Regulation of eukaryotic phospholipid metabolism

Phospholipids have diverse and critical roles in cellular metabolism and function. Questions about the mechanisms of regulation of phospholipid synthesis are being investigated with a variety of systems and approaches. For example, the yeast Saccharomyces cerevisiae is an organism in which both biochemical and genetic analyses are used. Biochemical approaches have yielded considerable information on the regulatory properties of enzymes of phospholipid biosynthesis. Studies of the activity of purified phosphatidylserine synthase have suggested how that enzyme is influenced by membrane phospholipids in the cell. The enzyme that regulates mammalian phosphatidylcholine biosynthesis, CTP:phosphocholine cytidylyltransferase, is also influenced by phospholipids. In addition, the activity of this enzyme often correlates with its translocation to membranes. The location of such enzymes in the cell is of particular interest in light of the possibility that the enzymatic reactions may be efficiently coupled in vivo. Techniques to render cultured cells permeable to phosphorylated molecules indicated that the enzymes of phosphatidylcholine biosynthesis may exist in an organized compartment so that the precursors of phosphatidylcholine are efficiently channeled through the pathway. To ask how phospholipids are transported in the cell, a combined biochemical and genetic approach has been used. These studies have revealed that the phosphatidylinositol/phosphatidylcholine transfer protein, considered to mediate intracellular phospholipid transfer, is a critical component of the secretory pathway for proteins. These results have allowed formulation of a number of new questions on the regulation of phospholipid metabolism and its relationship to general membrane processes.     

DNA diagnosis for hereditary cerebral hemorrhage with amyloidosis (Dutch type)

Hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D) is tightly linked to the Alzheimer amyloid precursor protein gene on chromosome 21, which codes for the amyloid beta-protein. A point mutation detected at position 1852 of the amyloid precursor protein gene in four HCHWA-D patients was hypothesized to be the basic defect. This study proves that 22 HCHWA-D patients from three pedigrees all carry this point mutation, whereas the mutation is absent in escapees from the HCHWA-D families as well as in randomly selected Dutch individuals. A mutation-specific oligonucleotide is now available for the confirmation of the HCHWA-D diagnosis. Therefore, presymptomatic testing and prenatal evaluation of individuals at risk in the HCHWA-D families is now feasible.     

Induction of embryogenic Triticum aestivum L. calli. I. Quantification of genotype and culture medium effects

Somatic embryo (embryoid) formation from immature-embryo-derived calli was quantified in replicated experiments involving 10 Triticum aestivum L. genotypes. Several published media formulations, which had previously been optimized for wheat tissue culture, were tested for each genotype. Embryos from each plant were randomly assigned to each medium. Percentage precocious germination of immature embryos and mean percentage scutellar callus per explant were recorded. Embryoids per callus were determined by microscopic examination at 28 and 56 days. There were highly significant differences among genotypes, media, and individual plants from which explants were taken. A medium based on double the Murashige and Skoog (MS) inorganic salt concentration was significantly better than other media. Inclusion of all MS vitamins appeared essential for optimal response. Two genotypes were tested in a second experiment where both 3,6-dichloro-o-anisic acid (9.05 M) and 6-furfurylaminopurine (0.46 M) were substituted for 2,4-dichlorophenoxyacetic acid (4.52 M) in either double or normal MS medium. This substitution significantly increased embryoid formation at 28 days. Additions of either 6-furfurylaminopurine or coconut water increased precocious germination of both embryo explants and embryoids.This study was supported in part by NASA-Ames Cooperative Agreement No. NCC2-139. Contribution of the Utah Agricultural Experiment Station, Utah State University, Logan, UT, Journal Paper No. 3358.     

Induction of embryogenicTriticum aestivum L. calli. II. Quantification of organic addenda and other culture variable effects

Nine experiments were conducted to determine effects of various culture medium addenda on induction of embryogenic calli from immature embryos of a responsiveTriticum aestivum L. genotype (PCYT 10). Effects were quatified by counting somatic embryos (embryoids) per callus. Optimal auxin concentrations to induce and maintain somatic embryogenesis were 3.62 M 2,4-dichlorophenoxyacetic acid (2,4-D) or 9.05 M 3,6-dichloro-o-anisic acid (dicamba). In general, dicamba permitted formation of significantly more embryoids than 2,4-D. Kinetin (6-furfurylaminopurine) at 2.56 M or 4.65 M significantly increased percentage scutellar callus when added to 2,4-D or dicamba-containing medium, respectively. Kinetin at 4.65 M signficantly increased the numbers of embryoids formed when added to medium containing either synthetic auxin. Significantly fewer embryoids formed when cultures were incubated under diffuse light (16-h photoperiod). Casein hydrolysate (200 mgl^-1) or L-arginine (0.23 mM) had no effect on numbers of embryoids formed, whereas L-tryptophan (0.20 mM) enhanced such formation with 2,4-D and decreased such formation with dicamba. Two additional experiments generally demonstrated that response to auxin source in the genotypes ND 7532, PCYT 20, Yaqui 50, and Oasis was similar to that in PCYT 10. The higher molar concentration of dicamba required to induce embryogenic callus coupled with more evident embryoid precocious germination and a more rapid rate of tissue necrosis upon extended incubation without subculture suggests that dicamba is metabolized more rapidly than 2,4-D inT. aestivum callus cultures.This study was supported by NASA-Ames Cooperative Agreement No. NCC2-139. Contribution of the Utah Agricultural Experiment Station, Utah State University, Logan, UT, Journal Paper No. 3359.     

Phospholipids chiral at phosphorus. Steric course of the reactions catalyzed by phosphatidylserine synthase from Escherichia coli and yeast

The steric courses of the reactions catalyzed by phosphatidylserine (PS) synthase from Escherichia coli and yeast were elucidated by the following procedure. RP and SP isomers of 1,2-dipalmitoyl-sn-glycero-3-[17O,18O]phosphoethanolamine ([17O,18O]DPPE) were synthesized with slight modification of the previous procedure [Bruzik, K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754] and converted to (RP)- and (SP)-1,2-dipalmitoyl-sn-glycero-3-[16O,17O,18O]phosphoric acid ([16O,17O18O]DPPA), respectively, by incubating with phospholipase D. Condensation of [16O,17O,18O]DPPA with cytidine 5'-monophosphomorpholidate in pyridine gave the desired substrate for PS synthase, [17O,18O]cytidine 5'-diphospho-1,2-dipalmitoyl-sn-glycerol ([17O,18O]CDP-DPG), as a mixture of several isotopic and configurational isomers. Incubation of [17O,18O]CDP-DPG with a mixture of L-serine, PS synthase (which converted [17O,18O]CDP-DPG to phosphatidylserine), and PS decarboxylase (which catalyzes decarboxylation of phosphatidylserine) gave [17O,18O]DPPE. The configuration and isotopic enrichments of the starting [17O,18O]DPPE and the product were analyzed by 31P NMR following trimethylsilylation of the DPPE. The results indicate that the reaction of E. coli PS synthase proceeds with retention of configuration at phosphorus, which suggests a two-step mechanism involving a phosphatidyl-enzyme intermediate, while the yeast PS synthase catalyzes the reaction with inversion of configuration, which suggests a single-displacement mechanism. Such results lend strong support to the ping-pong mechanism proposed for the E. coli enzyme and the sequential Bi-Bi mechanism proposed for the yeast enzyme, both based on previous isotopic exchange experiments.     

Saccharomyces cerevisiae mutant with a partial defect in the synthesis of CDP-diacylglycerol and altered regulation of phospholipid biosynthesis.

A Saccharomyces cerevisiae mutant (cdg1 mutation) was isolated on the basis of an inositol excretion phenotype and exhibited pleiotropic deficiencies in phospholipid biosynthesis. Genetic analysis of the mutant confirmed that the cdg1 mutation represents a new genetic locus and that a defect in a single gene was responsible for the Cdg1 phenotype. CDP-diacylglycerol synthase activity in mutant haploid cells was 25% of the wild-type derepressed level. Biochemical and immunoblot analyses revealed that the defect in CDP-diacylglycerol synthase activity in the cdg1 mutant was due to a reduced level of the CDP-diacylglycerol synthase Mr-56,000 subunit rather than to an alteration in the enzymological properties of the enzyme. This defect resulted in a reduced rate of CDP-diacylglycerol synthesis, an elevated phosphatidate content, and alterations in overall phospholipid synthesis. Unlike wild-type cells, CDP-diacylglycerol synthase was not regulated in response to water-soluble phospholipid precursors. The cdg1 lesion also caused constitutive expression of inositol-1-phosphate synthase and elevated phosphatidylserine synthase. Phosphatidylinositol synthase was not affected in the cdg1 mutant.     

Regulation of phosphatidylinositol kinase activity in Saccharomyces cerevisiae.

The effects of growth phase and carbon source on membrane-associated phosphatidylinositol kinase in cell extracts of Saccharomyces cerevisiae were examined. Phosphatidylinositol kinase activity increased 2- and 2.5-fold in glucose- and glycerol-grown cells, respectively, in the stationary phase as compared with the exponential phase of growth. The increase in phosphatidylinositol kinase activity in the stationary phase of growth correlated with an increase in the relative amounts of phosphatidylinositol 4-phosphate, the product of the reaction. The increase in phosphatidylinositol kinase activity was not due to the presence of water-soluble effector molecules in cell extracts as indicated by mixing experiments. Phosphatidylinositol kinase activity decreased in cell extracts of exponential-phase cells preincubated under phosphorylation conditions which favor cyclic AMP-dependent protein kinase activity. Phosphatidylinositol kinase activity was not affected in cell extracts of stationary-phase cells preincubated under phosphorylation conditions.     

Purification and characterization of phosphatidate phosphatase from Saccharomyces cerevisiae

Membrane-associated phosphatidate phosphatase (EC 3.1.3.4) was purified 9833-fold from the yeast Saccharomyces cerevisiae. The purification procedure included sodium cholate solubilization of total membranes followed by chromatography with DE53, Affi-Gel Blue, hydroxylapatite, Mono Q, and Superose 12. The procedure resulted in the isolation of a protein with a subunit molecular weight of 91,000 that was apparently homogeneous as evidenced by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Phosphatidate phosphatase activity was associated with the purified 91,000 subunit. The molecular weight of the native enzyme was estimated to be 93,000 by gel filtration chromatography with Superose 12. Maximum phosphatidate phosphatase activity was dependent on magnesium ions and Triton X-100 at pH 7. The Km value for phosphatidate was 50 microM, and the Vmax was 30 mumol/min/mg. The turnover number (molecular activity) for the enzyme was 2.7 x 10(3) min-1 at pH 7 and 30 degrees C. The activation energy for the reaction was 11.9 kcal/mol, and the enzyme was labile above 30 degrees C. Phosphatidate phosphatase activity was sensitive to thioreactive agents. Activity was inhibited by the phospholipid intermediate CDP-diacylglycerol and the neutral lipids diacylglycerol and triacylglycerol.     

Neonatal suppression with anti-Ia antibody. III. In vivo responses to the type 2 antigen TNP-Ficoll

We studied the response to thymus-independent type 2 (type 2) Ag in mice suppressed from birth with anti-Ia antibody. Although these mice have significantly reduced numbers of surface IgM+ cells and reduced or absent levels of Ia-restricted Th cell activity, their IgM antibody response to the type 2 Ag TNP-Ficoll was unaffected whereas that to the prototypic thymus-dependent Ag SRBC was predictably eliminated. These data suggest that an in vivo antibody response can be made to type 2 Ag in the absence of Ia-dependent cellular interactions. The surface IgM+IgD-Ia- B cells that are found in the anti-Ia antibody-suppressed mouse may represent an expanded population of Ia-independent, type 2 Ag-sensitive B cells normally present as a smaller proportion of the splenic lymphocyte population. Thymus-dependent responses, which have been shown to have an absolute requirement for an Ia-dependent interaction, are absent in these animals.     

Radioactive labeling of a natural assemblage of marine sedimentary bacteria and microalgae for trophic studies: An autoradiographic study

Autoradiography was used to examine critical questions for trophic studies concerning the uptake of radioactive tracers by a natural assemblage of sedimentary microorganisms. Labeled organic substrates ([³H]-acetate and [³H]-thymidine) were taken up only by heterotrophic bacteria, and [¹⁴C]-bicarbonate was taken up only by microalgae. Only approximately 2% of the bacterial assemblage took up detectable quantities of either [³H]-acetate or [³H]-thymidine, regardless of whether labeled substrates were delivered to sediments via slurries or by injection with a microliter syringe. Significantly more diatoms were labeled when [¹⁴C]-bicarbonate was delivered to sediments by the injection method (75%) as compared to the slurry method (50%). These results indicate that radio-active tracers can be used in natural sediments to selectively label potential microbial food of invertebrate grazers. Only a small proportion of bacteria, however, may actually use a labeled substrate, which introduces a large uncertainty into the conversion of radioactivity in grazers to the number of bacteria consumed. Finally, the use of disruptive methods (e.g., slurries) to deliver labels to sediments does not increase the proportion of microorganisms that become labeled. Thus, given the variety of artifacts that may be associated with the use of sediment slurries, it is probably advisable to use nondisruptive methods to deliver substrates to sediments.