Malate dehydrogenase isoenzymes in division synchronized cultures of euglena

Sucrose density gradient centrifugation of broken cell suspensions of autotrophically grown Euglena gracilis Klebs. has allowed the separation of chloroplasts, mitochondria, and peroxisomes. Chlorophyll was taken as a marker for chloroplasts, fumarase and succinate dehydrogenase for mitochondria, and glycolate oxidoreductase for peroxisomes. Peaks of malate dehydrogenase (l-malate-NAD oxidoreductase, EC 1.1.1.37) activity were found in the mitochondrial and peroxisomal fractions. Acrylamide gel electrophoresis showed specific isoenzymes in the mitochondrial and peroxisomal fractions and a third isoenzyme in the supernatant. The mitochondrial isoenzyme which had a Km (oxaloacetate) of 30μm was inhibited by oxaloacetate concentrations above 0.17 mm, an inhibition of 50% being given by 0.9 mm oxaloacetate. The peroxisomal isoenzyme had a Km (oxaloacetate) of 24 μm, was inhibited by oxaloacetate concentrations above 0.13 mm, 50% inhibition being given by 0.25 mm oxaloacetate. Malate dehydrogenase activity in the supernatant did not show inhibition by increasing oxaloacetate concentration, the Km (oxaloacetate) being 91 μm.     

Maturation in liver mitochondria of Ruthenium Red-sensitive calcium-ion-transport activity and the influence of glucagon administration in vivo and in utero

The maturation of Ca²⁺ transport in mitochondria isolated from rat liver was examined, from 5 days before birth. The mitochondria used were isolated from liver homogenates by centrifugation at 22000g-min. Ca²⁺ transport by mitochondria isolated from foetal liver is energy-dependent and Ruthenium Red-sensitive. The transmembrane pH gradient in these mitochondria is higher by about 7mV and the membrane potential lower by about 20mV than in adult mitochondria. The inclusion of 2mm-P_i in the incubation medium enhances the protonmotive force by approx. 30mV. The rate of Ca²⁺ influx in foetal mitochondria measured in buffered KCl plus succinate is low until about 2–3h after birth, when it increases to about 60% of adult values; approx. 24h later it has reached near-adult values. Higher rates of Ca²⁺ influx are observed in the presence of 2mm-P_i; 3–5 days before birth the rates are about one-third of adult values and decline slightly as birth approaches. By 2–3h post partum they have reached adult values. The inclusion of 12.5μm-MgATP with the P_i stimulates further the initial rate of Ca²⁺ influx in foetal mitochondria. The rates observed are constant over the prenatal period examined and are 50–60% of those observed in adult mitochondria. Mitochondria isolated from foetal livers 4–5 days before birth retain the accumulated Ca²⁺ for about 50min in the presence of 2mm-P_i. In the period 2 days before birth to birth, this ability is largely lost, but by 2–3h after birth Ca²⁺ retention is similar to that of adult mitochondria. The presence of 12.5μm-MgATP progressively enhances the Ca²⁺ retention time as development proceeds until 2–3h after birth, when it becomes less sensitive to added MgATP. Glucagon administration to older foetuses in utero enhances both the rate of mitochondrial Ca²⁺ influx assayed in the presence of 2mm-P_i and the time for which mitochondria retain accumulated Ca²⁺ in the presence of 12.5μm-MgATP and 2mm-P_i. Its administration to neonatal animals leads to an increase in mitochondrial Ca²⁺ retention similar to that seen in adult mitochondria. The data provide evidence that the Ruthenium Red-sensitive Ca²⁺ transporter is potentially as active in foetal mitochondria 5 days before birth as it is in adult mitochondria. They also show that foetal mitochondria have an ability to retain accumulated Ca²⁺ reminiscent of mitochondria from tumour cells and from hormone-challenged rat liver.     

Amino ketone formation and aminopropanol-dehydrogenase activity in rat-liver preparations

1. Rat tissue homogenates convert dl-1-aminopropan-2-ol into aminoacetone. Liver homogenates have relatively high aminopropanol-dehydrogenase activity compared with kidney, heart, spleen and muscle preparations. 2. Maximum activity of liver homogenates is exhibited at pH9·8. The K_m for aminopropanol is approx. 15mm, calculated for a single enantiomorph, and the maximum activity is approx. 9mμmoles of aminoacetone formed/mg. wet wt. of liver/hr.at 37°. Aminoacetone is also formed from l-threonine, but less rapidly. An unidentified amino ketone is formed from dl-4-amino-3-hydroxybutyrate, the K_m for which is approx. 200mm at pH9·8. 3. Aminopropanol-dehydrogenase activity in homogenates is inhibited non-competitively by dl-3-hydroxybutyrate, the K_i being approx. 200mm. EDTA and other chelating agents are weakly inhibitory, and whereas potassium chloride activates slightly at low concentrations, inhibition occurs at 50–100mm. 4. It is concluded that aminopropanol-dehydrogenase is located in mitochondria, and in contrast with l-threonine dehydrogenase can be readily solubilized from mitochondrial preparations by ultrasonic treatment. 5. Soluble extracts of disintegrated mitochondria exhibit maximum aminopropanol-dehydrogenase activity at pH9·1 At this pH, K_m values for the amino alcohol and NAD⁺ are approx. 200 and 1·3mm respectively. Under optimum conditions the maximum velocity is approx. 70mμmoles of aminoacetone formed/mg. of protein/hr. at 37°. Chelating agents and thiol reagents appear to have little effect on enzyme activity, but potassium chloride inhibits at all concentrations tested up to 80mm. dl-3-Hydroxybutyrate is only slightly inhibitory. 6. Dehydrogenase activities for l-threonine and dl-4-amino-3-hydroxybutyrate appear to be distinct from that for aminopropanol. 7. Intraperitoneal injection of aminopropanol into rats leads to excretion of aminoacetone in the urine. Aminoacetone excretion proportional to the amount of the amino alcohol administered, is complete within 24hr., but represents less than 0·1% of the dose given. 8. The possible metabolic role of amino alcohol dehydrogenases is discussed.     

Inability of tributyltin-induced chloride/hydroxyl exchange to stimulate calcium transport in mitochondria isolated from flight muscle of the sheep blowfly Lucilia cuprina

Tributyltin in the concentration range 1–4μm failed to stimulate Ca²⁺ transport by Lucilia flight-muscle mitochondria in a medium containing KCl and respiratory substrate but devoid of P_i, despite its promotion of a rapid Cl⁻/OH⁻ exchange. When 2mm-P_i was present, concentrations of tributyltin greater than 1μm inhibited the initial rate of Ca²⁺ transport and induced efflux of the ion from the mitochondria in Cl⁻- or NO₃⁻-containing media. Lower concentrations had little effect. Oligomycin added at up to 10μg/mg of mitochondrial protein had no effect on Ca²⁺ transport. By contrast, approx. 0.3μm-tributyltin completely inhibited respiration supported by α-glycerophosphate in either the presence or absence of added ADP. The data suggest that tributyltin can inhibit Ca²⁺ transport in Lucilia flight-muscle mitochondria other than by facilitating a Cl⁻/OH⁻ exchange or producing an oligomycin-like effect.     

Substrate and inhibitor specificity of the cholesterol oxidase in bovine adrenal cortex

1. Cholesteryl 3β-sulphate is oxidized in vitro by preparations of bovine adrenal-cortex mitochondria to pregnenolone sulphate and isocaproic acid (4-methyl-pentanoic acid) without hydrolysis of the ester linkage. 2. Free cholesterol is the preferred substrate for adrenal-cortex cholesterol oxidase; the apparent K_m for cholesteryl sulphate is 500μm and for free cholesterol 50μm under the same conditions. 3. Cholesteryl 3β-acetate is hydrolysed by bovine adrenal-cortex mitochondria in vitro to free cholesterol, which is subsequently oxidized to more polar steroids and isocaproic acid. Evidence was obtained that other cholesterol esters behave similarly. Cholesterol esters may thus act as precursors of steroid hormones. 4. Cholest-4-en-3-one is only poorly oxidized to isocaproic acid and more polar steroids and thus is probably not a significant precursor of steroid hormones. 5. Cholesteryl esters inhibit the oxidation of cholesterol competitively (K_i for cholesteryl phosphate 28μm, for cholesteryl sulphate 110μm, for cholesteryl acetate 65μm) but pregnenolone esters do not inhibit this system. 6. Pregnenolone and 20α-hydroxycholesterol (both metabolites of cholesterol in this system) inhibit the oxidation of cholesterol non-competitively. K_i for pregnenolone is 130μm and K_i for 20α-hydroxycholesterol is 17μm. 7. 25-Oxo-27-norcholesterol inhibits cholesterol oxidation non-competitively (K_i16μm). A number of other Δ⁵-3β-hydroxy steroids inhibit cholesterol oxidation and evidence was obtained that the 3β-hydroxyl group was necessary for inhibitory activity. 8. Pregnenolone, 20α-hydroxycholesterol and 25-oxo-27-norcholesterol inhibit oxidation of cholesteryl sulphate by this system but their sulphates do not. 9. 3β-Hydroxychol-5-enoic acid, 3α-hydroxy-5β-cholanic acid and 3β-hydroxy-22,23-bisnorchol-5-enoic acid stimulated formation of isocaproic acid from cholesterol. 10. No evidence was obtained that phosphorylation or sulphation are obligatory steps in cholesterol oxidation by adrenal-cortex mitochondria. 11. The cholesteryl 3β-sulphate sulphatase of bovine adrenal cortex was found mostly in the microsomal fraction and was inhibited by inorganic phosphate.     

Vanadate inhibits blue light-stimulated swelling of vicia guard cell protoplasts

When supplied under low chloride concentrations, vanadate inhibits the blue light-stimulated swelling of Vicia faba L. guard cell protoplasts in a dose-dependent fashion. The volume of guard cell protoplasts incubated in 10 mm K-imino-diacetic acid, 0.4 m mannitol, and 1 mm CaCl₂ remained essentially constant under 1000 μmol m⁻² s⁻¹ red light, but increased an average of 27% after 8 min of the addition of 50 μmol m⁻² s⁻¹ blue light to the background red light. At 500 μm, vanadate completely inhibits the response to blue light. Vanadate also inhibits the swelling of guard cell protoplasts stimulated by the H⁺-ATPase agonist fusicoccin. The vanadate sensitivity of the blue light-stimulated swelling implicates a proton-pumping ATPase as a component of the sensory transduction of blue light in guard cells.     

Translocation of iron citrate and phosphorus in xylem exudate of soybean

Soybean plants, Glycine max (L.) Merrill, in standard solution received 2.5 μm ferric ethylenediamine di(o-hydroxyphenylacetate (FeEDDHA) and 0 to 128 μm phosphorus. Their stem exudates contained: 32 to 52 μm Fe, 120 to 5000 μm P, and 120 to 165 μm citrate. Electrophoresis of exudates with high P caused Fe trailing that precluded identification of any major form of Fe. Exudate with low P gave an anodic band of Fe citrate as the major Fe compound. Phosphate added to exudate in vitro depressed the Fe citrate peak and cause Fe trailing. EDDHA added to exudate in vitro pulled Fe from Fe citrate; citrate then migrated as a slower form and Fe migrated as FeEDDHA. A modified preculture system, involving 2-day renewals of 0.2 μm FeEDDHA with 3.2, 9.6, or 16 μm P and low levels of other ions, controlled pH depression and produced considerable change in citrate and P levels. The exudates contained: 45 to 57 μm Fe, 200 to 925 μm P, and 340 to 1025 μm citrate. The high citrate was from plants grown with low P. The major form of Fe in the exudates was Fe citrate. This is probably the form translocated in the plants.     

Inhibition of chloroplast reactions with phenylmercuric acetate

Phenylmercuric acetate is a selective inhibitor of the photosynthetic activities of isolated spinach (Spinacia oleracea) chloroplasts. At 5 μm concentration of phenylmercuric acetate, photophosphorylation is inhibited. At 33 μm phenylmercuric acetate, ferredoxin is inactivated. Ferredoxin-NADP oxidoreductase is 50% inhibited at 100 μm phenylmercuric acetate. Photosystem II reactions are 50% inhibited at 150 μm phenylmercuric acetate and very much higher cooncentrations—500 μm—are needed to approach complete inhibition. Phenylmercuric acetate inhibition of photosystem II appears to be selective, blocking a site between the 3-(3,4-dichlorophenyl)-1,1-dimethyl urea sensitive site and the site inactivated by high concentrations of tris buffer.     

Purification and comparative properties of microsomal and glyoxysomal malate synthase from castor bean endosperm

Sucrose density gradient centrifugation was employed to separate microsomes, mitochondria, and glyoxysomes from homogenates prepared from castor bean (Ricinus communis) endosperm. In the case of tissue removed from young seedlings, a significant proportion of the characteristic glyoxysomal enzyme malate synthase was recovered in the microsomal fraction. Malate synthase was purified from both isolated microsomes and glyoxysomes by a procedure involving osmotic shock, KCI solubilization, and sucrose density gradient centrifugation. All physical and catalytic properties examined were identical for the enzyme isolated from both organelle fractions. These properties include a molecular weight of 575,000, with a single subunit type of molecular weight 64,000, a pH optimum of 8, apparent K_m for acetyl-CoA of 10 μm and glyoxylate of 2 mm. Microsomal and glyoxysomal malate synthases showed identical responses to various inhibitors. Adenine nucleotides were competitive inhibitors with respect to acetyl-CoA, and oxalate (K_i 110 μm) and glycolate (K_i 150 μm) were competitive inhibitors with respect to glyoxylate. Antiserum raised in rabbits against purified glyoxysomal malate synthase was used to confirm serological identity between the microsomal and glyoxysomal enzymes, and was capable of specifically precipitating ³⁵S-labeled malate synthase from KCI extracts of both microsomes and glyoxysomes isolated from [³⁵S]methionine-labeled endosperm tissue.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5′-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7·8 and 25° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K_i for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.     

Terminal Oxidases of Chlorella pyrenoidosa

In studies of the kinetics of oxygen uptake by glucose-stimulated Chlorella pyrenoidosa, two terminal oxidases could be distinguished. The cytochrome oxidase of Chlorella has a Km (O₂) of 2.1 ± 0.3 μm, while the second oxidase has a Km (O₂) of 6.7 ± 0.5 μm, and a maximum capacity about one-quarter of that of the cytochrome system. The identity of the second oxidase is unknown, but it is not inhibited by carbon monoxide, 1 mm cyanide, 0.1 mm thiocyanate, or 1 mm 8-hydroxyquinoline. In fresh cultures, the second oxidase accounts for at most 35% of the total oxygen uptake.     

Evidence for β-adrenergic activation of Na+-dependent efflux of Ca2+ from isolated liver mitochondria

The existence of a Na⁺-dependent mechanism for Ca²⁺ efflux from isolated rat liver mitochondria was confirmed. The activity of this system is decreased by 60% in mitochondria isolated from perfused livers. The Na⁺-dependent activity is fully restored by infusion of either 1μm-adrenaline or 1μm-isoprenaline, but the α-adrenergic agonist phenylephrine is ineffective.     

Effectors of rat-heart hexokinases and the control of rates of glucose phosphorylation in the perfused rat heart

1. The kinetic properties of the soluble and particulate hexokinases from rat heart have been investigated. 2. For both forms of the enzyme, the K_m for glucose was 45μm and the K_m for ATP 0·5mm. Glucose 6-phosphate was a non-competitive inhibitor with respect to glucose (K_i 0·16mm for the soluble and 0·33mm for the particulate enzyme) and a mixed inhibitor with respect to ATP (K_i 80μm for the soluble and 40μm for the particulate enzyme). ADP and AMP were competitive inhibitors with respect to ATP (K_i for ADP was 0·68mm for the soluble and 0·60mm for the particulate enzyme; K_i for AMP was 0·37mm for the soluble and 0·16mm for the particulate enzyme). P_i reversed glucose 6-phosphate inhibition with both forms at 10mm but not at 2mm, with glucose 6-phosphate concentrations of 0·3mm or less for the soluble and 1mm or less for the particulate enzyme. 3. The total activity of hexokinase in normal hearts and in hearts from alloxan-diabetic rats was 21·5μmoles of glucose phosphorylated/min./g. dry wt. of ventricle at 25°. The temperature coefficient Q₁₀ between 22° and 38·5° was 1·93; the ratio of the soluble to the particulate enzyme was 3:7. 4. The kinetic data have been used to predict rates of glucose phosphorylation in the perfused heart at saturating concentrations of glucose from measured concentrations of ATP, glucose 6-phosphate, ADP and AMP. These have been compared with the rates of glucose phosphorylation measured with precision in a small-volume recirculation perfusion apparatus, which is described. The correlation between predicted and measured rates was highly significant and their ratio was 1·07. 5. These findings are consistent with the control of glucose phosphorylation in the perfused heart by glucose 6-phosphate concentration, subject to certain assumptions that are discussed in detail.     

Photoreduction and Photophosphorylation with Tris-Washed Chloroplasts

The artificial electron donor compounds p-phenylenediamine (PD), N, N, N′, N′-tetramethyl-p-phenylenediamine (TMPD), and 2,6-dichlorophenol-indophenol (DCPIP) restored the Hill reaction and photophosphorylation in chloroplasts that had been inhibited by washing with 0.8 m tris (hydroxymethyl) aminomethane (tris) buffer, pH 8.0. The tris-wash treatment inhibited the electron transport chain between water and photosystem II and electron donation occurred between the site of inhibition and photosystem II. Photoreduction of nicotinamide adenine dinucleotide phosphate (NADP) supported by 33 μm PD plus 330 μm ascorbate was largely inhibited by 1 μm 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) while that supported by 33 μm TMPD or DCPIP plus ascorbate was relatively insensitive to DCMU. Experiments with the tris-washed chloroplasts indicated that electron donors preferentially donate electrons to photosystem II but in the presence of DCMU the donors (with the exception of PD at low concentrations) could also supply electrons after the DCMU block. The PD-supported photoreduction of NADP showed the relative inefficiency in far-red light characteristic of chloroplast reactions requiring photosystem II. With phosphorylating systems involving electron donors at low concentrations (33 μm donor plus 330 μm ascorbate) photophosphorylation, which occurred with P/e₂ ratios approaching unity, was completely inhibited by DCMU but with higher concentrations of the donor systems, photophosphorylation was only partially inhibited.     

The activities and kinetic properties of purine phosphoribosyltransferases in developing mouse liver

1. The total activity of adenine phosphoribosyltransferase/liver of mice remained constant from 1 to 16 days after birth despite a fourfold increase in liver weight. The total activity of this enzyme increased fivefold from 16 to 36 days and then remained relatively constant at least until 96 days after birth. Total hypoxanthine-phosphoribosyltransferase activity/liver steadily increased between 1 and 57 days after birth. 2. The mean K_m of 5-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase was 10·1μm between 3 and 11 days, at 64 days and at 96 days after birth. Between 17 and 51 days the mean K_m value was 3·0μm. The K_m of 5-phosphoribosyl pyrophosphate with hypoxanthine phosphoribosyltransferase remained constant at 28·2μm between 2 and 64 days. 3. Adenine-phosphoribosyltransferase activity was stimulated between 15 and 83% by 60μm-ATP when extracts were made between 3 and 11 days, at 64 days or at 96 days after birth. Between 17 and 51 days ATP had little stimulatory effect on the activity of this enzyme. 4. AMP competed with 5-phosphoribosyl pyrophosphate in the reaction catalysed by adenine phosphoribosyltransferase. Liver extracts containing enzyme with a low value of K_m for 5-phosphoribosyl pyrophosphate (3μm) had a K_m/K_i ratio approximately half that of extracts with a high value of K_m (10μm). 5. The results indicate that two different forms of adenine phosphoribosyltransferase can exist in mouse liver at different stages of development. The physiological significance of these findings is discussed.     

Partial Purification of the NADH-Nitrate Reductase Complex from Chlorella pyrenoidosa

The nitrate reductase complex from Chlorella pyrenoidosa has been purified by a procedure which includes as main steps, ammonium sulfate fractionation, polyethylene glycol treatment, and DEAE-cellulose chromatography. The Michaelis constants for NADH, FAD, and NO₃⁻ in the NADH-nitrate reductase assay are 10 μm, 2.6 μm, and 0.23 mm, respectively. Heat treatment exerts varying effects on the enzymatic activities associated with the nitrate reductase complex.     

Effect of l-Canavanine on Nitrate Reductase in Corn Roots

l-Canavanine inhibits the appearance of nitrate reductase (NADH-nitrate oxidoreductase, EC 1.6.6.1) in both root tips and mature root sections of corn (Zea mays L.). Ten-fold more canavanine was required to cause a 50% reduction in the level of nitrate reductase activity (NRA) in root tips than in mature root sections. For example with one particular batch of seeds 500 μm canavanine was effective in root tips whereas only 50 μm was required in mature root sections. In root tips arginine (1 mm) completely reversed the effect of 1 mm canavanine. In mature root sections higher concentrations of arginine (approximately 5 mm) were required for a complete reversal of the canavanine effect. Additions of canavanine to roots after a period of 3 hours with 5 mm KNO₃ resulted in a loss of NRA. NO₃⁻ protected nitrate reductase from this inactivation in both root tip and mature root sections.     

T-2 toxin decreases logarithmic growth rates of tobacco callus tissues

T-2 toxin, a mycotoxin produced by Fusarium tricinctum, decreases logarithmic growth rates of tobacco (Nicotiana tabacum L.) pith callus tissues. Toxin concentrations as low as 0.003 μm will decrease growth rates; a concentration of 0.081 μm will halt growth completely. Additional exogenous cytokinin will reduce the inhibition by toxin only when the initial cytokinin and toxin concentrations are quite low (about 0.01 μm). When inhibited tissues are transferred to media lacking toxin, they assume the faster, control rates almost immediately. Maximal yields of tissue (yields at the point at which no sugar was detected in the medium) are not affected by toxin concentrations of 0.01 to 0.036 μm.     

Characterization of an Electron Transport Pathway Associated with Glucose and Fructose Respiration in the Intact Chloroplasts of Chlamydomonas reinhardtii and Spinach

The role of an electron transport pathway associated with aerobic carbohydrate degradation in isolated, intact chloroplasts was evaluated. This was accomplished by monitoring the evolution of ¹⁴CO₂ from darkened spinach (Spinacia oleracea) and Chlamydomonas reinhardtii chloroplasts externally supplied with [¹⁴C]fructose and [¹⁴C]glucose, respectively, in the presence of nitrite, oxaloacetate, and conventional electron transport inhibitors. Addition of nitrite or oxaloacetate increased the release of ¹⁴CO₂, but it was shown that O₂ continued to function as a terminal electron acceptor. ¹⁴CO₂ evolution was inhibited up to 30 and 15% in Chlamydomonas and spinach, respectively, by 50 μm rotenone and by amytal, but at 500- to 1000-fold higher concentrations, indicating the involvement of a reduced nicotinamide adenine dinucleotide phosphate-plastoquinone oxidoreductase. ¹⁴CO₂ release from the spinach chloroplast was inhibited 80% by 25 μm 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone. ¹⁴CO₂ release was sensitive to propylgallate, exhibiting approximately 50% inhibition in Chlamydomonas and in spinach chloroplasts of 100 and 250 μm concentrations, respectively. These concentrations were 20- to 50-fold lower than the concentrations of salicylhydroxamic acid (SHAM) required to produce an equivalent sensitivity. Antimycin A (100 μm) inhibited approximately 80 to 90% of ¹⁴CO₂ release from both types of chloroplast. At 75 μm, sodium azide inhibited ¹⁴CO₂ evolution about 50% in Chlamydomonas and 30% in spinach. Sodium azide (100 mm) combined with antimycin A (100 μm) inhibited ¹⁴CO₂ evolution more than 90%. ¹⁴CO₂ release was unaffected by uncouplers. These results are interpreted as evidence for a respiratory electron transport pathway functioning in the darkened, isolated chloroplast. Chloroplast respiration defined as ¹⁴CO₂ release from externally supplied [1-¹⁴C]glucose can account for at least 10% of the total respiratory capacity (endogenous release of CO₂) of the Chlamydomonas reinhardtii cell.     

Cellular Models of Aggregation-dependent Template-directed Proteolysis to Characterize Tau Aggregation Inhibitors for Treatment of Alzheimer Disease

Alzheimer disease (AD) is a degenerative tauopathy characterized by aggregation of Tau protein through the repeat domain to form intraneuronal paired helical filaments (PHFs). We report two cell models in which we control the inherent toxicity of the core Tau fragment. These models demonstrate the properties of prion-like recruitment of full-length Tau into an aggregation pathway in which template-directed, endogenous truncation propagates aggregation through the core Tau binding domain. We use these in combination with dissolution of native PHFs to quantify the activity of Tau aggregation inhibitors (TAIs). We report the synthesis of novel stable crystalline leucomethylthioninium salts (LMTX®), which overcome the pharmacokinetic limitations of methylthioninium chloride. LMTX®, as either a dihydromesylate or a dihydrobromide salt, retains TAI activity in vitro and disrupts PHFs isolated from AD brain tissues at 0.16 μm. The K_i value for intracellular TAI activity, which we have been able to determine for the first time, is 0.12 μm. These values are close to the steady state trough brain concentration of methylthioninium ion (0.18 μm) that is required to arrest progression of AD on clinical and imaging end points and the minimum brain concentration (0.13 μm) required to reverse behavioral deficits and pathology in Tau transgenic mice.