Activity In Membrane Fraction Research Articles

Guanine nucleotide activation of adenylate cyclase in saponin permeabilized glioma cells

We have compared the regulation of adenylate cyclase activity in membrane fractions from C6 glioma cells and in monolayer cultures of C6 cells that had been permeabilized with saponin. Guanine nucleotides (GTP and GTPγS) and isoproterenol increase adenylate cyclase activity in C6 membranes and in permeabilized C6 cells. In C6 membranes, guanine nucleotides activate adenylate cyclase in the presence or absence of isoproterenol; in permeabilized cells, however, guanine nucleotides increase adenylate cyclase activity only in the presence of isoproterenol. We suggest that the properties of the permeabilized cells more closely resemble those of intact cells, and that some component which is present in permeabilized cells but is lost following cell disruption may be important for the normal regulation of adenylate cyclase activity.

Adenylate cyclase activity in membrane fractions of adrenal tissue of human anencephalic fetuses.

There is greater basal and ACTH-stimulated adenylate cyclase activity in membrane fractions prepared from the neocortex of human fetal adrenal (HFA) tissue than in similar preparations from the fetal zone. In this study, the specific activity of adenylate cyclase was determined in membrane preparations of adrenal tissue obtained from anencephalic fetuses (n = 5) varying in gestational age from 17-43 weeks. The basal adenylate cyclase activity in membrane fractions of adrenals of anencephalics was 2.9 +/- 2.1 (mean +/- SEM) pmol mg protein-1 min-1, 3-5% of the average specific activity in membrane preparations of fetal zone or neocortex of normal fetuses. ACTH (10(-10)-10(-4) M) in the incubation mixture stimulated adenylate cyclase activity 2- to 5-fold in whole HFA membrane fractions. In contrast, ACTH, when added to adrenal membrane preparations of the anencephalics, did not stimulate adenylate cyclase activity. Furthermore, sodium fluoride or forskolin stimulated adenylate cyclase activity markedly in HFA membrane preparations of normal fetuses, but did not affect enzyme activity in adrenal membrane preparations of the anencephalics. In conclusion, the basal activity of adenylate cyclase in adrenal membrane preparations of anencephalics was low and unresponsive to brief exposure to ACTH, sodium fluoride, or forskolin. These findings as well as those of our previous investigations suggest that the expression of HFA adenylate cyclase may be regulated in part by ACTH.

George E. Brown memorial lecture. Role of atrial peptides in body fluid homeostasis.

Extracts of mammalian atria, but not ventricles, induce marked diuresis, natriuresis, and reduction in blood pressure when infused systemically in rats and dogs. These extracts also inhibit aldosterone biosynthesis and renal renin release. Natriuretic peptides, 21 amino acids and longer, have been isolated from atria of rodents and man, and share a nearly homologous amino acid sequence at the carboxyterminus. Natriuretic activity resides in a 17-amino acid ring formed by a disulfide bridge, and the C-terminal Phe-Arg appears necessary for full biological potency. The deoxyribonucleic acid-encoding atrial natriuretic peptides have been cloned and the gene structure elucidated. Reduction of the diuretic and natriuretic responses to an acute volume load by right atrial appendectomy first suggested a role for atrial peptides in the physiological response to plasma volume expansion. Subsequently, release of peptides with natriuretic and spasmolytic properties from isolated heart preparations in response to right atrial distension was demonstrated by bioassay and radioimmunoassay. The presence of these peptides in normal rat and human plasma in concentrations of 20-100 pM, and the findings of increased levels in response to acute and chronic plasma volume expansion, rapid atrial tachyarrhythmias, systemic hypertension, congestive heart failure, and renal insufficiency imply that they play an important role in body fluid homeostasis. The mechanisms by which atrial peptides increase renal salt and water excretion are as yet unclear. Renal vascular effects have been consistently demonstrated, and limited evidence for direct actions on tubule ion transport has also been reported recently. In vitro, these peptides cause precontracted vascular and nonvascular smooth muscle to relax, mediated by a direct action on smooth muscle cells. Specific receptors for these peptides have been characterized in crude membranes prepared from whole kidney homogenates and adrenal glomerulosa cells, in intact glomeruli and cultured glomerular mesangial cells, and in intact bovine aortic smooth muscle and endothelial cells. Natriuretic peptides stimulate cyclic guanosine monophosphate accumulation in target tissues, and augment particulate guanylate cyclase activity in membrane fractions, suggesting that cyclic guanosine monophosphate is the second messenger mediating their cellular action.

Carotenoid synthesis and pleiotropic effects in carotenoid-deficient seedlings of maize

Plastid-envelope membranes from seedlings ofZea mays L. made carotenoid-deficient by either norflurazon treatment or mutation lack an activity permitting conversion of phytoene to β-carotene. This activity in membrane fractions was measured by coincubation in vitro with a soluble system from spinach chloroplasts capable of converting [(14)C]isopentenyl pyrophosphate into phytoene. When grown in light, the carotenoid-deficient seedlings lack many soluble chloroplast proteins, including NADP-dependent malic enzyme (EC 1.1.1.40), pyruvate phosphate dikinase (EC 2.7.9.1), and ribulose-1,5-bisphosphate carboxylase (EC 4.1.1.39), but apparently still contain the soluble activities permitting synthesis of phytoene.

Opposite effects of ethanol on the activation of adenylyl cyclase in human corpus luteum membranes

The influence of an acute exposure to ethanol on adenylyl cyclase activity in membrane fractions prepared from human corpus luteum was investigated. Ethanol up to a concentration of 5% (v/v) was without effect on basal luteal adenylyl cyclase activity, but markedly potentiated stimulation of NaF and hCG in a dose-dependent manner. In contrast, ethanol progressively inhibited forskolin stimulation at the same range of ethanol concentrations. Maximal NaF and hCG responsiveness of adenylyl cyclase activity was observed at 5% ethanol and reached values 80% and 100% higher than controls without ethanol, respectively. However, at the same ethanol concentration, forskolin-stimulated enzymatic activity was reduced by 40% relative to controls. Equilibrium binding studies involving [ 125I]hCG interaction with luteal membranes in the presence of the concentration of ethanol showing maximal hCG responsiveness indicated that ethanol slightly affected (15% increase) the hCG binding compared to controls, without any appreciable change on the K d for the hormone. This minor effect of ethanol on gonadotropin binding sites contrasted greatly with the extent at which ethanol maximally potentiated the gonadotropin-stimulated adenylyl cyclase. GTP was found to be less effective than GMP-P(NH)P in sustaining ethanol potentiation, suggesting that ethanol is unlikely to act by inhibiting GTPase activity. These data indicate that the acute effects of ethanol inhibit forskolin-stimulated adenylyl cyclase at concentrations potentiating stimulatory effects of NaF and of hCG, and that the synergistic interaction of ethanol and gonadotropin stimulation of adenylyl cyclase is, at least in part, due to an increase in the functional coupling of the occupied hCG-receptor complex with the components of the enzyme system.

Interaction of the pneumococcal amidase with lipoteichoic acid and choline

The choline-containing lipoteichoic acid (LTA, Forssman Antigen) of Streptococcus pneumoniae suppresses the activity of the pneumococcal autolysin, an N-acetyl-muramoyl-L-alanine-amidase (amidase) in aqueous solution [Höltje and Tomasz (1975) Proc. Natl Acad. Sci. USA 72, 1690-1694]. The interaction between LTA and enzyme was used to establish a purification by affinity chromatography on LTA-Sepharose. The amidase could be eluted from the column with choline only. This implies that binding of the enzyme to LTA is mediated via the choline residues of the LTA. Upon binding to the LTA-Sepharose, the amidase converted from the applied E-form (an inactive form of the amidase) to the active C-form, a process which up to now was known to be mediated only by the pneumococcal choline-containing wall teichoic acid. Similar interactions between LTA and amidase seemed to occur in membrane fractions derived from choline-grown cells: the membrane-associated enzyme was present in the C-form and could be detached completely with choline, suggesting that the amidase is bound to the membrane attached LTA rather than being a membrane protein itself. This was supported by the absence of amidase activity in membrane fractions derived from ethanolamine-grown pneumococci, in which choline containing LTA is absent. The LTA-Sepharose-associated amidase was not inhibited, but retained its activity. The enzyme was also not inhibited by lipase-digested LTA. Both are conditions where the LTA is not present in micelles, unlike in aqueous solution. Therefore, mere binding to the LTA is probably not responsible for the inhibitory effect, but inhibition is a manifestation of an inaccessibility of the substrate for the amidase when bound to micellar LTA. When the interactions between choline and amidase were investigated, it was found that high choline concentrations (2%) inhibited the enzyme completely. Even in vivo, 2% choline in the culture medium led to phenotypically amidase-deficient pneumococci. Furthermore, in vitro, low choline concentrations (0.1%) suppressed the wall-mediated conversion. On the other hand, with high choline concentrations (2%) conversion took place in the absence of cell walls. Depending on how the amidase has been converted, the apparent Mr of the resulting C-amidase was different: the cell-wall-converted enzyme was of high Mr, whereas the choline-converted and the LTA-Sepharose-eluted enzyme showed an apparent low molecular mass known for the E-form, when analyzed on sucrose gradients.(ABSTRACT TRUNCATED AT 400 WORDS)

The hydrolysis of the alkenyl group from enthanolamine-plasmalogen by oligodendroglial cell-enriched fractions

The rate of hydrolysis of the 1-0-alkenyl group of sn-1-alk-1?-enyl-2-acyl-glycerylphosphorylethanolamine (alkenyl, acyl-GPE; ethanolamine plasmalogen) by plasmalogenase is higher in oligodendroglial cell-enriched fractions from bovine brain compared with fractions enriched in neuronal perikarya and astroglia. The distribution of plasmalogenase activity in membrane fractions isolated from bovine oligodendroglia has been compared with that of 'marker' enzymes. The highest specific activity was in a fraction enriched in plasma membranes, whilst most activity was recovered in an endoplasmic reticulum membrane fraction. In bovine oligodendroglial cell homogenates, the enzyme had a neutral pH optimum, had no requirement for divalent cations and its activity towards 1-alkenyl-GPE (lysoplasmalogen) was half that with alkenyl, acyl-GPE. C(16) alkenyl groups were hydrolysed more rapidly than C(18) alkenyl groups. With (3)H-labelled alkenyl, acyl-GPE as substrate, radioactivity in released aldehydes appeared in fatty acids esterified in phospholipid while the oxidation of fatty aldehydes was blocked by the addition of NADH. An NAD-dependent aldehyde dehydrogenase was found to be present in oligodendroglia which exhibited highest activity towards C(14)?C(18) aldehydes (K(m), 2 ?M).

In vitro studies on prolactin release and adenylate cyclase activity in human prolactin-secreting pituitary adenomas. Different sensitivity of macro- and microadenomas to dopamine and vasoactive intestinal polypeptide.

A number of human PRL-secreting pituitary adenomas were tested for their in vitro responsiveness to dopamine (DA) and vasoactive intestinal polypeptide (VIP). PRL release from tumor fragments and adenylate cyclase (AC) activity in membrane fractions were evaluated. Both the hormone release and the enzyme activity were found to be inhibited by DA and stimulated by VIP. Significant differences between microadenomas (diameter less than 10 mm) and macroadenomas have been demonstrated: 1) the basal secretory activity (calculated per milligram of tissue protein) was higher in macro- than in microprolactinomas; 2) PRL release and AC activity were inhibited by a given concentration of DA to a higher extent in macro- than in microadenomas; 3) the dose-effect curves of DA both on PRL release and AC activity showed that the potency, and, in some cases, also the efficacy of the catecholamine was greater in macro- than in microadenomas; 4) PRL release and AC activity were stimulated by a given concentration of VIP to ...

Triton-stimulated nucleoside diphosphatase activity: Subcellular localization in corn root homogenates

The distribution of latent UDPase activity (cold storage-activated) is similar to Triton-stimulated UDPase activity in membrane fractions separated by differential centrifugation as well as fractions purified by linear sucrose density centrifugation. The Triton-stimulated UDPase activity appears to be a specific marker for Golgi membranes in corn root homogenates. Detergent-activated UDPase activity provides a more reliable, less cumbersome way to monitor Golgi membranes compared to cold storage-activation and this marker can be used on fresh preparations.

Differentiation of factor V-like coagulant activity from catalytic phospholipid-like surface activity in membrane fractions derived from human platelets

To investigate the role of the platelet membrane as a catalytic surface in thrombin generation, a relatively physiologic clotting assay was devised that was based on the kaolin-activated partial thromboplastin time. Frozen-and-thawed lysed platelets from normal and Factor V-deficient donors were used as a model for maximally activated normal or V-deficient platelets. An aqueous dispersion of brain phospholipids was employed as an analogue of the lipid portion of a platelet membrane. This test system made it possible to distinguish quantitatively between the contributions of an intrinsic platelet-associated Factor V-like activity (platelet factor 1 or PF1) and phospholipid-like catalytic surface activity (platelet factor 3 or PF3). The degree to which a particular membrane preparation shortened the clotting time was related to its overall catalytic surface activity which was the sum of the contributions of PF1 and PF3. In platelet preparations, both PF1 and PF3 were associated with a membrane-rich fraction that was not easily sedimentable at 12,000g-min. The log-log slope obtained for dilutions of a membrane preparation was taken as catalytic surface effectiveness and was dependent upon the parametric level of Factor V in the test system. The observed dependence was different for each membrane preparation. Effectiveness appeared to be a combined function of both the availability and occupancy of Factor V binding sites as well as the specific affinity of such sites for Factor V(Va).

Stimulation by calmodulin of Ca 2+ uptake and (Ca 2+-Mg 2+) ATPase activity in membrane fractions from ox neurohypophyses

Calmodulin stimulated 45Ca 2+ uptake into a plasma membrane enriched fraction from ox neurohypophysial nerve endings and into a microsome fraction. The 45Ca 2+ uptake and the (Ca 2+-Mg 2+) ATPase activity in the plasma membrane fraction exhibited similar pCa and calmodulin sensitivities, suggesting that the enzyme activity is the biochemical expression of a high affinity Ca 2+ pump. Calmodulin thus seems to play a role in regulation of the intracellular free Ca 2+ concentration in the neurohypophysis.

The effects of phospholipids on the properties of hepatic 5'-nucleotidase.

Arrhenius plots of 5'-nucleotidase activity in microsomes or plasma membranes from rat liver exhibited transitions at approximately 35 degrees C. The enzyme was purified from homogenates after solubilization in 2% Triton X-100 and 1% sodium deoxycholate. After the initial steps of the purification, the enzyme was recovered in membranes, as judged by both thin section and freeze-fracture electron microscopy, which contained sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine. The purest fractions of 5'-nucleotidase were enriched approximate 3,000-fold, consisted of similar membranes, but only contained sphingomyelin. Thermal transitions were detected in Arrhenius plots of 5'-nucleotidase after detergent solubilization, in the membranes which contained the three phospholipids, but not in the purified fraction which contained only sphingomyelin; transitions were also detected after reassociation of the purified enzyme with microsomal or plasma membrane lipids and phosphatidylcholine but not with phosphatidylethanolamine. Phosphatidylcholines containing specific fatty acids all affected the energy of activation of 5'-nucleotidase, and the detergent Sarkosyl, which has been shown to dissociate phospholipids from 5'-nucleotidase (Evans, W. H., and Gurd, J. W. (1973) Biochem. J. 133, 189-199), caused a marked decrease in the stability of the enzyme to heating. Inhibition of 5'-nucleotidase by concanavalin A followed by reactivation with alpha-methyl-D-mannoside resulted in linear Arrhenius plots of 5'-nucleotidase activity in membrane fractions, and in lower transition temperatures for the detergent, solubilized enzyme. It is concluded that in situ, 5'-nucleotidase interacts with both sphingomyelin and phosphatidylcholine; the first apparently influences the stability of the enzyme and the second, the energy of activation. In addition, the lipid environment of the enzyme seems to be altered as a result of lectin binding.

Proline transport activity in Escherichia coli membrane vesicles of different buoyant densities

Cytoplasmic membrane vesicles prepared by lysis of Escherichia coli W 3110 spheroplasts in a French press at 0° C are heterogeneous with respect to density due to membrane protein aggregation as a result of lateral phase separation of membrane phospholipids and to the presence of more or less outer membrane. These different vesicle classes can be separated on isopycnic density gradients. Assays for various membrane-associated functions show that the membranes differ not only with respect to density and structure but also with respect to function. The proline transport system (as detected by uptake experiments with the artificial electron donor ascorbate-phenazine methosulfate) shows maximal activities in membrane fractions that have considerably higher densities than the normal cytoplasmic membrane. This is always the case, whether vesicles are isolated from membranes that exhibit a temperature-induced protein aggregation or not. A correlation between high proline transport activity and the presence of vesicles with double membranes (consisting of outer and inner membrane) has been established. The possibility that the outer membrane protects the transport system in the cytoplasmic membrane during the isolation of vesicles is discussed.

Adenosine 3':5'-monophosphate and hormone interrelationships in the mammary gland of the rat during pregnancy and lactation.

The activities of adenyl cyclase, the phosphodiesterases of low and high Km and the tissue content of adenosine 3′:5′‐monophosphate (3′: 5′‐AMP) in the mammary glands of rats at different stages of the lactation cycle have been measured. The effect of in vitro F−, Ca2+ and of the hormones progesterone, β‐estradiol, hydrocortisone, insulin and prolactin on the adenyl cyclase activity of membrane fractions isolated from the mammary glands of pregnant and lactating rats has been investigated. The results show that there is a coordinated change of the activities of the enzymes adenyl cyclase and 3′:5′‐AMP phosphodiesterase and the tissue content of 3′:5′‐AMP at different stages of the lactation cycle with the activity of adenyl cyclase and the level of the cyclic nucleotide rising to a peak at the end of pregnancy and then falling sharply after parturition. The reverse pattern was observed for the 3′:5′‐AMP phosphodiesterase activity. F−(1–10 mM) consistently produced an inhibition (40%) of adenyl cyclase activity of mammary glands from pregnant rats while having no effect on the enzyme from the lactating gland. Low concentrations of Ca2+ (1 μM) activated the enzyme from pregnant glands and from those of early lactation by about 40% but had little effect on the glands taken from rats of mid‐lactation. Increasing the Ca2+ concentration led to a decline of this effect and eventual inhibition at 5 mM, the effect being most marked on the fully lactating gland. Progesterone and β‐estradiol consistently caused a considerable stimulation (80 and 50%, respectively) of the adenyl cyclase activity of the pregnant gland; hydrocortisone, insulin and prolactin did not. None of the hormones tested activated the adenyl cyclase activity of the lactating gland and insulin appeared to inhibit it severely. The results, which suggest that the growth and development of the mammary gland is related to the tissue content of 3′: 5‐AMP while the initiation and scale of lactation may be related to its removal, are discussed in relation to development, lactogenesis and milk ejection and the complex endocrine control of these processes.

Properties of ATP-ase activities of membrane fractions from the sheath of squid giant axons

Properties of ATP-ase activities of membrane fractions from the sheath of squid giant axons