Characterization of β-hydroxybutyrate transport in rat erythrocytes and thymocytes

A method was developed for study of β-hydroxybutyrate transport in erythrocytes and thymocytes. Critical to the method was a centrifugal separation of cells from medium which took advantage of β-hydroxybutyrate transport's temperature dependence and inhibition by phloretin and methylisobutylxanthine, all of which are demonstrated in this work. These properties suggested mediated transport, as did saturation kinetics and inhibition by several agents including pyruvate and α-cyanocinnamate. Most conclusive in this regard was a 2-fold preference for d- over l-β-hydroxybutyrate. Entry was not Na⁺ dependent. It was stimulated by substitution of SO₄^2? for most of the Cl^?. The equilibrium β-hydroxybutyrate space was much higher than the Cl^? space of thymocytes, suggesting that β-hydroxybutyrate entry is not associated with net inward negative current and is not coupled to outward Cl^? or inward K⁺ movement (assuming that K⁺ is at electrochemical equilibrium). Coupling to H⁺ entry or OH^? exit is compatible with the result. These findings are consistent with β-hydroxybutyrate entry by the carboxylate transport site which has been studied extensively with pyruvate and lactate as permeants. The Cl^?/HCO₃^? exchange carrier did not appear to contribute significantly to β-hydroxybutyrate transport.     

Effect of inhibitors on the sigmoidicity of the calcium ion transport kinetics in rat liver mitochondria

The kinetic plot (initial rate of Ca²⁺ transport versus concentration) of mitochondrial Ca²⁺ transport is hyperbolic in a sucrose medium. The plot becomes sigmoidal in the presence of competitive inhibitors of Ca²⁺ binding to low affinity sites of the membrane surface such as Mg²⁺ and K⁺. The plot also becomes sigmoidal in the presence of Ba²⁺. Ba²⁺ is a competitive inhibitor of both Ca²⁺ transport and Ca²⁺ binding to the low affinity sites. The K_i for the inhibition of Ca²⁺ transport by Ba²⁺ increases in the presence of K⁺ and Mg²⁺, which suggests a competition for the low affinity sites between the cations. The plot is still hyperbolic in the presence of La³⁺, which inhibits Ca²⁺ transport competitively. Ruthenium red which is a pure non-competitive inhibitor of mitochondrial Ca²⁺ transport, does not affect the shape of the kinetic plot. These results indicate that the surface potential, which depends on the ions bound to the low affinity sites, determines whether the kinetics of Ca²⁺ uptake in mitochondria is sigmoidal or hyperbolic.     

Isolation of calcium pump system and purification of calcium ion-dependent ATPase from heart muscle

The procedure for the isolation of the highly active fraction of sarcoplasmic reticulum from pigeon and dog hearts is described. The method is based on the partial loading of heart microsomes with calcium and oxalate ions and the precipitation of loaded vesicles in sucrose and potassium chloride concentration gradients. Preparations obtained possess high activity of Ca²⁺-dependent ATPase and are also able to accumulate up to 10 μmol Ca²⁺ per mg protein. Purification of sarcoplasmic reticulum membranes is accompanied by a decrease in concentration of cytochrome a+a₃ and an increase in the content of [³²P]phosphoenzyme. The basic components in “calcium-oxalate preparation” from hearts are proteins with molecular weights of about 100 000 (Ca²⁺-dependent ATPase) and 55 000 Calcium-oxalate preparation from pigeon hearts was used for subsequent purification of Ca²⁺-dependent ATPase. Specific activity of purified enzyme from pigeon hearts is 12–16 μmol P_i/min per mg protein. Enzyme activity of purified Ca²⁺-dependent ATPase is inhibited by EGTA and is not sensitive to azide, 2,4-dinitrophenol and ouabain. The data obtained demonstrate the similarity of calcium pump systems and Ca²⁺-dependent ATPases isolated from heart and skeletal muscles.     

Enzymatic esterification of vitamin a in the pigment epithelium of bovine retina

The kinetic properties and subcellular distribution of an esterifying enzyme in the pigment epithelium of bovine retina have been studied using both [1-³H]retinol and [³H]retinol bound to cellular retinol-binding protein as substrates. The most active esterifying fraction in pigment epithelial cell preparations was the microsomes, but the lysosome plus mitochondria fraction also showed some activity, probably due to endoplasmic reticulum present as an impurity. The microsomal enzyme showed optimum activity at pH 7.5, and the reaction was linear up to 30 μg protein and for the first 10–15 min. The apparent K_m values were 16.6 · 10^?6 and 5.5 · 10^?6 M for [³H]retinol and bound [³H]retinol, respectively. This is the first time that retinol bound to cellular retinol-binding protein has been shown to undergo metabolic stransformation. The microsomal esterifying activity was destroyed by boiling for 1 min, or after freezing for 2 months. No clear requirement for ATP, CoA or fatty acid could be demonstrated.Of all the other tissues examined under the same experimental conditions as those used for the pigment epithelium, onlt intestine showed measurable activity. With larger amounts of tissue protein and longer incubation periods, activity was also detectable in microsomes of liver, testis and retina     

The effect of charged synthetic polymers on proteoglycan synthesis and sequestration in chick embryo fibroblast cultures

The polycation, poly(l-lysine), repressed the synthesis of glycosaminoglycans in secondary cultures of chick embryo skin fibroblasts and caused sequestration of glycosaminoglycans around the cells. The synthesis of chondroitin sulphate, dermatan sulphate, hyaluronic acid and a fourth component, thought to be heparan sulphate, were all inhibited to the same extent but the sequestration of the sulphated polymers was greater than that of the unsulphated. The sequestered material was retained around and not within the cells. Incubations with the polyanion, poly(l-glutamate), showed a slight stimulation of glycosaminoglycan synthesis and in these and control incubations (no additions to medium), most of the glycosaminoglycan synthesised appeared in the culture medium. The subsequent addition of poly(l-glutamate) to incubations containing poly(l-lysine) reversed the inhibitory and sequestering effect of the polycation. It was concluded that the inhibition of synthesis by poly(l-lysine) was either a direct effect of poly(l-lysine) on the cell membrane or a result of the high local pericellular concentration of sequestered proteoglycan.     

Sensitivity of progesterone receptors to aurintricarboxylic acid: Inhibition of nuclear uptake,ATP and DNA binding

When hen oviduct cytosol samples containing progesterone receptor complexed to [³H]progesterone were included with isolated nuclei in presence of 0.2 mM aurintricarboxylic acid, more than 50% inhibition occurred in the uptake of progesterone receptor by the nuclei. The activated form of progesterone receptor appeared to be more sensitive to the presence of aurintricarboxylic acid since pretreatment of non-activated progesterone receptor with the inhibitor and the subsequent removal of the latter prior to activation did not result in the inhibition of receptor uptake by the nuclei. Also, the binding of progesterone receptor to columns of DNA-cellulose or ATP-Sepharose was abolished under simmilar conditions. When nuclei, ATP-Sepharose or DNA-cellulose were preincubated with the inhibitor prior to the addition of receptor preparations, no such inhibition resulted indicating that the inhibitor may be interacting with the receptor protein and not complexing to ATP, DNA or sites in the nuclei. The steroid binding properties of progesterone receptor, however, remained intact under these conditions. Both A and B forms of progesterone receptor are equally sensitive to aurintricarboxylic acid presence when tested for their nuclear uptake. Aurintricarboxylic acid was also found to be very effective at low concentrations (0.25 mM) in eluting the receptor complexes off ATP-Sepharose columns without disrupting the steroid binding properties of progesterone receptor. Our results suggest that auintricarboxylic acid is an effective inhibitor of progesterone receptor and that it may be acting by interfering with a site(s) on progesterone receptor which may be exposed upon activation and are involved in such processes as ATP binding, nuclear uptake and DNA binding. These observations suggest the use of aurintricarboxylic acid as a chemical probe for the analysis of progesterone receptor.     

Variations in mitochondrial monoamine oxidase during progressive starvation in the brain of developing rats

Effects of progressive starvation of 12, 24, 48 and 60 h upon brain mitochondrial monoamine oxidase activity were studied. The enzyme activity was determined by three different substrates: ¹⁴C-labeled tryptamine, dopamine and kynuramine. With dopamin as substrate, the enzyme activity showed decline during 24 and 48 h starvation. Monoamine oxidase when determined by tryptamine as the substrate, showed a decreased after 60 h of starvation. The use of kynuramine as substrate also produced a decrease in enzyme activity after 48 and 60 h of starvation. Refeeding the 60-h-starved rats for the following 24 h resulted in further decrease of monoamine oxidase activity of brain mitochondria from the 60 h starved values. The results suggest that oxidative deamination of biogenic amines is greatly inhibited during progressive starvation and remains low even after feeding the 60 h starved rats for 24 h.     

Characterization of a progestin-binding component in lactating rat mammary glands

Experiments were carried out to identify progestin-binding receptors in the mammary gland where casein synthesis is known to be inhibited by this hormone. A progestin-binding component with high affinity, low capacity and a sedimentation coefficient of 8.8 S was isolated from the cytosol of lactating rat mammary glands. This component strongly bound [³H]R5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) with a dissociation constant of 3.9 · 10^?9 M under low-salt conditions and with that of 8.2 · 10^?10 M in the presence of 0.3 M KCl. Specificity studies showed a higher degree of progestin specificity under high salt conditions. In the absence of KCl, binding of [³H]-R5020 was inhibited by unlabeled glucocorticoid in the same degree as unlabeled progestin, but the inhibition by glucocorticoid was greatly diminished by the presence of 0.3 M KCl. These observations suggest that the [³H]R5020-binding-component is the progestin receptor and that its function may be regulated by the concentration of glucocorticoid and salt.     

Evaluation of genetic risks of alkylating agents. III. Alkylation of haemoglobin after metabolic conversion of ethene to ethene oxide in vivo

Male CBA mice, exposed to air contaminated with [14C] labelled ethene, were able to metabolize this olefine to ethene oxide. The amount of epoxide formed was quantitatively determined from the degree of alkylation of cysteine and histidine in haemoglobin. These hydroxyethylated amino acids were determined by ion-exchange chromatography of the labelled products. In a separate experiment the formation of S-(2-hydroxyethyl) cysteine was verified by gas chromatography--mass spectrometry. In addition this cysteine derivative was determined in urine by thin-layer chromatography. For unknown reasons, uninduced mice varied strongly in the extent to which they converted ethene to epoxide.     

Metabolic responses of folic acid and related compounds to thyroxine in rats

This study deals with the effects of thyroidectomy and feeding thyroid powder on histidine and folic acid metabolism. Normal rats maintained on a soy protein diet, low in methionine but supplemented with vitamin B-12, oxidize approx. 10% of an injected dose of [2-¹⁴C]histidine in 3 h and excrete low levels of formiminoglutamic acid. Addition of methionine increases histidine oxidation to approx. 20%. The feeding of thyroid powder or the injection of high levels of thyroxine decreases histidine oxidation and increases formiminoglutamic acid excretion. Surgical thyroidectomy at weaning increases histidine oxidation to approx. 45% and, thus, resembles the effect of methionine in promoting histidine oxidation and decreasing formiminoglutamic acid excretion. The feeding of methionine to the thyroidectomized animal further increases histidine oxidation to 65%. The distribution of folate forms in the liver was determined by column chromatography following administration of a dose of tritiated folic acid. In the normal animal, tetrahydrofolate accounts for 38% of the total folate present. The feeding of methionine increases this to 48%, which is consistent with the observed increase in histidine metabolism. Thyroidectomy increases the percentage of tetrahydrofolate to 63% and the feeding of methionine further increases it to 68%. The percentage of tetrahydrofolate relative to total folate is in proportion to the observed rate of histidine metabolism. The action of thyroidectomy in increasing histidine oxidation may be accounted for by its effect in increasing the proportion of tetrahydrofolate.     

Regulation of ovarian 17β-hydroxysteroid dehydrogenase activity by gonadotropin

Serum testosterone levels are elevated prior to the lutropin surge, and decline abruptly following the release of endogenous lutropin. To investigate this phenomenon, the activity of 17β-hydroxysteroid dehydrogenase, the enzyme directly related to testosterone production form androstenedione, was measured. This was done in immature rats in which follicular maturation and ovulation were induced by pregnant mare serum gonadotropin administration. It appears that the effect of the gonadotropin on the enzyme activity is sharply divided into two phases that match with the follicular and the luteal phases. One day following gonadotropin administration, there was already a 7.67-fold increase in the original activity which further increased 48 h following hormone administration. At the peak of the lutropin surge, when follicular development is at its maximum, a 18.44-fold increase was measured. The activity fell abruptly 10 h following ovulation, at a time when fresh corpora lutea are already present in the ovary.It seems that the evaluation of serum testosterone followed by its abrupt decline, is directly related to the increased and decreased ovarian 17β-hydroxysteroid dehydrogenase activity. The possible importance of the observed changes to the mechanisms of the onset of puberty are discussed.     

Biphasic toxicity of diethyldithiocarbamate, a metal chelation, to T lymphocytes and polymorphonuclear granulocytes: reversal by zinc and copper.

A new type of toxicity biphasically dependent on concentration was observed with diethyldithiocarbamate, a metal chelator utilized in medicine. As judged by cell survival and [³H]Urd incorporation, diethyldithiocarbamate was maximally toxic to T lymphocytes and polymorphonuclears at 2.5×10^?5 M (first phase) and at higher than 2.5×10^?3 M (second phase), but was not toxic at intermediate concentrations around 2.5×10^?4 M. The response of chelator treated T lymphocytes to phytohemagglutinin was also biphasic. The first toxic phase was partially reversed by 2.5×10^?5 M ZnCl₂, while the second phase was partially reversed by 10^?2 M CuCl₂. This suggests that inhibition of Zn-metalloenzymes in the first phase and of Cu-metalloenzymes in the second may play a crucial role in the mechanism of toxicity. The second toxic phase may be in part due to the observed inhibition of superoxide dismutase rendering the cells susceptible to oxygen toxicity, like obligate anaerobes.     

Utilization of [2-14C]tetrahydropteroylglutamic acid and 5-[G-3H]methyltetrahydropteroylglutamic acid as substrates for folate polyglutamate synthesis in fruit bats: Effect of vitamin B-12-deficiency

[2-¹⁴C]Tetrahydropteroylglutamic acid and 5-[G-³H]methyltetrahydropteroylglutamic acid were given intraperitoneally to fruit bats. Folate polyglutamates were formed in the liver from both substrates in different amounts and at different rates. The methylfolate pool appeared to remain separate from the tetrahydrofolate pool. More polyglutamate was formed from tetrahydropteroylglutamic acid than from 5-methyltetrahydropteroylglutamic acid. There was a fall in the folate content of the liver in the vitamin B-12-deficient bat and a more rapid incorporation of folates into polyglutamates but thereafter a more rapid loss of the labelled folate from liver.     

Kinetic characterization and substrate requirement for the Ca2+ uptake system in platelet membrane

An ATP-dependent mechanism for Ca²⁺ uptake in human platelet membrane fractions has been identified and characterized. Ca²⁺ uptake into a membrane fraction is shown to be stimulated at low concentrations of ATP and Ca²⁺ and to require magnesium ions. Initial rate kinetics, using Eadie-Scatchard analysis, indicated a single class of calcium uptake sites in the presence of ATP, with a $\text{K}{}_{\mathrm{<mtext>d</mtext>}}$ for free [Ca²⁺] of 0.145 μM. Ca²⁺ uptake in the presence of several ATP concentrations demonstrates that ATP binds to at least two sites, representing high and low affinities of 3.21 and 80.1 μM, respectively. The neuroleptic drug fluphenazine inhibited ATP-stimulated calcium uptake ($\text{IC}{}_{50}\text{= 55 μ}\text{M}$), suggesting this ATP-dependent Ca²⁺ uptake system may provide a useful ion-transport model with which to study neuroleptic therapy in humans.     

Correction of timing errors in photomultiplier tubes used in phase-modulation fluorometry

The measurement of fluorescence lifetimes is known to be hindered by the wavelenght-dependent and photocathode area-dependent time response of photomultiplier tubes. A simple and direct method is described to minimize the effects in photomultiplier tubes for phase-modulation fluorometry. Reference fluorophores of known lifetime were used in place of the usual scattering reference. The emission wavelenghts of the reference and sample were matched by either filters or a monochromator, and the use of a fluorophore rather than a scatter decreases the differences in spatial distribution of light emanating from the reference and sample. Thus photomultiplier tube artifacts are minimized. Five reference fluorophores were selected on the basis of availability, ease of solution preparation, and constancy of lifetime with temperature and emission wavelenght. These compounds are p-terphenyl, PPO, PPD, POPOP and dimethyl POPOP. These compounds are dissolved in ethanol to give standard solutions that can be used over the temperature range from ?55 to +55°C. Purging with inert gas is not necessary. The measured phase and modulation of the reference solution is used, in conjunction with the known reference, lifetime, to calculate the actual phase and modulation of the exictation beam. The use of standard fluorophores does not require separate experiments to quantify photomultiplier effects, and does not increase the time required for the measurement of fluorescence lifetimes. Examples are presented which demonstrate the elimination of artifactual photomultiplier effects in measurements of the lifetimes of DADH (0.4 ns) and indole solutions quenched by iodide. In addition, the use of these reference solutions increases the accuracy of fluorescence lifetime measurements ranging ranging to 30 ns. We judge this method to provide more reliable lifetime measurements by the phase and modulation method. The test solutions and procedures we describe may be used by other laboratories to evaluate the performance of their phase fluorometers.