The effects of deuterium oxide (2H2O) on the polymerization of tubulin in vitro

The initial rate and final extent of polymerization of both bovine brain tubulin and sea urchin egg tubulin were enhanced in the presence of ²H₂O. The yields were increased in association with the elevation of the ²H₂O concentration. ²H₂O also reduced the critical concentration for polymerization of brain tubulin. Thermodynamic analysis was attempted using the temperature dependence of the critical concentration for polymerization in the presence of ²H₂O. We obtained linear van 't Hoff plots and calculated thermodynamic parameters which were positive and were increased with the elevation of the ²H₂O concentration. The enhancement of the polymerization of tubulin by ²H₂O could, therefore, be the result of the strenghening of intra-and/or inter-molecular hydrophobic interactions of the tubulin molecules. We believe that the increase in lenghth and number of microtubules of the mitotic spindles in the dividing cells of the eukaryotes with ²H₂O may be caused by the direct involvement of ²H₂O in the polymerization of tubulin.     

Presence of calmodulin in human platelet cytoskeletons and its concentration change upon activation of platelets

We found that a small, reproducible amount of calmodulin is present in the cytoskeleton of human platelets. Triton-insoluble materials (cytoskeletons), which were prepared by cetrifugation at 1000 × g for 10 min of platelets after lysis by Triton X-100, stimulated cyclic AMP phosphodiesterase activity in the presence of Ca²⁺ but not in the presence of the calcium chelator, EGTA, or the calmodulin antagonist, trifluoperazine. The activation of the enzyme was also obtained after heating Triton-insoluble materials. An alkaline glycerol polyacrylamide gel electrophoresis of fractions obtained after gel fitration of solubilized Triton residues showed a protein band which had a faster electrophoretic mobility in the absence than in the presence of Ca²⁺. Upon thrombin activation of platelets, calmodulin in the Triton-insoluble cytoskeletons increased rapidly parallel to actin, actin-binding protein and myosin. With other stimulants such as collagen, epinephrine and ADP, similar results were obtained but with slower association of these proteins with cytoskeletons. However, after treatment with the Ca²⁺-inophore A23187, calmodulin, actin and actin-binding protein in Triton residues decreased rapidly, whereas the association of myosin increased. Thus, calmodulin seems to be associated with actin filaments rather than myosin filaments, and may be involved in the generation of contractile force in the cell.     

Lipid phase transitions in model biomembranes: The effect of ions on phosphatidylcholine bilayers

Differential scanning calorimetry has been used to study the endothermic phase behaviour of some model biomembranes (i.e. phosphatidylcholine-water systems) in the presence of a wide range of alkaline, alkaline earth and heavy metal salts. Studies and comparisons were made of both cation and anion effects. Shifts occur in the temperatures of both the pre-transition and main transition endotherms. The observed shifts are smaller than those which have been reported for charged lipids, and no evidence has been found for the formation of specific complexes. Electron microscopic studies on freeze-fractured dispersions of phosphatidylcholine-water-salt systems show that with some salts the typical rippled surface observed with l-α-dimyristoyl phosphatidylcholine, when in the gel state, is replaced by a smooth surface.     

Highly purified sarcoplasmic reticulum vesicles are devoid of Ca2+-independent (‘basal’) ATPase activity

On solubilization with Triton X-100 of sarcoplasmic reticulum vesicles isolated by differential centrifugation, the Ca²⁺-ATPase is selectively extracted while approximately half of the initial Mg²⁺-, or ‘basal’, ATPase remains in the Triton X-100 insoluble residue. The insoluble fraction, which does not contain the 100 000 dalton polypeptide of the Ca²⁺-ATPase, contains high levels of cytochrome c oxidase. Furthermore, its Mg²⁺-ATPase activity is inhibited by specific inhibitors of mitochondrial ATPase, indicating that the ‘basal’ ATPase separated from the Ca²⁺-ATPase by detergent extraction originates from mitochondrial contaminants.To minimize mitochondrial contamination, sarcoplasmic reticulum vesicles were fractionated by sedimentation in discontinuous sucrose density gradients into four fractions: heavy, intermediate and light, comprising among them 90–95% of the initial sarcoplasmic reticulum protein, and a very light fraction, which contains high levels of Mg²⁺-ATPase. Only the heavy, intermediate and light fractions originate from sarcoplasmic reticulum; the very light fraction is of surface membrane origin. Each fraction of sarcoplasmic reticulum origin was incubated with calcium phosphate in the presence of ATP and the loaded fractions were separated from the unloaded fractions by sedimentation in discontinuous sucrose density gradients. It was found that vesicles from the intermediate fraction had, after loading, minimal amounts of mitochondrial and surface membrane contamination, and displayed little or no Ca²⁺-independent basal ATPase activity. This shows conclusively that the basal ATPase is not an intrinsic enzymatic activity of the sarcoplasmic reticulum membrane, but probably originates from variable amounts of mitochondrial and surface membrane contamination in sarcoplasmic reticulum preparations isolated by conventional procedures.     

Some properties of the purified (Ca2+ + Mg2+-ATPase from human red cell membranes

The (Ca²⁺ + Mg²⁺-ATPase from red cell membranes, purified by means of a calmodulin-containing affinity column according to the method of Gietzen et al. (Gietzen, K., Tej?ka, M. and Wolf, H.U. (1980) Biochem. J. 189, 81–88) with either phosphatidylcholine or phosphatidylserine as phospholipid is characterized. The phosphatidylcholine preparation can be activated by calmodulin, while the phosphatidylserine preparation is fully activated without calmodulin. The enzyme shows a biphasic ATP dependence with two K_m values of 3.5 and 120 μM. The enzyme is phosphorylated by ATP in the presence of Ca²⁺ only.     

Catecholamine-induced phosphorylation of cardiac muscle proteins

The catecholamine-induced phosphorylation of cardiac muscle protein was investigated using a rat ventricular muscle slice preparation. Slices 0.5 mm thick and weighing 40–50 mg were incubated for 40 min in oxygenated bathing medium containing ³²P to partially label intracellular ATP. Subsequent addition of 10^?5 M isoproterenol for 10 min resulted in a 44–63% (based on protein) or a 63–70% (based on inorganic phosphate) increase in ³²P incorporation into 100 000 × g particulate and 100 000 × g supernatant (soluble) fractions without an increase into homogenates, 1000 and 29 000 × g particulate fractions prepared from the slices. The catecholamines also produced a 93% increase in ³²P incorporation ans a 27% increase in inorganic phosphate in trichloroacetic acid-insoluble protein that was obtained from ventricular slice homogenates. A significant increase in the incorporation of ³²P occurred in the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein within 2 and 1 min, respectively. While the β-adrenergic blocking agent propanolol had no effect by itself on ³²P incorporation, it prevented the isoproterenol-induced incorporation of ³²P into the 100 000 × g particulate and supernatant fractions and the acid-insoluble protein. Removal of isoproterenol from the bathing medium eliminated the differences in ³²P incorporation, indicating that the effects of the catecholamine were reversible. Norepinephrine and ipinephrine at 10^?5 M caused phosphorylation effects similar to that of isoproterenol. When the slices were bathed under anoxic conditions isoproterenol failed to enhance the incorporation of ³²P into proteins of the 100 000 ×g particulate and supernatant fractions or acid-insoluble protein. SDS gel eloectrophoresis of ventricular slice homogenates revealed that isoproterenol enhanced the ³²P incorporation into several myocardial proteins having molecular weights of 155, 94 (glycogen phosphorylase), 79, 68–77, and 54–59 · 10³ and decreased the incorporation into a 30 · 10³ dalton protein(s). These results are consistent with the notion that catecholamines may increase the phosphorylation of myocardial proteins in the intact myocardium which in turn may play a role in catecholamine-induced glycogenolysis and augmentation of contractility.     

Regulation of calcium accumulation and efflux from platelet vesicles: Possible role for cyclic-AMP-dependent phosphorylation and calmodulin

Calcium-accumulating vesicles were isolated by differential centrifugation of sonicated platelets. Such vesicles exhibit a $\text{(Ca}{}^{\mathrm{2+}}\text{+ Mg}{}^{\mathrm{2+}}\text{)-ATPase}$ activity of about 10 nmol (min·mg)^?1 and an ATP-dependent Ca²⁺ uptake of about 10 nmol (min·mg)^?1. When incubated in the presence of Mg[γ-³²P]ATP, the pump is phosphorylated and the acyl phosphate bond is sensitive to hydroxylamine. The [³²P]phosphate-labeled Ca²⁺ pump exhibits a subunit molecular weight of 120 000 when analyzed by lithium dodecyl sulfate-polyacrylamide gel electrophoresis. Platelet calcium-accumulating vesicles contain a 23 kDa membrane protein that is phosphorylatable by the catalytic subunit of cAMP-dependent protein kinase but not by protein kinase C. This phosphate acceptor is not phosphorylated when the vesicles are incubated in the presence of either Ca²⁺ or Ca²⁺ plus calmodulin. The latter protein is bound to the vesicles and represents 0.5% of the proteins present in the membrane fraction. Binding of ¹²⁵I-labeled calmodulin to this membrane fraction was of high affinity (16 nM), and the use of an overlay technique revealed four major calmodulin-binding proteins in the platelet cytosol ($\text{M}{}_{\mathrm{r}}\text{= 94 000, 87 000, 60 000 and 43 000}$). Some minor calmodulin-binding proteins were enriched in the membrane fractions ($\text{M}{}_{\mathrm{r}}\text{= 69 000, 57 000, 39 000 and 37 000}$). When the vesicles are phosphorylated in the presence of MgATP and of the catalytic subunit of cAMP-dependent protein kinase, the rate of Ca²⁺ uptake is essentially unaltered, while the Ca²⁺ capacity is diminished as a consequence of a doubling in the rate of Ca²⁺ efflux. Therefore, the inhibitory effect of cAMP on platelet function cannot be explained in such simple terms as an increased rate of Ca²⁺ removal from the cytosol. Calmodulin, on the other hand, was observed to have no effect on the initial rate of calcium efflux when added either in the absence or in the presence of the catalytic subunit of the cyclic AMP-dependent protein kinase, nor did the addition of 0.5 μM calmodulin result in increased levels of vesicle phosphorylation.     

Biosynthesis of mouse erythrocyte membrane proteins by friend erythroleukemia cells

The synthesis of mouse erythrocyte membrane proteins by Friend erythroleukemia cells during dimethyl sulfoxide-induced differentiation was studied. Untreated and dimethyl sulfoxide-treated cells were incubated with l-[³H] leucine and the incorporation of radioactivity into total trichloroacetic acid-insoluble proteins and into proteins immunoprecipitated with a multivalent rabbit antibody to mouse erythrocyte membranes was determined. The immunoprecipitated membrane proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and radioactivity was detected by fluorography. The incorporation of l-[³H]leucine into total cell proteins was linear for 20 min in both untreated and treated cells. Exposure of the cells to dimethyl sulfoxide had an inhibitory effect on protein synthesis, with a significant decrease noted on the fourth day of treatment and a continued decline occurring until the seventh day when protein synthesis was 42% that of untreated cells. The synthesis of erythrocyte membrane proteins was 0.49% that of total cell proteins in untreated cells, was increased to 1.27% by the third day of treatment and remained at about 1% of total protein synthesis from the fourth to the seventh day. Untreated cells synthesized low levels of spectrin, bands 5 and 6 proteins. Treatment with dimethyl sulfoxide caused a staggered increase in synthesis of a number of erythrocyte membrane proteins. Spectrin synthesis increased 4-fold by the third day of treatment and declined thereafter. The synthesis of membrane proteins with electrophoretic mobilities similar to bands 3 and 4 was increased 2–3-fold by the fourth day, while bands 6 and 5 proteins attained maximal synthesis (4-fold) on the fifth and sixth days of treatment.     

Vesiculation of platelet plasma membranes. Dilauroylglycerophosphocholine-induced shedding of a platelet plasma membrane fraction enriched in acetylcholinesterase activity

Incubation of washed rabbit platelets with suspensions of dilauroylglycerophosphocholine resulted in the shedding of vesicles without causing any appreciable leakage of cytoplasmic marker (lactate dehydrogenase) or organelle marker ([¹⁴C]serotonin). The response was dependent on incubation time, concentration of dilauroylglycerophosphocholine and reaction temperature. Vesicles were separated from platelets and exogenous dilauroylglycerophosphocholine by a series of centrifugation steps. An average diameter of vesicles was 100–200 nm on scanning electron microscopy. Vesicles were enriched 5-fold in plasma membrane marker enzyme, acetylcholinesterase, whereas specific activities of lactate dehydrogenase and intracellular membrane marker enzyme, NADH-cytochrome c reductase were decreased in vesicles. Protein analysis by SDS-polyacrylamide gel electrophoresis showed that actin and actin-binding protein were present, while myosin was barely detectable in vesicles. Vesicles contained all phospholipid species of intact platelets and cholesterol but almost 50% of phospholipids in vesicles was dilauroylglycerophosphocholine. The phospholipid to protein ratio in vesicles was about 6.5-times higher than in intact platelets.     

Mechanisms of hormonal modulation of ion transport in the toad's urinary bladder: Subcellular localization of aldosterone-induced proteins

A dual-label isotope technique was used to study the effects of aldosterone upon the incorporation of amino acids into proteins of the in vitro toad urinary bladder. Following labeling, the mucosal cells were disaggregated and the mitochondria-rich and granual cells were separated. Proteins with an elevated isotope ratio were found in a plasma membrane fraction (170 000, 110 000 and 85 000 daltons) and in the cytosol (36 000 and 6 000 daltons) of the preparations enriched in mitochondria-rich cells. These effects of aldosterone were blocked by cycloheximide. There was no evidence that aldosterone had induced the incorporation of labeled amino acids into carbonic anhydrase isolated from the soluble fraction by affinity chromatography. The results suggests that the physiologic response of the toad bladder to aldosterone is related to the synthesis of both soluble and plasma membrane proteins.     

Histone phosphorylation in phorbol ester stimulated and β-adrenergically stimulated mouse epidermis in vivo and characterization of an epidermal protein phosphorylation system

Under certain physiological conditions a change i n the phosphorylation of histones in mouse epidermis in vivo was observed. Thus a single local application of the tumor-promoting mitogen 12-O-tetradecanoylphorbol-13-acetate caused a long-lasting increase of histone H1 phosphorylation which paralleled stimulated cell proliferation. Injection of the antimitotic β-adrenergic agonist isoproterenol led to a temporatory decrease in the rate of phosphorylation of H1, H2A and H2b immediately after cyclic AMP accumulation. A complete protein phosphorylation system could be demonstrated in mouse epidermis homogenates. The following enzyme activities were partially purified and characterized: a cyclic AMP-dependnet histone kinase; a ‘casein kinase’ and an ‘unsopecific’ protein kinase; a histone-specific protein phosphatase; and two ‘unspecific’ phosphoprotein phosphatases. In addition, a stimulatory effect of cyclic GPM on histone phosphorylation was observed. The enzymes were found to be predominantly localized in the 105 000 × g supernatant, but a small proportion of protein kinase and phosphatase activity could be regularly demonstrated in cell nuclei.     

Maturation dependent decline of adenylate cyclase in rabbit red blood cell membranes

Adenylate cyclase (EC 4.6.1.1) was studied in membrane preparations of reticulocyte-rich blood obtained from phenylhydrazine-treated rabbits and compared to that of untreated animals.Basal and fluoride-stimulated activities were decreased 2- and 4-fold, respectively, during the process of maturation.Catalytic parameters such as time course, protein, ATP, Mg²⁺ concentration curves and K_m have been determined and were found to be similar in the reticulocyte and the erythrocyte.Adenylate cyclase was sensitive to GTP, 5′-guanylyl imidodiphosphate, prostaglandin E₁ and prostaglandin E₂. Activation by prostaglandin E₁ was higher than that produced by prostaglandin E₂. Only additive effect was found when 5′-guanylyl imidodiphosphate or GTP was added to hormone-stimulated activity. The sensitivity of the enzyme to these effectors was decreased over the transition reticulocyte-erythrocyte.In either cell the enzyme was not activated by catecholamines (epinephrine, norepinephrine, isoproterenol).     

Evidence for two types of β-adrenergic-sensitive adenylate cyclase activities in bovine cerebellum

A latent, as well as an expressed form of adenylate cyclase coupled to β-adrenergic receptors is present in intact crude synaptosomal preparations from bovine cerebellum. The latent adenylate cyclase activity was assayed in Krebs-Ringer buffer by [³H]adenine labeling and was found to be coupled to a β₁-like adrenergic receptor. The externally accessible adenylate cyclase assayed in the same with [³H]ATP was stimulated via β₂-adrenergic receptors.     

Site-directed mutagenesis of the His residues of the rat mitochondrial carnitine/acylcarnitine carrier: Implications for the role of His-29 in the transport pathway

The mitochondrial carnitine/acylcarnitine carrier (CAC) of Rattus norvegicus contains two His, His-29 and His-205. Only the first residue is conserved in all the members of the CAC subfamily and is positioned before the first of the three conserved motifs. In the homology model of CAC, His-29 is located in H1 close to the bottom of the central cavity. His-205 is the first amino acid of H5 and it is exposed towards the cytosol. The effect of substitution of the His residues on the transport function of the reconstituted mutant CACs has been analysed, in comparison with the wild-type. H29A showed very low activity, H29K and H29D were nearly inactive, whereas H205A, H205K and H205D showed activities similar to that of the wild-type. His-29 has also been substituted with Gln, Asn, Phe and Tyr. All the mutants showed very low transport function and, similarly to H29A, higher Km, reduced Vmax and altered selectivity towards (n)acylcarnitines, with the exception of H29Q, which exhibited functional properties similar to those of the wild-type. The experimental data, together with a comparative analysis of the carnitine acyltranferase active sites, indicated that His-29 forms an H-bond with the β-OH of carnitine. The substitution of His-205 led to a change of response of the CAC to the pH. The results are discussed in terms of relationships of His-29 with the molecular mechanism of translocation of the CAC.     

An improved purification and further characterization of the messenger ribonucleic acid for the fatty acid synthetase from rat liver

High purity fatty acid synthetase mRNA has been prepared from rat liver. The translational purity of the mRNA preparation was at least 27% as judged by the percentage of the radioactivity incorporated into acid-insoluble material that was precipitated by anti-fatty acid synthetase antibody. The specific activity of the mRNA was 220-times greater than that reported previously from this laboratory [1]. The large increase in the specific activity was achieved by the repeated use of high resolution linear-log sucrose density gradient centrifugation and the removal of 28 S rRNA by Sepharose 4B chromatography, as well as by the optimization of the K⁺ concentration (160 mM) in the reticulocyte lysate translation system. The mRNA preparation showed a single major band on agarose gel electrophoresis under denaturing conditions, and the translational activity of the fatty acid synthetase mRNA on the gel was found to coincide with this band. The molecular weight of the fatty acid synthetase mRNA is 2.5·10⁶ Da. The mRNA directed the synthesis of fatty acid synthetase with a molecular weight indistinguishable from that of the authentic enzyme subunit (M_r = 240 000). The copurification of the translation product and authentic enzyme revealed that the fatty acid synthetase polypeptides synthesized in the reticulocyte lysate system are assembled in vitro into dimers, the native form of the enzyme.     

Crosslinking of platelet glycoprotein Ib by N-succinimidyl(4-azidophenyldithio)propionate and 3,3′-dithiobis(sulfosuccinimidyl propionate)

To examine the relationship between glycoprotein Ib and other proteins in the platelet membrane and the interaction of this protein with thrombin, platelets were crosslinked by two cleavable reagents, SADP (N-succinimidyl(4-azidophenyldithio)propionate) and DTSSP (3,3′-dithiobis(sulfosuccinimidyl propionate)). Two-dimensional, unreduced-reduced sodium dodecyl sulphate (SDS)-polyacrylamide electrophoresis and staining by silver or wheat germ agglutinin-conjugated peroxidase, after protein transfer to nitrocellulose, demonstrated that SADP intramolecularly crosslinked glycoprotein Ib and formed intermolecular complexes of glycoprotein IIb and some high molecular weight proteins. DTSSP intermolecularly crosslinked glycoprotein Ib, glycoprotein IIb, and other high molecular weight proteins. With a low concentration of ¹²⁵I-labeled TLCK-thrombin (6 nM), crosslinking with SADP yielded a 200 000 Da complex containing radioactive-labeled thrombin, and high TLCK-thrombin concentration (0.1 μM) gave the complex and a 167 000 band. α- and TLCK-thrombin crosslinking with DTSSP also yielded the 200 000 complex, with the remaining radioactivity in a band corresponding to a highly crosslinked complex. The 200 000 complex formed by reaction with SADP or DTSSP was markedly reduced by preincubation of platelets with excess unlabeled TLCK-thrombin and had a pI similar to glycoprotein Il. These results suggest that glycoprotein Il is one of the proteins composing the high affinity receptor for thrombin.     

Interaction of guanine nucleotides with adenylate cyclase in normal and spontaneously transformed RL-PR-C cloned rat hepatocytes

Spontaneous transformation of RL-PR-C hepatocytes leads to alterations in the adenylate cyclase complex which include a lower than normal basal level of activity, a loss of sensitivity to exogenous GTP, and a decreased sensitivity to isoproterenol. Both normal and transformed membranes posses substantial TGPase activity. Treatment of transformed hepatocyte membranes with either isoproterenol plus GMP or with cholera toxin, under conditions that displace tightly bound GDP, restored the GTP effect on adenylate cyclase, and eliminated the lag in the activation by guanyl-5′-yl-imidodiphosphate. Such pretreatment also enhanced guanine nucleotide effects on the adenylate cyclase of normal hepatocytes. These results are explainable on the basis that transformation increases adenylate cyclase-associated GTPase activity, and increase occupancy of nuceotide regulatory sites by inactive or inhibitory guanine nucleotides, e.g., GDP. Seemingly, both catecholamines and cholera toxin promote an exchange reaction at the regulatory sites, resulting in clearance of these sites of inhibitory nucleotides.     

Studies on the mechanism of regulation of the red-cell Ca2+ pump by calmodulin and ATP

(1) The effects of calmodulin binding on the rates of Ca²⁺-dependent phosphorylation and dephosphorylation of the red-cell Ca²⁺ pump, have been tested in membranes stripped of endogenous calmodulin or recombined with purified calmodulin. (2) In Mg²⁺-containing media, phosphorylation and dephosphorylation rates are accelerated by a large factor (at 0°C), but the steady-state level of phosphoenzyme is unaffected by calmodulin binding (at 0°C and 37°C). In Mg²⁺-free media, slower rates of phosphoenzyme formation and hydrolysis are observed, but both rates and the steady-state phosphoenzyme level are raised following calmodulin binding. (3) At 37°C and 0°C, the rate of (Ca²⁺ + Mg²⁺)-ATPase activity is stimulated maximally by 6–7-fold, following calmodulin binding. At 37°C the apparent Ca²⁺ affinity for sustaining ATP hydrolysis is raised at least 20-fold, K_m(Ca) ? 10 μM (—calmodulin) and K_m(Ca) < 0.5 μM (+ calmodulin), but at 0°C the apparent Ca²⁺ affinity is very high in calmodulin-stripped membranes and little or no effect of calmodulin is observed (K_m(Ca) ? 3–4 · 10^-8 M). (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin activated membranes and at saturating ATP levels, is sharply inhibited by addition of calcium in the range 50–2000 μM. (4) A systematic study of the effects of the nucleotide species MgATP, CaATP and free ATP on (Ca²⁺ + Mg²⁺)-ATPase activity in calmodulin-activated membranes reveals: (a) In the 1–10 μmolar concentration range MgATP, CaATP and free ATP appear to sustain (Ca²⁺ + Mg²⁺)-ATPase activity equally effectively. (b) In the range 100–2000 μM, MgATP accelerates ATP hydrolysis (K_m(MgATP) ? 360 μM), and CaATP is an inhibitor (K_i(CaATP) ? 165 μM), probably competing with MgATP fo the regulatory site. (5) The results suggest that calmodulin binding alters the conformational state of the Ca²⁺- pump active site, producing a high (Ca²⁺ + Mg²⁺)-ATPase activity, high Ca²⁺ affinity and regulation of activity by MgATP.     

Proline hydroxylation by cell free extract of a streptomycete

Free L-proline was hydroxylated to free L-hydroxyproline by cell free extract of Streptomyces griseoviridus P8648. The hydroxylation reaction required ferrous ion, 2-ketoglutarate and ascorbate. Zinc ion, ethylenediaminetetraacetic acid and alpha,alpha'-dipyridyl inhibited the reaction. Optimum temperature and pH were 25.0 degrees C and 7.5, respectively.