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K Keegstra

Publications and source records attributed to K Keegstra.

At least 73 records · Page 4Linked to original sources
PubMed1985

Final step of phosphatidic Acid synthesis in pea chloroplasts occurs in the inner envelope membrane.

The second enzyme of phosphatidic acid synthesis from glycerol-3-phosphate, 1-acylglycerophospate acyltransferase, was localized to the inner envelope membrane of pea chloroplasts. The activity of this enzyme was measured by both a coupled enzyme assay and a direct enzyme assay. Using the coupled enzyme assay, phosphatidic acid phosphatase was also localized to the inner envelope membrane, although this enzyme has very low activity in pea chloroplasts. The addition of UDP-galactose to unfractionated pea chloroplast envelope preparations did not result in significant conversion of newly synthesized diacylglycerol to monogalactosyldiacylglycerol. Thus, the envelope synthesized phosphatidic acid may not be involved in galactolipid synthesis in pea chloroplasts.

Journal Article↗
PubMed1984

Thermolysin is a suitable protease for probing the surface of intact pea chloroplasts.

Several proteases, i.e., pronase, a mixture of trypsin and chymotrypsin, and thermolysin were screened as potential surface probes of isolated intact pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Of these, only thermolysin met the criteria of a suitable probe. Thermolysin destroyed outer envelope polypeptides, but did not affect inner envelope polypeptides, envelope permeability properties or such chloroplast activities as metabolite transport and O(2) evolution.

Journal Article↗
PubMed1984

Characterization and localization of the ATPase associated with pea chloroplast envelope membranes.

Chloroplast envelope membranes isolated from Pisum sativum seedlings have been found to contain a Mg-ATPase activity (specific activity 50-175 nanomoles per minute per milligram protein). The ATPase had a broad pH optimum between 7.0 and 9.5. The activity was not inhibited by oligomycin, N,N'-dicyclohexylcarbodiimide, ouabain, or antibodies directed against chloroplast coupling factor 1; nor was the activity stimulated by monovalent cations. However, the ATPase was inhibited by vanadate, molybdate, and adenylyl imidodiphosphate.The ATPase hydrolyzed a broad range of nucleoside triphosphates, but did not hydrolyze ADP, AMP, or pyrophosphate. The K(m) for Mg-ATP was determined to be 0.2 millimolar. The ATPase was found to be distinct from ADPase and pyrophosphatase activities also present in pea envelope membranes.The ATPase was determined to be located on the inner membrane of the envelope after resolution of inner and outer membranes by sucrose density gradient centrifugation.

Journal Article↗
PubMed1983

An adenine nucleotide-phosphoenolpyruvate counter-transport system in C3 and C4 plant chloroplasts.

A rapid counter-exchange between ATP and phosphoenolpyruvate (PEP) has been demonstrated in pea and maize mesophyll chloroplasts. Chloroplasts preloaded with either [14C] ATP or [14C] PEP readily exchange the radioactive compound with the externally added anions, ATP or PEP, whereas, cold external Pi counter-transports only with internal [14C] PEP. Flooding the system with cold Pi, however, will significantly reduce the counter-transport of external cold PEP with internal [14C] ATP. This ATP-PEP exchange is also markedly decreased by lowering the incubation temperature. The results indicate that the ATP-PEP counter-exchange could represent a key transport system in plant chloroplasts and may be particularly important in the photosynthesis of C4 plants. Furthermore, they provide information required to elucidate the mechanism of the ATP-PEP counter-transport system.

Adenosine Triphosphate↗
PubMed1983

Galactosyltransferases involved in galactolipid biosynthesis are located in the outer membrane of pea chloroplast envelopes.

The galactosylation steps in the biosynthesis of galactolipids involve two different enzymes; a UDP-Gal:diacylglycerol galactosyltransferase and a galactolipid:galactolipid galactosyltransferase. Previous localization studies have shown that in spinach these enzymes are located in the chloroplast envelope. Our results with peas (Pisum sativum var Laxton's Progress No. 9) confirm these results and extend the localization by providing evidence that the galactosyltransferases are in the outer membrane of the envelope. The specific activity of UDP-Gal:diacylglycerol galactosyltransferase in outer membrane preparations was 6 to 10 times greater than that exhibited by inner membrane preparations. In addition, using quantitative sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it was possible to show that the UDP-Gal:diacylglycerol galactosyltransferase activity associated with inner membrane preparations could be accounted for by outer membrane contamination. It is concluded from these results that this enzyme is located predominantly, if not exclusively, in the outer membrane of the envelope. An analysis of the galactolipid products synthesized by the highly purified outer membrane showed that the galactolipid:galactolipid galactosyltransferase is also present, suggesting that this enzyme is also an outer membrane enzyme. The implication of these results is that the final assembly of galactolipids is carried out on the outer membrane of the chloroplast envelope.

Journal Article↗
PubMed1983

Acyl-CoA Synthetase Is Located in the Outer Membrane and Acyl-CoA Thioesterase in the Inner Membrane of Pea Chloroplast Envelopes.

Both acyl-CoA synthetase and acyl-CoA thioesterase activities are present in chloroplast envelope membranes. The functions of these enzymes in lipid metabolism remains unresolved, although the synthetase has been proposed to be involved in either plastid galactolipid synthesis or the export of plastid-synthesized fatty acids to the cytoplasm. We have examined the locations of both enzymes within the two envelope membranes of pea (Pisum sativum var Laxton's Progress No. 9) chloroplasts. Inner and outer envelope membranes were purified from unfractionated envelope preparations by linear density sucrose gradient centrifugation. Acyl-CoA synthetase was located in the outer envelope membrane while acyl-CoA thioesterase was located in the inner envelope membrane. Thus, it seems unlikely that the synthetase is directly involved in galactolipid assembly. Instead, its localization supports the hypothesis that it functions in the transport of plastid-synthesized fatty acids to the endoplasmic reticulum.

Journal Article↗
PubMed1983

Analysis of pea chloroplast inner and outer envelope membrane proteins by two-dimensional gel electrophoresis and their comparison with stromal proteins.

Analysis of inner and outer pea (Pisum sativum var. Laxtons Progress No. 9) chloroplast envelope membranes by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that, although the two membranes have distinct polypeptide compositions, there are several comigrating polypeptides in the two membrane fractions. To determine whether these comigrating polypeptides were identical by criteria other than molecular weight, the membrane proteins were analyzed by two-dimensional gel electrophoresis. The results demonstrated that an 86-kilodalton band found in both membranes represents at least two different polypeptides, one an outer membrane protein and the other an inner membrane protein. Several other polypeptide bands found in both membranes appear to be of stromal origin. Two of these polypeptides were shown to be the large and small subunits of ribulose 1,5-bisphosphate carboxylase. The large subunit was identified by two-dimensional electrophoresis of envelope membranes to which stromal proteins were added. Additionally, the large and small subunits of ribulose 1,5-bisphosphate carboxylase were immunologically identified using an electrophoretic transfer procedure coupled with an enzyme-linked immunosorbent assay. Various treatments, including sonication, resulted in no significant loss of the stromal polypeptides from the outer envelope membranes. Based on these results, it is suggested that the stromal proteins are not simply bound to the outer surface of the vesicles.

Journal Article↗
PubMed1982

Immunocytochemical localization of wheat germ agglutinin in wheat.

Immunocytological techniques were developed to localize the plant lectin, wheat germ agglutinin (WGA), in the tissues and cells of wheat plants. In a previous study we demonstrated with a radioimmunoassay that the lectin is present in wheat embryos and adult plants both in the roots and at the base of the stem. We have now found, using rhodamine, peroxidase, and ferritin-labeled secondary antibodies, that WGA is located in cells and tissues that establish direct contact with the soil during germination and growth of the plant In the embryo, WGA is found in the surface layer of the radicle, the first adventitious roots, the coleoptile, and the scutellum. Although found throughout the coleorhiza and epiblast, it is at its highest levels within the cells at the surface of these organs. In adult plants, WGA is located only in the caps and tips of adventitious roots. Reaction product for WGA was not visualized in embryonic or adult leaves or in other tissues of adult plants. At the subcellular level, WGA is located at the periphery of protein bodies, within electron-translucent regions of the cytoplasm, and at the cell wall-protoplast interface. Since WGA is found at potential infection sites and is known to have fungicidal properties, it may function in the defense against fungal pathogens.

Cell Wall↗
PubMed1982

Evidence that Envelope and Thylakoid Membranes from Pea Chloroplasts Lack Glycoproteins.

Envelope and thylakoid membranes from pea (Pisum sativum var. Laxton's Progress No. 9) chloroplasts were analyzed for the presence of glycoproteins using two different approaches. First, the sugar composition of delipidated membrane polypeptides was measured directly using gas chromatographic analysis. The virtual absence of sugars suggests that plastid membranes lack glycoproteins. Second, membrane polypeptides separated by sodium dodecyl sulfate gel electrophoresis were tested for reactivity toward three different lectins: Concanavalin A, Ricinus communis agglutinin, and wheat germ agglutinin. In each case, there was no reactivity between any of the lectins and the plastid polypeptides. Microsomal membranes from pea tissues were used as a positive control. Glycoproteins were readily detectable in microsomal membranes using either of the two techniques. From these results it was concluded that pea chloroplast membranes do not contain glycosylated polypeptides.

Journal Article↗
PubMed1981

Separation and characterization of inner and outer envelope membranes of pea chloroplasts.

A procedure for purifying the chloroplast envelope subfractionates it into two membrane fractions of comparable quantities. This procedure differs from previous ones in that the chloroplasts are ruptured by freezing and thawing in hypertonic medium rather than by osmotic shock. The two membrane fractions have qualitatively similar polar lipid compositions but differ in their content of individual lipids, specifically monogalactosyldiacylglycerol and phosphatidylcholine. The two fractions also differ in their constituent polypeptides and in their appearance when examined by electron microscopy. The light (density = 1.08 g/ml) and heavy (density = 1.13 g/ml) membrane fractions have been tentatively identified as the outer and inner envelope membranes, respectively.

Journal Article↗
PubMed1980

Lipopolysaccharide Composition of the Wilt Pathogen, Pseudomonas solanacearum: CORRELATION WITH THE HYPERSENSITIVE RESPONSE IN TOBACCO.

In the induction of the hypersensitive response (HR) in tobacco by Pseudomonas solanacearum, the recognition between host and pathogen is thought to involve an interaction between plant lectins and bacterial lipopolysaccharide (LPS). The LPS of a series of strains of P. solanacearum were examined to determine if there are structural differences that might account for the ability or inability of these strains to induce the hypersensitive response. Analysis of the components of LPS by gas chromatography indicates a clear difference in sugar composition between the HR-inducing and non-HR-inducing strains, especially in terms of the percentage of glucose, xylose and rhamnose. Sodium dodecyl sulfate polyacrylamide gel electrophoresis shows there are two distinct kinds of LPS, differing greatly in size, which correspond to rough and smooth LPS in other systems. In addition, a phage, CH154, was isolated which lyses non-HR-inducing bacteria and which is inactivated by LPS from these bacterial strains. Therefore, differences in LPS structure correlate strongly with host recognition of Pseudomonas solanacearum.

Journal Article↗
PubMed1980

Distribution of wheat germ agglutinin in young wheat plants.

A liquid phase, competition-binding radioimmunoassay for wheat germ agglutinin, with a detection limit of 10 nanograms, was developed in order to determine the distribution of this lectin in young wheat plants. Affinity columns for wheat germ agglutinin removed all antigenically detectable activity from crude extracts of wheat tissue; thus, the antigenic cross-reactivity detected by the assay possesses sugar-binding specificity similar to the wheat germ-derived lectin. The amount of lectin per dry grain is approximately 1 microgram, all associated with the embryo. At 34 days of growth, the level of lectin per plant was reduced by about 50%, with approximately one-third in the roots and two-thirds in the shoot. The data also indicate that actively growing regions of the plant (the bases of the leaves and rapidly growing adventitious roots) contain the highest levels of lectin. Half of the lectin associated with the roots could be solubilized by washing intact roots in buffer containing oligomers of N-acetylglucosamine, whereas the remainder is liberated only upon homogenization of the tissue.

Journal Article↗
PubMed1979

Carbohydrate structure of Sindbis virus glycoprotein E2 from virus grown in hamster and chicken cells.

Sindbis virus was used as a probe to examine glycosylation processes in two different species of cultured cells. Parallel studies were carried out analyzing the carbohydrate added to Sindbis glycoprotein E2 when the virus was grown in chicken embryo cells and BHK cells. The Pronase glycopeptides of Sindbis glycoprotein E2 were purified by a combination of ion-exchange and gel filtration chromatography. Four glycopeptides were resolved, ranging in molecular weight from 1,800 to 2,700. Structures are proposed for each of the four glycopeptides, based on data obtained by quantitative composition analyses, methylation analyses, and degradation of the glycopeptides using purified exo- and endoglycosidases. The largest three glycopeptides (S1, S2, and S3) have similar structures but differ in the extent of sialylation. All three contain N-acetylglucosamine, mannose, galactose, and fucose, in a structure similar to oligosaccharides found on other glycoproteins. Glycopeptide S1 has two residues of sialic acid, whereas glycopeptides S2 and S3 contain 1 and 0 residues of sialic acid, respectively. The smallest glycopeptide, S4, contains only N-acetyglucosamine and mannose, and is also similar to mannose-rich oligosaccharides found on other glycoproteins. Each of the complex glycopeptides (S1, S2, or S3) from virus grown in BHK cells is indistinguishable from the corresponding glycopeptides derived from virus grown in chicken cells. Glycopeptide S4 is also very similar in size, composition, and sugar linkages from virus derived from the two hosts. These results suggest that chicken cells and BHK cells have similar glycosylation mechanisms and glycosylate Sindbis glycoprotein E2 in nearly identical ways.

Amino Acids↗
PubMed1977

Comparison of the carbohydrate of Sinbis virus glycoproteins with the carbohydrate of host glycoproteins.

The carbohydrate portions of the Sindbis virus glycoproteins were compared with the carbohydrate portions of cell surface glycoproteins from uninfected host cells. Comparisons of the size of glycopeptides were made using gel filtrations. Comparisons of sugar linkages were made by methylation analysis. The conclusion was that the Sindbis carbohydrate is similar to a portion of the host carbohydrate. Thus, the Sindbis carbohydrate structures appear to be structures normally made in the uninfected host cell, but which are added to the Sindbis glycoproteins in virus-infected cells.

Animals↗