Physiological roles of animal succinate thiokinases Specific association of the guanine nucleotide-linked enzyme with haem biosynthesis

The discovery of two distinct succinate thiokinases in mammalian tissues, one (G-STK) specific for GDP/GTP and the other (A-STK) for ADP/ATP, poses the question of their differential metabolic roles. Evidence has suggested that the A-STK functions in the citric acid cycle in the direction of succinyl-CoA breakdown (and ATP formation) whereas one role of the G-STK appears to be the re-cycling of succinate to succinyl-CoA (at the expense of GTP) for the purpose of ketone body activation. A third metabolic participation of succinyl-CoA is in haem biosynthesis. This communication shows that in chemically induced hepatic porphyria, when the demand for succinyl-CoA is increased, it is the level of G-STK only which is elevated, that of A-STK being unaffected. The results implicate G-STK in the provision of succinyl-CoA for haem biosynthesis, a conclusion which is further supported by the observation of a high G-STK/A-STK ratio in bone marrow.     

Studies on well-coupled Photosystem I-enriched subchloroplast vesicles — energy-dependent switching between two different active states of the proton-translocation adenosine triphosphatase

Single-turnover flash-induced ATP synthesis coupled to natural cyclic electron flow in Photosystem I-enriched subchloroplast vesicles (from spinach) was continuously followed by the luciferin-luciferase luminescence. The ATP yield per flash was maximal (1 ATP per s per 1000 Chl) around a flash frequency of 0.5–2 Hz. It decreased both at lower and higher flash frequencies. The decrease at high flash frequency was due to limitation by the electron-transfer rate, while the decrease at low flash frequency was directly due to intrinsic properties of the ATPase itself. Carbonylcyanide-p-trifluoromethoxyphenylhydrazone (FCCP) decreased the yield at low frequency more than at high frequency. The same behaviour was observed if electron transfer was artificially mediated by pyocyanin. If the ADP concentration was increased from 40 to at least 80 μM, or if the vesicles were preincubated with 5 mM dithiothreitol (DTT), the decrease of the yield at flash frequencies below 0.5 Hz was no longer observed. Incubation with DTT increased the rates of ATP hydrolysis and synthesis at any flash frequency. The decrease of the yield could be elicited again by addition of 50 nM FCCP. It is concluded that at low levels of the protonmotive force (Δ gm_H⁺), the ATPase is converted into an active ATP-hydrolyzing state in which ATP synthesis activity is decreased due to a decreased affinity towards ADP and/or to a decreased release of newly synthesized ATP, that can be cancelled by increasing the ADP concentration or by addition of DTT in the absence of uncoupler.     

Highly selective affinity labelling of RNA polymerase B (II) from wheat germ

DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(β-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH₄. Reaction of the modified enzyme with [-³²P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with M_r 140000 by covalently linked ApU. Labelling was inhibited by 1μg/ml -amanitin.     

Developmental Pattern for Phosphatidylserine Decarboxylase in Rat Brain

In adult rats, a significant portion of brain ethanolamine glycerophospholipids are synthesized by a pathway involving phosphatidylserine decarboxylase, a mitochondrial enzyme. We have now examined whether this enzyme plays a particularly prominent role during development. Activities for both phosphatidylserine decarboxylase and succinate dehydrogenase (another mitochondrial enzyme) were determined in brain homogenates from rats 5 days of age to adulthood. Succinate dehydrogenase activity, expressed on a per unit brain protein basis, increased markedly during development. This pattern has been reported previously and is as expected from the postnatal increase in oxidative metabolism. In contrast, phosphatidylserine decarboxylase activity decreased 40% from 5 to 30 days of age. The apparent Km for brain phosphatidylserine decarboxylase was 85 microM in both young (8- and 20-day-old) and adult animals. Parallel studies in vivo were carried out to determine the contribution of the phosphatidylserine decarboxylase pathway, relative to pathways utilizing ethanolamine directly, to the synthesis of brain ethanolamine glycerophospholipids. Animals were injected intracranially with a mixture of L-[G-3H]serine and [2-14C]ethanolamine and incorporation into the base moieties of the phospholipids determined. The 3H/14C ratio of ethanolamine glycerophospholipids decreased about 50% during development. Our studies in vitro and in vivo both suggest that phosphatidylserine decarboxylase plays a significant role in the synthesis of brain ethanolamine glycerophospholipids at all ages, although it is relatively more prominent early in development.     

The frequency of mutants in human fibroblasts UV-irradiated at various times during S-phase suggests that genes for thioguanine- and diphtheria toxin-resistance are replicated early

Human cells deficient in rate of excision repair of DNA damage induced by UV-radiation, i.e., xeroderma pigmentosum (XP) cells, are much more sensitive to the mutagenic effect of UV than are cells from normal persons. The lower frequency of mutants in the latter cells has been attributed to the fact that, unlike XP cells, they excise most of the potentially mutagenic lesions before these can be converted into mutations. If semi-conservative DNA synthesis on a template still containing unexcised lesions is responsible for introducing mutations and if replication of the gene of interest, e.g., hypoxanthine (guanine)phosphoribosyltransferase (HPRT) for thioguanine resistance or the elongation factor 2 (EF-2) for diphtheria toxin resistance, occurs at a particular time during S-phase, it should be possible to shorten the time available for such repair by synchronizing cells and irradiating them just as the gene is to be replicated. The predicted result would be a much higher frequency of mutants at one part in the S-phase than at other times. To test this, cells were synchronized using the alpha-polymerase inhibitor aphidicolin, which blocks cells at the G1/S border. Autoradiography, cytofluorimetry, and incorporation of tritiated thymidine studies showed that DNA synthesis started immediately after release from aphidicolin and was completed in 8-10 h. Cells irradiated with 6 J/m2 at various times post-release were assayed for survival and mutations. The frequency of thioguanine- or diphtheria toxin-resistant cells in the population was highest in cells irradiated during the first fifth of the S-phase, i.e., 0-1.5 h post-release. It was significantly lower in cells irradiated at later times. In contrast, UV-induced cytotoxicity showed no significant time dependence during S-phase. These data suggest that the HPRT and EF-2 genes are replicated early in S-phase.     

Translocation of photoassimilate from leaves of two popyploid genotypes of tall fescue differing in photosynthetic rates

The photosynthetic rate of a decaploid genotype (1-16-2) of tall fescue ( Festuca arundinacea Schreb.) is about twice that of a common hexaploid genotype (V6-802) (Plant Physiol. 72: 16–21, 1983). Translocation of photosynthate out of the leaves is a possible means of regulating carbon assimilation. To evaluate this possibility, we have examined a) translocation velocity, b) time course of translocation from leaves, c) photoassimilate partitioning pattern into whole plants in pulse and chase experiments, and d) interveinal distances between two ploidy genotypes. Most of the ¹⁴C accumulated in sucrose, and the labelled carbon moved down the leaf blades at similar velocities (6 to 10 cm h⁻¹) in both genotypes. Recent ¹⁴C assimilate was rapidly translocated from the fed area of the leaf blade. For example, the decaploid and the common hexaploid had translocated 40 and 26% of the ¹⁴C, respectively, at 6 h, and 79 and 49% of the ¹⁴C, respectively, at 24 h. Partitioning of ¹⁴C among plant organs was considerably different between the genotypes after a 24 h chase. For example, out of the total ¹⁴C recovered from the whole plant, the decaploid had retained 40% in the labelled leaf with 10, 33 and 29% in other leaves, stem bases and roots, respectively; whereas the hexaploid had retained 91% in the labelled leaf with 4, 3 and 2% in other leaves, stem bases and roots, respectively. However, the higher rate of translocation was correlated with greater interveinal distances in the decaploid genotype. These results suggested that the higher translocation percentage in the decaploid than the hexaploid genotype was due to greater sink activity.     

Resumption of cellular activity induced by cytokinin and gibberellin treatments in tomato flowers targeted for abortion in unfavorable light conditions

Mitotic activity and nuclear DNA synthesis in tomato ( Lycopersicon esculentum Mill., cv. King plus) flowers targeted for abortion under unfavorable light conditions are completely stopped 6 days after macroscopic appearance of the inflorescence. Ovular cells are arrested at the G₁ (80%) and G₂ (20%) stages of the cell cycle. Exogenous applications of a mixture of N⁶-benzyladenine (BA) and gibberellins A₄₊₇ (GA) directly on the inflorescence may prevent its failure. Nuclear DNA synthesis and mitoses resume in ovules of the flower 16 to 20 h after the BA+GA treatment. When applied alone, BA and GA are able to mimic the effect of the mixture upon the progression of ovular cells through their cycle. Sporogenesis processes are also set in motion by the exogenous plant growth regulators. The mechanism of action of cytokinins and gibberellins in the control of floral development is discussed.     

Experimental Methyl Mercury Neurotoxicity: Locus of Mercurial Inhibition of Brain Protein Synthesis In Vivo and In Vitro

Brain cell-free protein synthesis is inhibited by methyl mercury chloride (MeHg) following in vivo or in vitro administration. In this report, we have identified the locus of mercurial inhibition of translation. Intraperitoneal injection of MeHg (40 nmol/g body wt) induced variable inhibition of amino acid incorporation into the post-mitochondrial supernatant (PMS) harvested from the brain of young (10-20-day-old) rats. No mercurial-induced disaggregation of brain polyribosomes nor change in the proportion of 80S monoribosomes was detected on sucrose density gradients. No difference in total RNA was found in the PMS. Initiation complex formation was stimulated by MeHg, as detected by radiolabelled methionine binding to 80S monoribosomes following continuous sucrose density gradient centrifugation. After micrococcal nuclease digestion of endogenous mRNA, both in vivo and in vitro MeHg inhibited polyuridylic acid-directed incorporation of [3H]phenylalanine. However, the in vivo inhibition was no longer observed when [3H]phenylalanyl-tRNAPhe replaced free [3H]phenylalanine in the incorporation assay. The formation of peptidyl[3H]puromycin revealed no difference from controls. There was significant mercurial inhibition of phenylalanyl-tRNA Phe synthetase activity in pH 5 enzyme fractions derived from brain PMS of MeHg-poisoned rats. These experiments revealed that the apparent MeHg inhibition of brain translation in vivo and in vitro is due primarily to perturbation in the aminoacylation of tRNA and is not associated with defective initiation, elongation, or ribosomal function.     

Improved preservation of the form and contents of wall vesicles and the golgi apparatus in freeze substituted hyphae ofSaprolegnia

Summary Secretory vesicles involved in cell wall synthesis (wall vesicles) and the Golgi apparatus have been compared in conventionally fixed and freeze substituted hyphae of the oomycete fungusSaprolegnia ferax. Wall vesicles freeze substituted in various fluids range from spherical to tubular and contain an intensely staining, phosphorous rich matrix. In contrast diverse conventional fixations cause artefactual constrictions in most tubular vesicles and loss of their intensely staining contents. These data are interpreted to show the existence of an intravesicular skeletal system, with cellular regulation, to determine vesicle morphology and intravesicular synthesis of a hypothetical phosphorylated glycolipid cell wall precursor. Whilst freeze substitution gives superior preservation of wall vesicle morphology, it does not demonstrate any preferential association between wall vesicles and microtubules thus suggesting that microtubules are only indirectly involved in wall vesicle transport. Freeze substitution is superior to conventional fixation for analysis of the Golgi apparatus because it uniquely reveals both differentiation of a specific single cisterna in each Golgi body and greater differences in membrane thicknesses throughout the endomembrane system.     

The regulation of the fatty-acid composition of the triacylglycerols in microsomal preparations from avocado mesocarp and the developing cotyledons of safflower

The utilisation of [¹⁴C]glycerol 3-phosphate and [¹⁴C]linoleoyl-CoA in the synthesis of triacylglycerol has been studied in the microsomal preparations of developing cotyledons of safflower seed. The results confirm that the glycerol backbone, which flows towards triacylglycerol from phosphatidic acid through the Kennedy pathway, can enter phosphatidylcholine from diacylglycerol. The equilibration between diacylglycerol and phosphatidylcholine offers a mechanism for the return of oleate to phosphatidylcholine for desaturation to linoleate. We have established that the oleate entering position 1 of sn-phosphatidylcholine from diacylglycerol is desaturated in situ to linoleate. The results indicate that the diacylglycerol phosphatidylcholine interconvertion coupled to the acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine brings about the continuous enrichment of the glycerol backbone with C₁₈-polyunsaturated fatty acids and hence these enzymes are of major importance in regulating the acyl quality of the accumulating triacylglycerols. Microsomal preparations from avocado mesocarp, however, did not have detectable acyl exchange between acyl-CoA and phosphatidylcholine or diacylglycerol phosphatidylcholine interconversion despite the high activity of the enzymes of the Kennedy pathway. A scheme is presented which incorporates many of the observations on triacylglycerol synthesis and provides a working model for the regulation of acyl quality in linoleate-rich vegetable oils.Abbreviation BSA bovine serum albumin