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E Heinz

Publications and source records attributed to E Heinz.

At least 37 records · Page 2Linked to original sources
PubMed1998

A sphingolipid desaturase from higher plants. Identification of a new cytochrome b5 fusion protein.

A recently cloned cDNA from sunflower codes for a fusion protein composed of an N-terminal cytochrome b5 and a domain similar to membrane-bound acyl lipid desaturases. For a functional identification, homologous cDNAs from Brassica napus and Arabidopsis thaliana were expressed in Saccharomyces cerevisiae, and sphingolipid long chain bases were analyzed. The expression of the heterologous enzyme results in significant proportions of new Delta8, 9-cis/trans-phytosphingenines that accompany the residual C18-phytosphinganine predominating in wild-type yeast cells. These results represent the first identification of a gene coding for a sphingolipid desaturase and for a stereounselective desaturase showing trans-activity from any organism. Furthermore, this fusion protein is a new member of the cytochrome b5 superfamily. The formation of the two regioisomeric phytosphingenines in the transformed yeast sheds new light on the factors controlling regioselectivity.

Amino Acid Sequence↗
PubMed1998

A UDP glucosyltransferase from Bacillus subtilis successively transfers up to four glucose residues to 1,2-diacylglycerol: expression of ypfP in Escherichia coli and structural analysis of its reaction products.

We have isolated the ypfP gene (accession number P54166) from genomic DNA of Bacillus subtilis Marburg strain 60015 (Freese and Fortnagel, 1967) using PCR. After cloning and expression in E. coli, SDS-PAGE showed strong expression of a protein that had the predicted size of 43.6 kDa. Chromatographic analysis of the lipids extracted from the transformed E. coli revealed several new glycolipids. These glycolipids were isolated and their structures determined by nuclear magnetic resonance (NMR) and mass spectrometry. They were identified as 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylgl ycerol, 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl]-1,2-diacylglycerol and 3-[O-beta-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl-(1-->6)-O-bet a-D-glucopyranosyl-(1-->6)-O-beta-D-glucopyranosyl]-1,2-diacylglycerol. The enzymatic activity expected to catalyse the synthesis of these compounds was confirmed by in vitro assays with radioactive substrates. In these assays, one additional glycolipid was formed and tentatively identified as 3-[O-beta-D-glucopyranosyl]-1,2-diacylglycerol, which was not detected in the lipid extract of transformed cells. Experiments with some of the above-described glycolipids as 14C-labelled sugar acceptors and unlabelled UDP-glucose as glucose donor suggest that the ypfP gene codes for a new processive UDP-glucose: 1,2-diacylglycerol-3-beta-D-glucosyl transferase. This glucosyltransferase can use diacylglycerol, monoglucosyl-diacylglycerol, diglucosyl diacylglycerol or triglucosyl diacylglycerol as sugar acceptor, which, apart from the first member, are formed by repetitive addition of a glucopyranosyl residue in beta (1-->6) linkage to the product of the preceding reaction.

Bacillus subtilis↗
PubMed1998

Identification of a novel delta 6-acyl-group desaturase by targeted gene disruption in Physcomitrella patens.

The moss Physcomitrella patens contains high levels of arachidonic acid. For its synthesis from linoleic acid by desaturation and elongation, novel delta 5- and delta 6-desaturases are required. To isolate one of these, PCR-based cloning was used, and resulted in the isolation of a full-length cDNA coding for a putatively new desaturase. The deduced amino acid sequence has three domains: a N-terminal segment of about 100 amino acids, with no similarity to any sequence in the data banks, followed by a cytochrome b5-related region and a C-terminal sequence with low similarity (27% identify) to acyl-lipid desaturases. To elucidate the function of this protein, we disrupted its gene by transforming P. patens with the corresponding linear genomic sequence, into which a positive selection marker had been inserted. The molecular analysis of five transformed lines showed that the selection cartridge had been inserted into the corresponding genomic locus of all five lines. The gene disruption resulted in a dramatic alteration of the fatty acid pattern in the knockout plants. The large increase in linoleic acid and the concomitant disappearance of gamma-linolenic and arachidonic acid in all knockout lines suggested that the new cDNA coded for a delta 6-desaturase. This was confirmed by expression of the cDNA in yeast and analysis of the resultant fatty acids by GC-MS. Only the transformed yeast cells were able to introduce a further double bond into the delta 6-position of unsaturated fatty acids. To our knowledge, this is the first report of a successful gene disruption in a multicellular plant resulting in a specific biochemical phenotype.

Amino Acid Sequence↗
PubMed1997

Membrane lipids of Rhodopseudomonas viridis.

In search of the precyanobacterial origin of the typical thylakoid lipids found in cyanobacteria and chloroplasts, we analyzed the polar lipids of the anaerobic phototrophic bacterium Rhodopseudomonas viridis. Glycolipids (monogalactosyl-, digalactosyl- and glucuronosyl diacylglycerol), phospholipids (phosphatidyl choline, -ethanolamine, -glycerol and cardiolipin) and an ornithine lipid were isolated and identified by NMR (1H, 13C, 31P) and mass spectrometry. Positional distribution and pairing of fatty acids in molecular species show small, but significant differences between glyco- and phospholipids. In this context, a new enzymatic method is described for assigning the enantiomeric structure of the diacylglycerol moiety in glyco- and phospholipids. 14C-Labelling studies suggest that monogalactosyl diacylglycerol is formed by galactosylation of diacylglycerol as in chloroplasts and not by glucosylation followed by epimerization as in cyanobacteria. The two 1,6-linked galactopyranose residues of digalactosyl diacylglycerol are both in beta-linkage and thus differ from the corresponding chloroplast lipid with its alpha-beta-sequence. R. viridis does not contain the sulfolipid, and even phosphate starvation does not induce the synthesis of this most characteristic thylakoid lipid, which on the other hand is present in other anaerobic phototrophic bacteria.

Cardiolipins↗
PubMed1997

Brassica napus cDNAs encoding fatty acyl-CoA synthetase.

From a cDNA library of developing siliques of rapeseed (Brassica napus L.) we have isolated five full-length clones encoding polypeptides of the AMP-binding protein family. Two cDNAs encode fatty acyl-CoA synthetase activity (EC 6.2.1.3). The deduced polypeptides share about 52% identical amino acids. After expression in Escherichia coli the predicted enzymatic activity was confirmed by in vitro assays and product analysis. The enzymatic activity for one of the clones was characterized in detail by determination of the K(m) for oleic acid ( 10.4 microm) and the pH optimum (between 7 and 8). For the three additional clones no enzymatic activities could be demonstrated after expression in E. coli, although two of them exhibit similarity to either eukaryotic or prokaryotic acyl-CoA synthetases. The sequences are compared to a number of related expressed sequence tags from Brassica and Arabidopsis. Potential subcellular locations and functions of the deduced polypeptides within plant cells are discussed.

Adenosine Monophosphate↗
PubMed1997

UDP-glucose:sterol glucosyltransferase: cloning and functional expression in Escherichia coli.

Steryl glucosides are characteristic lipids of plant membranes. The biosynthesis of these lipids is catalyzed by the membrane-bound UDP-glucose:sterol glucosyltransferase (EC 2.4.1.173). The purified enzyme (Warnecke and Heinz, Plant Physiol 105 (1994): 1067-1073) has been used for the cloning of a corresponding cDNA from oat (Avena sativa L.). Amino acid sequences derived from the amino terminus of the purified protein and from peptides of a trypsin digestion were used to construct oligonucleotide primers for polymerase chain reaction experiments. Screening of oat and Arabidopsis cDNA libraries with amplified labeled DNA fragments resulted in the isolation of sterol glucosyltransferase-specific cDNAs with insert lengths of ca. 2.3 kb for both plants. These cDNAs encode polypeptides of 608 (oat) and 637 (Arabidopsis) amino acid residues with molecular masses of 66 kDa and 69 kDa, respectively. The first amino acid of the purified oat protein corresponds to the amino acid 133 of the deduced polypeptide. The absence of these N-terminal amino acids reduces the molecular mass to 52 kDa, which is similar to the apparent molecular mass of 56 kDa determined for the purified protein. Different fragments of these cDNAs were expressed in Escherichia coli. Enzyme assays with homogenates of the transformed cells exhibited sterol glucosyltransferase activity.

Amino Acid Sequence↗
PubMed1995

Accumulation of UDP-sulfoquinovose in a sulfolipid-deficient mutant of Rhodobacter sphaeroides.

The sulfolipid 6-sulfo-alpha-D-quinovosyl diacylglycerol is found in the photosynthetic membranes of all plants and most photosynthetic bacteria. Progress toward the elucidation of the pathway for sulfolipid biosynthesis has been slow in the past. However, the recent isolation of three genes of the photosynthetic bacterium Rhodobacter sphaeroides known to be involved in sulfolipid biosynthesis provides promising new opportunities. Two of the genes flank an open reading Rhodobacter sphaeroides known to be involved in sulfolipid biosynthesis provides promising new opportunities. Two of the genes flank an open reading frame predicted to encode a protein with amino acid sequence similarity to sugar nucleotide-dependent glycosyltransferases. The UDP-sulfoquinovose:diacylglcerol sulfoquinovosyltransferase thought to catalyze the last step of sulfolipid biosynthesis belongs to this group of glycosyltransferases. To test whether this open reading frame encodes the sulfoquinovosyltransferase of R. sphaeroides, it was inactivated by gene replacement avoiding polar mutagenesis. The resulting sulfolipid-deficient mutant defines a new gene, designated sqdD. Mutant cells grown in the presence of [35S]sulfate accumulate a water-soluble 35S-labeled compound. The purified compound was tentatively identified by co-chromatography with standards and enzymatic conversion as UDP-sulfoquinovose, the final precursor of sulfolipid biosynthesis. This result strongly suggests that the inactivation of sqdD causes a metabolic block in the last step of sulfolipid biosynthesis.

Bacterial Proteins↗
PubMed1995

A cytochrome-b5-containing fusion protein similar to plant acyl lipid desaturases.

The similarity between oleate and linoleate desaturase sequences from several plants was used to construct degenerate oligonucleotide primers for PCR experiments with cDNA transcribed from mRNA of ripening sunflower embryos. A DNA fragment was amplified and sequenced. Specific primers derived from this partial sequence were used for rapid amplification of the 3'- and 5'-ends of this cDNA. With appropriate primers derived from these sequences, a full-length clone of 1377 bp was amplified by PCR which, after sequencing, showed an open reading frame of 458 amino acids corresponding to a putative protein of about 52 kDa. Comparison with other desaturases showed the conserved three histidine boxes and the characteristic hydropathy profile of membrane-bound desaturases, but the amino acid identity was restricted to 18% and the N-terminal region was about 100 amino acids longer. This N-terminal extension showed high similarity with cytochrome b5 and, accordingly, the whole sequence can be considered as coding for a fusion protein between cytochrome b5 and a desaturase-like enzyme. Furthermore, we detected a similar cytochrome b5 fold in the previously sequenced delta 9 acyl-CoA desaturase from yeast, but in this enzyme it was located at the C-terminus. An alignment of these fusion proteins with other heme-binding proteins revealed desaturases to be novel members of the cytochrome b5 superfamily. A truncated DNA representing 366 bp of the 5'-end was amplified from the cDNA clone and expressed in Escherichia coli. The truncated cDNA coded for a soluble protein of about 12 kDa as shown by SDS/PAGE and N-terminal sequencing. The enriched recombinant protein exhibited redox absorbance spectra characteristic of plant microsomal cytochrome b5.

Amino Acid Sequence↗
PubMed1994

Purification and PCR-based cDNA cloning of a plastidial n-6 desaturase.

A plastidial membrane-bound n-6 desaturase from spinach (Spinacia oleracea) was purified from chloroplast envelope membranes by anion exchange, cation exchange and ferredoxin-affinity chromatography. The molecular mass of the protein was estimated by SDS-PAGE to be 40 kDa. The highest specific activity of the desaturase in the final preparation was 196 nmol/min per mg protein with free oleic acid as the substrate. The N-terminal amino acid sequence of the blotted protein was determined and used for the construction of a degenerated and inosine-containing oligonucleotide primer for PCR experiments with cDNA transcribed from leaf mRNA. A 3'-RACE experiment with this primer amplified a single band of 1500 bp that after sequencing showed an open reading frame of 382 amino acids corresponding to a protein of 43 kDa. The 5' end of the cDNA was amplified by a 5'-RACE experiment and isolated as a 500 bp fragment. Sequencing of this DNA revealed an additional 65 amino acids at the N-terminus of the native protein that are attributed to a plastidial leader peptide. With appropriate primers derived from these sequences a full-length clone was amplified by PCR and sequenced. Comparison of the plastidial oleate desaturase with the homologous enzyme from cyanobacteria showed about 50% amino acid homology. Comparison with other desaturases revealed three histidine boxes with the general sequence HXXXH that are highly conserved in all membrane-bound desaturases. These boxes might be involved in metal ion complexation required for reduction of oxygen.

Amino Acid Sequence↗
PubMed1994

The fadD gene of Escherichia coli K12 is located close to rnd at 39.6 min of the chromosomal map and is a new member of the AMP-binding protein family.

The fadD gene of Escherichia coli K12 was cloned and sequenced. The gene was identified by its ability to complement the corresponding mutant and by measuring the enzymatic activity after its expression in this mutant. The deduced polypeptide sequence exhibits similarity to other long chain acyl-CoA (coenzyme A) synthetases and a variety of other proteins, which together form a family of AMP-binding proteins. This family is extended by several new members and subdivided into four groups. fadD is assigned to a subgroup that does not include long chain acyl-CoA synthetases from eukaryotic organisms.

Adenosine Monophosphate↗
PubMed1993

In vivo desaturation of cis-delta 9-monounsaturated to cis-delta 9,12-diunsaturated alkenylether glycerolipids.

Plants convert lipid-bound cis-n-9 monoenoic to polyenoic fatty acid residues without involvement of corresponding CoA-thioesters. To provide additional evidence for this type of lipid-linked desaturation we incubated sn-1-O- and 2-O-(cis-9)octadecenylglycerol isomers with photoautotrophic cell cultures from tomato. After 14 days the fractions of phosphatidylcholine and monogalactosyldiacylglycerol were isolated and the incorporated glycerol ether backbones released by treatment with LiAlH4 (reduction of ester bonds) and short acid hydrolysis (cleavage of enol ether bonds). High performance liquid chromatography and mass spectroscopy of the products in appropriately derivatized form showed that the (cis-9)octadecenyl group in the sn-1 position of the phospholipid was nearly completely desaturated to a (cis-9,12)octadecadienyl residue having the same double bond arrangement as linoleic acid. In the galactolipid fraction the desaturation had progressed to octadecatrienyl residues. Similarly, the octadecenyl residue in the sn-2 position of the phospholipid was nearly completely desaturated to an octadecadienyl group. These results are unambiguous proof for lipid-linked desaturation by both microsomal and plastidial desaturase systems of plants.

Aluminum Compounds↗
PubMed1993

Isomeric sn-1-octadecenyl and sn-2-octadecenyl analogues of lysophosphatidylcholine as substrates for acylation and desaturation by plant microsomal membranes.

To provide supporting and independent evidence for lipid-linked desaturation of acyl groups in plant microsomal membranes, ether-analogous substrates were synthesized and used for in-vitro desaturation studies. The substrates included 1-O-(9-cis-octadecenyl)-sn-glycero-3-phosphocholine and 2-O-(9-cis-octadecenyl)-sn-glycero-3-phosphocholine as well as labelled 1-O-(9-cis-[9,10-3H2]octadecenyl)-sn-glycero-3-phosphocholine. In experiments with microsomal membranes from developing fruits of sunflower, it was shown that both isomeric alkenyl ether phospholipids were acylated with [14C]oleoyl-CoA and [14C]palmitoyl-CoA. In the presence of O2 and NADH, the oleoyl groups incorporated into both compounds, i.e. at the sn-1 and sn-2 positions of the glycerol backbone of the substrate, were desaturated to linoleoyl residues in similar proportions. Under the same conditions, an additional double bond, but not an enol-ether double bond, was introduced into the ether-linked side chain of acylated 1-O-(9-cis-[9,10-3H2]octadecenyl)-sn-glycero-3-phosphocholine. This represents the first demonstration of this type of desaturation with an alkenyl ether phospholipid and confirms previous conclusions that plants introduce second and further double bonds into lipid-linked acyl groups.

Acylation↗
PubMed1993

Direct desaturation of intact galactolipids by a desaturase solubilized from spinach (Spinacia oleracea) chloroplast envelopes.

In plants, polyenoic fatty acids are synthesized by desaturase enzymes which use acyl groups of membrane lipids as substrates. To provide direct 'in vitro' evidence for this reaction, we solubilized envelope membranes from spinach (Spinacia oleracea) chloroplasts with Triton X-100 to release a membrane-bound n-6 desaturase. In the presence of oxygen and reduced ferredoxin, the solubilized enzyme desaturated a variety of substrates, such as free oleic acid, free erucic acid, 1-oleoyl-sn-glycerol 3-phosphate and the three galactolipids 1-oleoyl-2-(7'-cis-hexadecenoyl)-3-beta-D-galactopyranosyl-sn-glycerol, 1,2-dioleoyl-3-beta-D-galactopyranosyl-sn-glycerol and the ether analogue 1,2-di-(9'-cis-octadecenyl)-3-beta-D-galactopyranosyl-sn- glycerol. The in vitro desaturation of these exogenously added complex lipids with ester- and ether-linked substrate chains is unambiguous evidence for lipid-linked desaturation. The enzyme measures the insertion of the new double bond from the methyl end and the existing (n-9)-cis-double bond of an appropriate acyl or alkyl chain. The distal part of the substrate group, normally the carboxy end of a fatty acyl residue, is of less importance and, in particular, its activation in thioester form is not required.

Chloroplasts↗
PubMed1992

Chilling sensitivity of Arabidopsis thaliana with genetically engineered membrane lipids.

Upon transfer of a genetically engineered Escherichia coli gene for glycerol-3-phosphate acyltransferase (plsB) to Arabidopsis thaliana (L.) Heynh., the gene is transcribed and translated into an enzymatically active polypeptide. This leads to an alteration in fatty acid composition of membrane lipids. From these alterations it is evident that the enzyme is located mainly inside the plastids. The amount of saturated fatty acids in plastidial membrane lipids increased. In particular, the fraction of high-temperature melting species of phosphatidylglycerol is elevated. These molecules are thought to play a crucial role in determining chilling sensitivity of plants. An increase in sensitivity could be observed in the transgenic plants during recultivation after chilling treatment. Implications for the hypothesis of phosphatidylglycerol-determined chilling sensitivity are discussed.

Arabidopsis↗
PubMed1991

Energetic coupling of Na-glucose cotransport.

(1) Energetic coupling in Na-linked glucose transport in renal brush border membrane vesicles has been studied in terms of various carrier models differing with respect to reaction order (random vs. ordered), and to rate limitation of steps within the routes of carrier-mediated solute transfer (translation across the membrane barrier vs. binding/release between carrier and bulk solution). (2) By computer simulation it was found that effective energetic coupling requires the leakage routes to be significantly, if not predominantly, rate-limited by their (barrier-crossing) translatory steps. This does not apply to the transfer route of the ternary complex, as coupling is possible whether or not this route is rate-limited by the translatory step. (3) The system transports glucose in the absence of Na+ (uniport) and the unidirectional flux is stimulated by unlabeled glucose on the trans side (negative tracer coupling). It is concluded that glucose binds to the carrier on either side without Na, as would be consistent with either a random system or one mode of ordered system with mirror symmetry (glucose binds before Na) but inconsistent with either mode of glide symmetry. The tracer coupling appears to indicate that the rate coefficient of carrier-mediated glucose transfer exceeds that of the empty carrier. (4) The Na-linked zero-trans flow of glucose in either direction is strongly trans-inhibited by Na. This consistent with a random system in which Na blocks or retards the translocation of the glucose-free carrier, thereby reducing 'slipping' through an internal leakage route. It is also consistent with the above mentioned ordered system, (i.e., in the absence of Na-transport without D-glucose) if it is assumed that trans Na interferes with the dissociation of the ternary complex, thereby slowing the release of glucose. (5) Minimum equilibrium exchange of glucose is stimulated in the presence of Na. This appears to indicate that Na expands the flow density of carrier-mediated glucose transfer. This expansion does not result from a 'velocity effect' (the ternary complex moving faster than the binary glucose carrier complex), as Na fails to stimulate maximum equilibrium exchange. It can instead be accounted for by an 'affinity effect' (the affinity of the carrier for glucose being increased by Na) as Na depresses the Michaelis constant of equilibrium exchange.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗