Biomedical subjects

R Premakumar

Publications and source records attributed to R Premakumar.

At least 19 recordsLinked to original sources

PubMed2005

Carbohydrate partitioning between upper and lower regions of the crown in oat and rye during cold acclimation and freezing.

Carbohydrates have long been recognized as an important aspect of freezing tolerance in plants but the association between these two factors is often ambiguous. To help clarify the relationship, the allocation of carbohydrates between specific tissues within the over wintering organ (crown) of winter cereals was measured. A winter-hardy and non-winter-hardy oat (Avena sativa L.), and a rye (Secale cereale L.) cultivar were grown and frozen under controlled conditions. Crown tissue was fractionated into an upper portion, called the apical region, and a lower portion, called the lower crown. These tissues were ground in liquid N and extracted with water. Extracts were analyzed by HPLC for the simple sugars, sucrose, glucose, fructose, and for fructan of various size classes. After 3 weeks of cold acclimation at 3 degrees C, carbohydrates accounted for approximately 40% of the dry weight of oats and 60% of the dry weight of rye. The apical region, which is the tissue within the crown that acclimates to the greatest extent, was generally 10% higher in total carbohydrates than the lower crown. During a mild freeze, various carbohydrates were allocated differently between specific tissues in the three genotypes. When frozen, fructan generally decreased to a greater extent in the lower crown than in the apical region but sugars increased more in the apical region than in the lower crown. Results suggest that to understand how carbohydrates relate to freezing tolerance, regions of the crown that endure freezing stress differently should be compared.

Acclimatization↗

PubMed2005

Carbohydrate concentrations in crown fractions from winter oat during hardening at sub-zero temperatures.

BACKGROUND AND AIMS: Contradictory results in correlation studies of plant carbohydrates with freezing tolerance may be because whole crown tissue is analysed for carbohydrates while differences exist in the survival of specific tissue within the crown. The aim of this study was to see if carbohydrate changes in tissue within oat crowns during second phase hardening (sub-zero hardening) are tissue specific. METHODS: The lower portion of oat (Avena sativa) crowns was exposed to mild grinding in a blender and the remaining crown meristem complex, consisting of tough root-like vessels, was ground in a device developed specifically for grinding cereal crown tissue. Carbohydrates were extracted by water and measured by HPLC. Carbohydrate concentrations were compared in the two regions of the crown before and after hardening at sub-zero temperatures. KEY RESULTS: Fructan of all size classes except DP>6 decreased during sub-zero hardening in both stems (base of leaf sheath) and crown meristem complex. Total simple sugar increase, including sucrose, was significantly higher in the crown meristem complex than in the stem. CONCLUSIONS: Results support the hypothesis that carbohydrate change in mildly frozen plants is tissue specific within crowns and underscore the need to evaluate specific tissue within the crown when correlating the biochemistry of plants with freezing tolerance.

Adaptation, Physiological↗

PubMed2000

Thermal effect of CO(2) on apoplastic ice in rye and oat during freezing.

Meristematic tissues from rye (Secale cereale) and oat (Avena sativa) were studied in an isothermal calorimeter at -3 degrees C. When the frozen tissue was placed in the calorimeter, the pressure increased within 4 d to 25 and 9 kPa above ambient pressure in the sample vessels containing crowns of rye and oat, respectively. Concurrently, the thermal output went down to -194 microW in rye over the 4-d period; this negative thermal activity could be accounted for by ice melting in the plants. When the pressure was released, the output from the calorimeter went from -194 to 229 microW within 1 h, suggesting that water had frozen in the plants. We propose that CO(2) from respiration had dissolved in the water in the plants and caused melting of ice (heat absorption) due to the colligative properties of solutions. When the pressure was released, the CO(2) came out of solution and the water froze (heat evolution). These thermal observations were duplicated in a simplified, non-biological system using a glycol/water mixture that was partially frozen at -3 degrees C.

Avena↗

PubMed1998

Regulation of the transcriptional activators AnfA and VnfA by metals and ammonium in Azotobacter vinelandii.

Transcription of the genes encoding molybdenum (Mo)-independent nitrogenases 2 and 3 of Azotobacter vinelandii requires the activators VnfA and AnfA, respectively. The effect of NH4+, Mo, or V (vanadium) was tested on the expression of vnfA-lacZ and anfA-lacZ transcriptional fusions. Mo repressed expression of both fusions whereas NH4+ and V repressed the anfA-lacZ fusion, but not the vnfA-lacZ fusion. Thus the repressive effect on transcription of the anfHDGKOR operon by NH4+, Mo, or V is mediated through their effect on transcription of anfA and the repressive effect of Mo on the vnfHFd and vnfDGK operons is mediated through Mo repression of vnfA transcription. Mo-dependent repression of anfA transcription is influenced but not entirely mediated by the Mo-responsive regulator ModE.

Acetates↗

PubMed1996

Characteristics of orf1 and orf2 in the anfHDGK genomic region encoding nitrogenase 3 of Azotobacter vinelandii.

In Azotobacter vinelandii, the anfHDGK operon encodes the subunits for the third nitrogenase complex. Two open reading frames (orf1 and orf2) located immediately downstream of anfK were shown to be required for diazotrophic growth under Mo- and V-deficient conditions. We have designated orf1 and orf2 anfO and anfR, respectively. Strains (CA115 and CA116) carrying in-frame deletions in anfO and anfR accumulate the subunits for nitrogenase 3 under Mo-deficient diazotrophic conditions. AnfO and AnfR are required for nitrogenase 3-dependent diazotrophic growth and 15N2 incorporation but not for acetylene reduction. AnfO contains a putative heme-binding domain that exhibits similarity to presumed heme-binding domains of P-450 cytochromes. Amino acid substitutions of Cys-158 show that this residue is required for fully functional AnfO as measured by diazotrophic growth under Mo- and V-deficient conditions. The nucleotide sequence of the region located immediately downstream of anfR has been determined. A putative rho-independent transcription termination site has been identified 250 bp from the 3' end of anfR. A third open reading frame (orf3), located downstream of anfR, does not appear to be required for diazotrophic growth under Mo- and V-deficient conditions.

Acetylene↗

PubMed1996

Phenotypic characterization of a tungsten-tolerant mutant of Azotobacter vinelandii.

A tungsten-tolerant mutant strain (CA6) of Azotobacter vinelandii first described in 1980 (P. E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980) has been further characterized. Results from growth experiments suggest that both nitrogenases 1 and 3 are utilized when CA6 grows in N-free medium containing Na2MoO4. Strain CA6.1.71, which lacks both nitrogenases 2 and 3, grew as well as strain CA in N-free medium containing Na2MoO4 after an initial lag. This indicates that nitrogenase 1 is fully functional in strain CA6. nifH-lacZ and anfH-lacZ transcriptional fusions were expressed in CA6 in the presence of Na2MoO4. Thus, in contrast to wild-type strain CA, transcription of the anfHDGK gene cluster in strain CA6 is not repressed by Mo. Expression of the vnfD-lacZ fusion was the same in both strains CA and CA6. In agreement with the results obtained with lac fusions, subunits of both nitrogenases 1 and 3 were found in protein extracts of CA6 cells grown in N-free medium containing Na2MoO4. However, CA6 cells, cultured in the presence of Na2WO4, accumulated nitrogenase 3 proteins without detectable amounts of nitrogenase 1 proteins. This indicates that expression of Mo-independent nitrogenase 3 is the basis for the tungsten tolerance phenotype of strain CA6. A measure of Mo accumulation as a function of time showed that accumulation by strain CA6 was slower than that for strain CA. When Mo accumulation was studied as a function of Na2MoO4 concentration, the two strains accumulated similar amounts of Mo in the concentration range of 0 to 1 microM Na2MoO4 during a 2-h period. Within the range of 1 to 5 microM Na2MoO4, Mo accumulation by strain CA increased linearly with increasing concentration whereas no further increases were observed for strain CA6. These results are consistent with the possibility that the tungsten tolerance mutation carried by CA6 is in a Mo transport system.

Azotobacter vinelandii↗

PubMed1995

The genes encoding the delta subunits of dinitrogenases 2 and 3 are required for mo-independent diazotrophic growth by Azotobacter vinelandii.

vnfG and anfG encode the delta subunits of alternative nitrogenases 2 and 3 in Azotobacter vinelandii, respectively. As a first step towards elucidating the role of these subunits, diazotrophic growth and acetylene reduction studies were conducted on mutants containing alterations in the genes encoding these subunits. Mutants containing a stop codon (C36stop) or an in-frame deletion in anfG were unable to grow in N-free, Mo-deficient medium (Anf-). Mutants in which cysteine 36 of AnfG (a residue conserved between VnfG and AnfG) was changed to Ala or Ser were Anf+. Thus, this conserved cysteine is not essential for the function of AnfG in dinitrogenase 3. A mutant with a stop codon in vnfG (C17stop) grew after a lag of 25 h in N-free, Mo-deficient medium containing V2O5. However, a Nif- Anf- strain with this mutation was unable to grow under these conditions. This shows that the vnfG gene product is required for nitrogenase 2-dependent growth. Strains with mutations in vnfG and anfG reduced acetylene to different degrees. This indicates that the delta subunits are not required for acetylene reduction by nitrogenases 2 and 3.

Acetylene↗

PubMed1994

Mo-independent nitrogenase 3 is advantageous for diazotrophic growth of Azotobacter vinelandii on solid medium containing molybdenum.

Competition experiments between wild-type Azotobacter vinelandii and a mutant lacking Mo-independent nitrogenase 3 indicate that nitrogenase 3 provides an advantage during diazotrophic growth on agar media containing 100 to 500 nM Na2MoO4 but not in liquid media under the same conditions. Expression of nitrogenase 3 in wild-type cells growing on agar surfaces was verified with an anfH-lacZ fusion and by detection of nitrogenase 3 subunits. These results show that nitrogenase 3 is important for diazotrophic growth on agar medium at molybdenum concentrations that are not limiting for Mo-dependent diazotrophic growth in liquid medium.

Azotobacter vinelandii↗

PubMed1994

Effect of amino acid substitutions in a potential metal-binding site of AnfA on expression from the anfH promoter in Azotobacter vinelandii.

AnfA, an activator required for transcription of the structural genes encoding nitrogenase 3 (anfHDGK) in Azotobacter vinelandii, has a potential metal-binding site [(S19)H(C21)FTGE(C26)R] in its N terminus. Growth studies and expression of an anfH-lacZ fusion in mutants containing amino acid substitutions in this site indicate that Ser-19 is not required for AnfA activity whereas Cys-21 and Cys-26 are required. Residual expression of the anfH-lacZ fusion in AnfA- mutants was found to be due to activation by VnfA, the activator required for expression of genes encoding nitrogenase 2.

Azotobacter↗

PubMed1992

Characterization of transcripts expressed from nitrogenase-3 structural genes of Azotobacter vinelandii.

Five major anfH-hybridizing mRNA species accumulated in Azobacter vinelandii cells derepressed for nitrogenase-3 (an alternative nitrogenase, which appears to lack Mo and V). Using anfH-, anfD-, anfG-, anfK-, and orflorf2-specific probes and mutant strains of A. vinelandii these mRNA species have been identified as encoding anfHDGKorflorf2 (6.0 kb), anfHDGK (4.3 kb), anfHD (2.6 kb), vnfHorfFd (1.3 kb), and vnfH and (or) anfH (1.0 kb). A 0.6-kb mRNA species, which hybridized only to the orflorf2-specific probe, and a 3.5-kb mRNA species, which hybridized to anfD or anfK, also accumulated under these conditions. Northern blot analysis and S1 nuclease mapping indicate that transcription of the anf structural gene cluster initiates at a unique nif consensus promoter situated 127 base pairs upstream from the anfH coding region. Observation of anfH-hybridizing mRNA species that accumulate in strains derepressed for nitrogen fixation demonstrates that transcription of the anfHDGKorflorf2 cluster is normally repressed by Mo, V, and NH4+, whereas transcription of the vnfHorfFd cluster does not require the presence of V and is repressed only by Mo, but not NH4+. Analysis of the accumulation of mRNAs in a tungsten-tolerant strain revealed that Mo and V repression of anf transcription must occur by different mechanisms.

Ammonia↗

PubMed1989

Nucleotide sequence and mutational analysis of the structural genes (anfHDGK) for the second alternative nitrogenase from Azotobacter vinelandii.

The nucleotide sequence of a region of the Azotobacter vinelandii genome exhibiting sequence similarity to nifH has been determined. The order of open reading frames within this 6.1-kilobase-pair region was found to be anfH (alternative nitrogen fixation, nifH-like gene), anfD (nifD-like gene), anfG (potentially encoding a protein similar to the product of vnfG from Azotobacter chroococcum), anfK (nifK-like gene), followed by two additional open reading frames. The 5'-flanking region of anfH contains a nif promoter similar to that found in the A. vinelandii nifHDK gene cluster. The presumed products of anfH, anfD, and anfK are similar in predicted Mr and pI to the previously described subunits of nitrogenase 3. Deletion plus insertion mutations introduced into the anfHDGK region of wild-type strain A. vinelandii CA resulted in mutant strains that were unable to grow in Mo-deficient, N-free medium but grew in the presence of 1 microM Na2MoO4 or V2O5. Introduction of the same mutations into the nifHDK deletion strain CA11 resulted in strains that grew under diazotrophic conditions only in the presence of vanadium. The lack of nitrogenase 3 subunits in these mutant strains was demonstrated through two-dimensional gel analysis of protein extracts from cells derepressed for nitrogenase under Mo and V deficiency. These results indicate that anfH, anfD, and anfK encode structural proteins for nitrogenase 3.

Amino Acid Sequence↗

PubMed1988

Purification of a second alternative nitrogenase from a nifHDK deletion strain of Azotobacter vinelandii.

A second alternative nitrogenase complex (nitrogenase 3) was purified from a nifHDK deletion strain of Azotobacter vinelandii. The active complex is made up of two components, dinitrogenase 3 and dinitrogenase reductase 3. Dinitrogenase 3 contains two protein subunits (alpha, Mr 58,000, and beta, Mr 50,000) which assemble into at least two active configurations: alpha 2 beta 2 (dinitrogenase 3s) and alpha 1 beta 2 (dinitrogenase 3F). Dinitrogenase 3s contains 24 Fe and 18 acid-labile S2-ions per Mr 216,000, and dinitrogenase 3F contains 11 Fe and 9 acid-labile S2-ions per Mr 158,000. Dinitrogenase reductase 3 is composed of two protein subunits of identical Mr (32,500) and contains four Fe and four acid-labile S2- ions per Mr 65,000. On two-dimensional gels, the protein subunits of the nitrogenase 3 complex comigrated with the four Mo-, V-, and NH4+-repressible proteins originally designated as N2ase B: the nitrogenase hypothesized to exist in the alternative N2 fixation system first described in 1980 (P.E. Bishop, D. M. L. Jarlenski, and D. R. Hetherington, Proc. Natl. Acad. Sci. USA 77:7342-7346, 1980). Neutron activation analysis indicated that the nitrogenase 3 complex lacked significant amounts of Mo, V, Cr, Re, and W. Some Zn, however, was found in the dinitrogenase 3S and dinitrogenase 3F preparations. The pattern of substrate reduction efficiency was H+ greater than N2 greater than C2H2. The maximum specific activity found for N2 reduction was 38 nmol of NH3 per min per mg of protein (dinitrogenase 3S). Nitrogenase 3 was found to be extremely sensitive to O2, and activities could not be reproducibly maintained during freezing and thawing.

Azotobacter↗

PubMed1986

Transcriptional regulation of nitrogen fixation by molybdenum in Azotobacter vinelandii.

Multiple genomic regions homologous to nifH were found in the diazotroph Azotobacter vinelandii. The nifHDK gene cluster, located on a 12.8-kilobase (kb) XhoI fragment and two additional XhoI fragments (7.4 and 8.4 kb) hybridized to a nifH-specific DNA template but the 7.4- and 8.4-kb fragments did not hybridize to nifD- or nifK-specific DNA probes. In vivo transcription of the nifHDK gene cluster was ammonia-repressible and required the presence of at least 50 nM molybdenum in the derepression medium. Three mRNA species were transcribed from the nifHDK gene cluster, a 4.2-kb transcript homologous to nifH-, nifD-, and nifK-specific DNA templates, a 2.6-kb transcript homologous to nifH- and nifD-specific DNA templates, and a 1.2-kb transcript homologous only to the nifH-specific DNA template. In strain CA11, a nifHDK deletion mutant, the nifHDK-specific transcripts were not produced and the strain was unable to grow in N-free medium in the presence of Na2MoO4 at concentrations of 50 nM or higher. However, at concentrations of 25 nM Mo or less, growth occurred in N-free medium. Under these conditions two nifH-homologous (but not nifD- or nifK-homologous) transcripts were observed (1.2 and 1.8 kb). Presumably these were transcribed from the additional nifH-homologous sequences present in the genome. These results are consistent with the existence of two N2 fixation systems in A. vinelandii which are regulated by molybdenum at the level of transcription.

Azotobacter↗

PubMed1986

Tn5-induced mutants of Azotobacter vinelandii affected in nitrogen fixation under Mo-deficient and Mo-sufficient conditions.

Mutants of Azotobacter vinelandii affected in N2 fixation in the presence of 1 microM Na2MoO4 (conventional system), 50 nM V2O5, or under Mo deficiency (alternative system) have been isolated after Tn5 mutagenesis with the suicide plasmid pSUP1011. These mutants can be grouped into at least four broad phenotypic classes. Mutants in the first class are Nif- under Mo sufficiency but Nif+ under Mo deficiency or in the presence of V2O5. A nifk mutant and a mutant apparently affected in regulation of the conventional system belong to this class. Mutants in the second class are Nif- under all conditions. An FeMo-cofactor-negative mutant (NifB-) belongs to this class, implying an involvement of nifB in both the conventional and the alternative N2 fixation systems. The third mutant class consists of mutants incapable of N2-dependent growth under Mo deficiency. Most of the mutants in this class are also affected in N2 fixation in the presence of 1 microM Na2MoO4, with acetylene reduction rates ranging from 28 to 51% of the rates of the wild type. Strains constructed by genetic transfer of the Kanr marker of mutants from this class into nifHDK or nifK deletion mutants showed N2-dependent growth only in the presence of V2O5, suggesting that growth in the presence of V2O5 and growth under Mo deficiency are independent phenomena. The only mutant in the fourth class shows wild-type nitrogenase activity under Mo sufficiency, but only 10% of the acetylene reduction activity of the wild type in the presence of 50 nM V2O5. The acetylene reduction rates of whole cells of this mutant are identical in Mo-deficient medium and in medium containing V2O5. The conventional nitrogenase subunits are expressed in this mutant even under Mo deficiency or in the presence of V2O5; however, the NH4+- and Mo-repressible proteins normally seen under these conditions could not be detected on two-dimensional gels. The Tn5 insertion carried by this mutant makes N2 fixation dependent solely on the conventional system and consequently abolishes the vanadium effect.

Acetylene↗

PubMed1985

S-Adenosylhomocysteine hydrolase from human placenta. Affinity purification and characterization.

S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) was purified to homogeneity from human placenta by using S-adenosylhomocysteine-agarose affinity chromatography. The enzyme is a tetramer with a native Mr of 189 000 and subunit Mr of 47 000-48 000; there were nine cysteine residues per subunit and no disulphide bonds. The pI was 5.7. H.p.l.c. analysis revealed that the enzyme contained four molecules of tightly bound cofactor (NAD) per tetramer, of which 10-50% was in the reduced form. The enzyme had four binding sites per tetramer for adenosine, of which 10-35% were found to be occupied. Two types of adenosine-binding sites could be distinguished on the basis of differences in rates of dissociation of the enzyme-adenosine complex, and by examining binding of adenosine at 0 degree C and 37 degrees C. The enzyme catalysed the interconversion of adenosine and 4',5'-dehydroadenosine; the equilibrium constant for this reaction was 2.1 and favoured 4',5'-dehydroadenosine formation. Variability in the specific activity of preparations of S-adenosylhomocysteine hydrolase was related to the NAD+/NADH ratio of the preparation. The capacity to bind radioactively labelled adenosine depended on the adenosine content of the purified enzyme. The rate of adenosine binding and the sensitivity of S-adenosylhomocysteine hydrolase to inactivation by adenosine were both diminished in the absence of dithiothreitol.

Adenosine↗

PubMed1980

Repression of nitrate reductase in Neurospora studied by using L-methionine-DL-sulfoximine and glutamine auxotroph gln-1b.

The effect of L-methionine-DL-sulfoximine, an inhibitor of glutamine synthetase, on the formation of nitrate reductase in the wild-type strain of Neurospora in the presence of ammonium ions and of glutamine was studied. Under conditions in which glutamine synthetase was inactivated, it was found that only glutamine could repress nitrate reductase. In a mutant of Neurospora, gln-1b, which requires glutamine for growth, only glutamine could repress nitrate reductase. These results suggest a direct role for glutamine as corepressor of nitrate reductase in Neurospora.

Enzyme Repression↗

PubMed1980

Physiological characterization of a Neurospora crassa mutant with impaired regulation of nitrate reductase.

This report describes the isolation and characterization of a Neurospora crassa mutant with an impaired regulation of nitrate reductase. Glutamine, which prevents the induction of nitrate reductase in N. crassa, did so relatively ineffectively in this mutant. The mutation did not affect the regulation of all enzymes regulated by "nitrogen metabolite regulation"; it did affect the regulation of nitrate reductase, nitrite reductase, histidase, and acetamidase, as well as that of thiourea sensitivity. The mutation was not allelic with nit-2, the gene controlling a general positive effector of nitrogen metabolite-regulated enzyme formation.

Enzyme Induction↗

PubMed1979

Nitrogen metabolite repression of nitrate reductase in Neurospora crassa.

The effect of different nitrogen compounds on the induction of reduced nicotinamide adenine dinucleotide phosphate-nitrate reductase was examined in Neurospora crassa. Whereas in the wild-type strain several amino acids and ammonia inhibit the formation of nitrate reductase, only glutamine, cysteine, and histidine are shown to inhibit the synthesis of nitrate reductase in a glutamine-requiring auxotroph. None of the amino acids inhibited nitrate reductase activity in vitro. The effects of cysteine and histidine are nonspecific, these amino acids being inhibitory of the growth of the organism. The effect of glutamine on the induction of nitrate reductase is not due to an inhibition of the uptake of the inducer nitrate. By the use of histidine-, pyrimidine-, and arginine-requiring auxotrophs, it was shown that glutamine appears to act per se and does not seem to be converted to another product in order to be effective in repression. The repression of nitrate reductase by ammonia appears, from the results described herein, to be indirect; ammonia has to be converted first to glutamine in order to be effective in repression.

Amino Acids↗