriptional oscillation, but does not abolish it, suggesting that it is at least partially controlled by a circadian clock. The oscillations are still visible after three days in complete darkness, and have a period very close to 24 h. The oscillator phase can be reset by out-of-phase light treatment.

Cell Nucleus↗
PubMed1988

Determination of 5-fluorouracil in plasma and whole blood by ion-pair high-performance liquid chromatography.

5-Fluorouracil in blood or plasma was determined by extraction and column liquid chromatography. Acetonitrile was added to blood or plasma and the mixture was stirred and centrifuged. Zinc sulfate addition was followed by stirring and centrifuging. The acetonitrile was salted out with ammonium sulfate, and an aliquot was evaporated with nitrogen. The residue was dissolved in mobile phase and chromatographed. The stationary phase was a styrene-divinylbenzene copolymer, and the mobile phase was 10 mM tetrabutylammonium hydroxide-methanol (74:26). 5-Fluorouracil was detected by UV absorption at 266 nm. Time of the assay was less than 30 min. The detection limit was 10 ng/ml and the relative standard deviation was 4 to 10% depending on the concentration.

Chromatography, High Pressure Liquid↗
PubMed1987

Determination of cyclophosphamide in whole blood and plasma by reversed-phase high-performance liquid chromatography.

A rapid, simple, and sensitive reversed-phase high-performance liquid chromatographic determination of the cytostatic drug cyclophosphamide in whole blood and plasma has been developed. The pre-chromatography isolation of the drug involves salting-out of acetonitrile with simultaneous extraction of cyclophosphamide from whole blood and plasma. A short column packed with 5-micron reversed-phase octadecylsilane (ODS) spherical particles was used with an isocratic elution of 5 mM potassium phosphate (pH 6.80)-acetonitrile (80:20, v/v). The cyclophosphamide was monitored at 190 nm and 0.40-0.002 a.u.f.s. At a flow-rate of 1.0 ml/min, the retention time of cyclophosphamide was ca. 9 min. The completion time for the assay was less than 20 min and the assay had a detection limit of 0.30 microgram/ml. This method was used to determine the stability of cyclophosphamide in plasma at room temperature and at -10 degrees C.

Chromatography, High Pressure Liquid↗