Regulation of phospholipid biosynthesis in Saccharomyces cerevisiae by inositol. Inositol is an inhibitor of phosphatidylserine synthase activity

The addition of inositol to the growth medium of Saccharomyces cerevisiae resulted in rapid changes in the rates of phospholipid biosynthesis. The partitioning of the phospholipid intermediate CDP-diacylglycerol was shifted to phosphatidylinositol at the expense of phosphatidylserine and its derivatives phosphatidylethanolamine and phosphatidylcholine. Serine at 133-fold greater concentrations than that of inositol shifted the partitioning of CDP-diacylglycerol to phosphatidylserine at the expense of phosphatidylinositol but to a much lesser degree. Kinetic experiments with pure phosphatidylserine synthase and phosphatidylinositol synthase indicated that the partitioning of CDP-diacylglycerol between phosphatidylserine and phosphatidylinositol was not governed by the affinities both enzymes have for their common substrate CDP-diacylglycerol. Instead, the main regulation of phosphatidylinositol and phosphatidylserine synthesis was through the exogenous supply of inositol. The Km of inositol (0.21 mM) for phosphatidylinositol synthase was 9-fold higher than cytosolic concentration of inositol (24 microM). The Km of serine (0.83 mM) for phosphatidylserine synthase was 3-fold below the cytosolic concentration of serine (2.6 mM). Therefore, inositol supplementation resulted in a dramatic increase in the rate of phosphatidylinositol synthesis, whereas serine supplementation resulted in little affect on the rate of phosphatidylserine synthesis. Inositol also contributed to the regulation of phosphatidylinositol and phosphatidylserine synthesis by having a direct affect on phosphatidylserine synthase activity. Kinetic experiments with pure phosphatidylserine synthase showed that inositol was a noncompetitive inhibitor of the enzyme with a Ki of 65 microM.     

Stereocontrolled routes to cis-hydroxyamino sugars, part VII: synthesis of daunosamine and ristosamine

The nitrogen of an allylic amine can serve as the fulcrum for stereocontrolled delivery of oxygen to an adjacent trigonal site, and cis-hydroxyamino sugars can thus be prepared. Methods for achieving the complementary procedure, namely, control of the delivery of nitrogen to an adjacent site by an allylic oxygen, are described. For example, treatment of methyl 2,3,6-trideoxy-- -erythro-hex-2-enopyranoside with trichloroacetonitrile gave an imidate ester which reacted with iodonium dicollidine perchlorate to give 2-trichloromethyl-(methyl 2,3,4,6-tetradeoxy-2-iodo-- -altropyranoside)-[3,4-d]-2-oxazoline. Exhaustive reductive dehalogenation of this product followed by hydrolysis led to methyl N-acetyl-- -ristosaminide. An analogous series of reactions was used to prepare the corresponding daunosaminide.     

Purification and characterization of a human pituitary growth factor

A growth factor has been purified to homogeneity from human pituitary glands. The pituitary growth factor (PGF) is trypsin-sensitive and acid- and heat-labile and has a molecular weight of 18,000 and an isoelectric point of 7.5. PGF was purified by heparin and copper affinity chromatography followed by carboxymethylcellulose 52. The amino-terminal amino acid sequence of PGF was established as PALPEXGGXGA and is identical with that of basic fibroblast growth factor at the identified amino acid residues. PGF was mitogenic for rabbit fetal chondrocytes and bovine corneal endothelial cells in the range of 0.015-15 ng mL-1. Heparin alone at low concentrations (0.5 microgram mL-1) was found to be weakly mitogenic for rabbit fetal chondrocytes. In combination with PGF a marked increase in cell growth was observed, which was inhibited by protamine sulfate. These data demonstrate the presence of a potent mitogen in human pituitaries that is structurally related to basic fibroblast growth factor and synergizes with heparin to promote cell growth.     

Superoxide Radical-Mediated Alteration of Synaptosome Membrane Structure and High-Affinity γ-[14C]Aminobutyric Acid Uptake

Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.     

Increased Acetylcholine Synthesis and Release in Brains of Cats with GM1 Gangliosidosis

Cholinergic processes were measured in motor cortex, hippocampus, and striatum of cats in the terminal stages of GM1 gangliosidosis and compared to those of control cats. The greatest difference observed was elevation in the rate of K+-stimulated release of acetylcholine (ACh) from brain slices prepared from affected cats. The K+-stimulated release of endogenous ACh was increased by 31-43% and of newly synthesized ACh by 19-80% in brain slices from different brain regions. All regions that were examined were affected but the greatest effects occurred in cortex. The rate of synthesis of ACh was elevated in cortical and hippocampal slices. Choline acetyltransferase activity in brain regions of cats with GM1 gangliosidosis was not significantly different from that in controls, whereas high-affinity choline transport in cortical synaptosomes was elevated. Muscarinic receptor binding sites were reduced in the cortex, hippocampus, and striatum of GM1 mutant cats, whereas the apparent affinity was not altered. These results indicate that there are major alterations of cholinergic function in the brains of cats with GM1 gangliosidosis.     

Photoaffinity labeling of the tetrabenazine binding sites of bovine chromaffin granule membranes

An azido derivative of tetrabenazine, a specific inhibitor of the monoamine carrier of chromaffin granule membranes, has been synthesized. In the dark, this compound, 3H-labeled N-(3-isobutyl-9,10-dimethoxy-1,2,3,4,6,7-hexahydro-11bH-benzo [a]quinolizin-2-yl)-4-[(4-azido-2-nitrophenyl)amino]butanamide+ ++ ([3H]TBA), bound reversibly to purified chromaffin granule membranes. Centrifugation through SP-Sephadex columns was used to separate bound and free [3H]TBA. This technique gave low levels of nonspecific binding and allowed recovery of [3H]TBA-membrane complexes. Scatchard analysis of the data indicated one class of sites with an equilibrium dissociation constant KD of 50 nM and a density of sites of 40-50 pmol/mg of protein, consistent with reported densities of reserpine and dihydrotetrabenazine binding sites. Competition experiments showed that TBA and tetrabenazine bound to the same site. Irradiation at 435 nm of [3H]TBA-membrane mixtures induced some irreversible binding of the probe to membranes. After irreversible binding of TBA, the number of dihydrotetrabenazine binding sites was decreased, indicating that the probe was covalently bound to the monoamine carrier. [3H]TBA-membrane complexes isolated by centrifugation through SP-Sephadex columns were irradiated, and their radioactivity was analyzed by electrophoresis on sodium dodecyl sulfate/polyacrylamide gels. A polypeptide with a molecular weight of 70 000 was labeled. This polypeptide was different from dopamine beta-hydroxylase, and it was not adsorbed on concanavalin A-Sepharose. It is proposed that the monoamine carrier of chromaffin granule membrane has an oligomeric structure, involving a 45K subunit [Gabizon, R., Yetinson, T., & Schuldiner, S. (1982) J. Biol. Chem. 257, 15145] and a 70K subunit.     

Spectroscopic studies of protein-heme interactions accompanying the allosteric transition in methemoglobins

Resonance Raman, optical absorption, and circular dichroism spectroscopic techniques have been used to examine the effect of the addition of inositol hexaphosphate (IHP) to a series of carp and human methemoglobin derivatives. Markers of spin equilibrium in the high-frequency region (1450-1650 cm-1) of the resonance Raman spectrum yield high/low-spin ratios consistent with direct magnetic susceptibility measurements. Changes in the low-frequency region (100-600 cm-1) of the resonance Raman spectrum appear to correlate with the quaternary structure transition. Changes in the ultraviolet absorption spectra and the circular dichroism spectra also appear to be related to the quaternary structure change. By using the resonance Raman spin markers, we find that those derivatives of carp methemoglobin which are in spin equilibrium have a larger ratio of high-spin to low-spin populations than the corresponding derivatives of human methemoglobin. Upon the addition of IHP to the methemoglobins the spin equilibrium is shifted toward a larger high-spin population. This change in equilibrium is larger for the carp protein than for the human protein. We obtain an IHP-induced change in the free energy difference between the high-spin and low-spin states of 300 cal/mol for those human methemoglobins in which a quaternary structure change occurs and 600 cal/mol for carp methemoglobins. Our data are consistent with a quaternary structure change induced by IHP in all the carp methemoglobins studied (F-, H2O, SCN-, NO2-, N3-, and CN-) and in the F-, H2O, and SCN- derivatives of the human protein but not in the NO2-, N3-, and CN- derivatives. The Fe-CN stretching mode has been identified by isotopic substitution and found to be unchanged in frequency in carp CN- metHb when the quaternary structure is changed. On the basis of our results we conclude that the protein forces at the heme due to the addition of IHP do not significantly affect the position of the iron atom with respect to the heme plane. Rather, the changes in spin equilibrium may be caused by protein-induced changes in the orientation of the proximal histidine or tertiary structure changes in the heme pocket which affect the porphyrin macrocycle. Either of these changes, or a combination thereof, leads to changes in the iron d orbital energies and concomitant changes in the spin equilibrium.     

Acid phosphatase localization in the digestive glands of Dionaea muscipula Ellis flytraps

The intracellular localization of acid phosphatases in stimulated digestive glands of Dionaea flytraps has been studied to provide evidence for the route taken by this enzyme during secretion. Previous studies have either included or excluded a role for the dictyosomes in this pathway. Both p-nitrophenyl phosphate and beta-glycerophosphate were used as substrates, and both gave similar localization patterns. Unstimulated glands contained little phosphatase activity in the endomembrane system, whereas 24 and 48 hr after stimulation, heavy deposits of lead were located in the endoplasmic reticulum cisternae, including the nuclear envelope, the dictyosome cisternae, and secretory vesicles. Since dictyosome activation, as judged by the presence of secretory vesicles in the cytoplasm, also coincides with gland stimulation, we conclude that secretion of the hydrolase enzymes occurs via this route and not, as suggested elsewhere, via direct endoplasmic reticulum to plasma membrane connections.     

The impact of zooplankton size structure on phosphorus cycling in field enclosures

Plastic bag field enclosures (1570 1) were used to investigate the relationship between zooplankton size distributions and the turnover rate of pelagic phosphorus. Water within the enclosures was filtered to simulate either low (200 µm mesh net) or high (80 µm) planktivory. The bags were then spiked with radiolabelled phosphorus and sampled 7 times during the following 24 h period. Zooplankton communities dominated by smaller size classes cycled more phosphorus than those composed of larger species. These experiments reveal that higher trophic level interactions, such as size selective predation, may have a significant impact upon nutrient regeneration rates and, hence, primary production.     

Cleavage of Rabbit Myelin Basic Protein by Plasmin: Isolation and Identification of the Major Products

Rabbit myelin basic protein (BP) was subjected to partial cleavage with plasmin, and 15 cleavage products were isolated by a combination of gel filtration and ion-exchange chromatography. Their identification was achieved by amino acid analysis and tryptic peptide mapping, supplemented in some instances by carboxy-terminal analyses with carboxypeptidases A, B, and Y and amino-terminal analyses with dipeptidyl aminopeptidase I. The results showed that major plasmic cleavage sites included the Lys89-Asn90, Lys133-Ser134, and Lys153-Leu154 bonds. Cleavages also occurred at the Arg31-His32, Lys53-Arg54, and Arg25-His26 bonds, but these appeared to be less extensive. A large number of additional peptides were produced in relatively low yield. The smaller of these were isolated from heterogeneous fractions by high-voltage electrophoresis-TLC. Amino acid analysis of these peptides showed that minor cleavage sites included the Arg9-His10, Lys13-Tyr14, Lys103-Gly104, Lys137-Gly138, Lys140-Gly141, and Arg160-Ser161 bonds. In spite of a lower selectivity toward peptide bonds in BP as compared with pepsin, cathepsin D, and thrombin, plasmin has the advantage over the former proteinases in that it does not cleave at or near the Phe44-Phe45 bond. Instead it cleaves at the Arg31-His32 and Lys53-Arg54 bonds, thus preserving the entire hydrophobic sequence Ile-Leu-Asp-Ser-Ile-Gly-Arg-Phe-Phe as well as short sequences to either side.