The alpha and beta subunits of lamb kidney Na,K-ATPase are both glycoproteins

We have determined the carbohydrate compositions of the protein components of lamb kidney Na,K-ATPase. The α subunit contains a total of about 16 monosaccharide residues per mol of protein, while the β subunit contains about 36 residues per mol. The γ protein, a proteolipid associated with the Na,K-ATPase, contains only traces of carbohydrate. A comparison of our results with those of others shows considerable variability in the carbohydrate compositions of α and β subunits from different species.     

(Na (+) + K (+)-stimulated ATPase of human kidney, normal and adenocarcinoma. Phosphorylation and inhibition by antitumor proteins

(Na⁺+K⁺)-ATPase was purified from human kidney of normal and tumor tissue with specific activities of 100.0 and 16.6 μmol Pi/mg/h, respectively. The antitumor proteins, macromomycin, largomycin, and NSC 327459 (50 μg/ml each) caused 70 to 90% inhibition of (Na⁺+K⁺)-ATPase from tumor tissue, whereas auromomycin had no effect. (Na⁺+K⁺)-ATPase from both sources could be phosphorylated by rabbit muscle protein kinase; there was 3 to 6-fold stimulation of phosphorylation by cyclic AMP. Phosphorylation resulted in 70 to 80% decrease in (Na⁺+K⁺)-ATPase activity, and caused the normal enzyme to become sensitive to inhibition by macromomycin.     

Minimal functional unit for transport and enzyme activities of (Na+ + K+)-ATPase as determined by radiation inactivation

Frozen aqueous suspensions of partially purified membrane-bound renal (Na⁺ + K⁺)-ATPase have been irradiated at –135°C with high-energy electrons. (Na⁺ + K⁺)-ATPase and K⁺-phosphatase activities are inactivated exponentially with apparent target sizes of $\text{184 ± 4 kDa and 125 ± 3 kDa}$, respectively. These values are significantly lower then found previously from irradiation of lyophilized membranes. After reconstitution of irradiated (Na⁺ + K⁺)-ATPase into phospholipid vesicles the following transport functions have been measured and target sizes calculated from the exponential inactivation curves: ATP-dependent Na⁺?K⁺ exchange, $\text{201 ± 4}\text{kDa}$; (ATP + P_i)-activated Rb⁺?Rb⁺ exchange, $\text{206 ± 7}\text{kDa}$ and ATP-independent Rb⁺?Rb⁺ exchange, $\text{117 ± 4}\text{kDa}$. The apparent size of the α-chain, judged by disappearance of Coomassie stain on SDS-gels, lies between 115 and 141 kDa. That for the β-glycoprotein, though clearly smaller, could not be estimated. We draw the following conclusions: (1) The simplest interpretation of the results is that the minimal functional unit for (Na⁺ + K⁺)-ATPase is αβ. (2) The inactivation target size for (Na⁺ + K⁺)-dependent ATP hydrolysis is the same as for ATP-dependent pumping of Na⁺ and K⁺. (3) The target sizes, for K⁺-phosphatase (125 kDa) and ATP-independent Rb⁺?Rb⁺ exchange (117 kDa) are indistinguishable from that of the α-chain itself, suggesting that cation binding sites and transport pathways, and the $\text{p-}\text{nitrophenyl}$ phosphate binding site are located exclusively on the α-chain. (4) ATP-dependent activities appear to depend on the integrity of an αβ complex.     

Kinetic studies on interaction of (Na,K)-ATPase with Cibacron Blue F3GA as probe of the nucleotide fold

Cibacron Blue F3GA (Cb) effectively and reversibly inhibits the activity of (Na,K)-ATPase. Its inhibitory effect does not occur through occupation of the ouabain binding site, but presumably results from Cb-occupation of one catalytic site not competitively attracting ATP. Cb also inhibits ouabain binding to (Na,K)-ATPase. Its inhibitory effect is competitively antagonized by ATP proving accomodation of Cb in the ATP binding site. - If one admits Cb as a suitable analytical tool for the detection of a supersecondary structure folding pattern, the findings suggest that the ATP binding site is lined by β-pleated sheets flanked by α-helices thus providing an environment that funnels ATP to the catalytic site.     

Stabilization of Na,K-ATPase by ionic interactions

The effect of ions on the thermostability and unfolding of Na,K-ATPase from shark salt gland was studied and compared with that of Na,K-ATPase from pig kidney by using differential scanning calorimetry (DSC) and activity assays. In 1 mM histidine at pH 7, the shark enzyme inactivates rapidly at 20 °C, as does the kidney enzyme at 42 °C (but not at 20 °C). Increasing ionic strength by addition of 20 mM histidine, or of 1 mM NaCl or KCl, protects both enzymes against this rapid inactivation. As detected by DSC, the shark enzyme undergoes thermal unfolding at lower temperature (T_m ≈ 45 °C) than does the kidney enzyme (T_m ≈ 55 °C). Both calorimetric endotherms indicate multi-step unfolding, probably associated with different cooperative domains. Whereas the overall heat of unfolding is similar for the kidney enzyme in either 1 mM or 20 mM histidine, components with high mid-point temperatures are lost from the unfolding transition of the shark enzyme in 1 mM histidine, relative to that in 20 mM histidine. This is attributed to partial unfolding of the enzyme due to a high hydrostatic pressure during centrifugation of DSC samples at low ionic strength, which correlates with inactivation measurements. Addition of 10 mM NaCl to shark enzyme in 1 mM histidine protects against inactivation during centrifugation of the DSC sample, but incubation for 1 h at 20 °C prior to addition of NaCl results in loss of components with lower mid-point temperatures within the unfolding transition. Cations at millimolar concentration therefore afford at least two distinct modes of stabilization, likely affecting separate cooperative domains. The different thermal stabilities and denaturation temperatures of the two Na,K-ATPases correlate with the respective physiological temperatures, and may be attributed to the different lipid environments.     

Radioiodination of brain tubulin with Bolton-Hunter reagent

Radio-iodination of tubulin can be achieved by Bolton-Hunter reagent both in the absence and presence of microtubule associated proteins. Specific radioactivities as high as 400 C_i/mmole tubulin dimer can be obtained, i.e. an average of 0.2 molecule of reagent is bound per molecule of tubulin. About 80 % of the [¹²⁵I]- labelled tubulin keeps its ability to assemble in microtubules and polymerizes with the same critical concentration as the native tubulin, which makes the method adequate for preparing tracer tubulin useful for in vivo and in vitro studies. Both α and β subunits are labelled, 60 % of the radiolabel being bound to the β subunit.     

Isolation and structure of T-kinin

The Ca2+- and calmodulin-dependent myosin light chain kinase of rabbit skeletal muscle was converted to a Ca2+-independent form by limited proteolysis with alpha-chymotrypsin. The conditions prevailing during proteolysis are important and the loss of Ca2+-dependence was achieved best by hydrolysis of the Ca2+-calmodulin-kinase complex. The lack of Ca2+- and calmodulin-dependence was found using both myosin and isolated light chains as substrates. The specific activity of the Ca2+-independent form (Mr approximately 65,000) was similar to that of the native enzyme, i.e., 2 to 5 mumol phosphate transferred min-1 mg-1 kinase. The 65,000-dalton fragment was phosphorylated by the catalytic subunit of the cAMP-dependent protein kinase and approximately 0.8 moles phosphate were incorporated per fragment.     

Metal requirement of the isolated red cell Ca-pump ATPase after elimination of calmodulin dependence by trypsin attack

Mild proteolysis by trypsin activates the purified (Ca²⁺ + Mg²⁺) - ATPase protein from human red cells in a way which is similar to the effect obtained by addition of calmodulin. The trypsin concentration required to reach half maximal effect in 3 minutes at 37°C is 2.5 – 3.5 μg/ml. SDS-poly-acrylamide gel electrophoresis reveals a degradation of the main protein (150'000 Dalton) into a large fragment (95'000 – 100'000 Dalton) and a small fragment (35'000 – 40'000 Dalton). Increasing ATPase activity correlates with the degree of proteolysis.The _Ca of the digested (Ca²⁺ + Mg²⁺)-ATPase is 0.85 ± 0.1 μM Ca²⁺ as compared to 8.0 ± 0.75 μM Ca²⁺ before digestion and is statistically significantly different from _Ca = 1.66 ± 0.22 μM Ca²⁺ observed in activation by a saturating calmodulin concentration. Addition of calmodulin to the trypsinized enzyme has neither an effect on the Ca²⁺-affinity nor achieves any large increase of the maximal rate.High Ca²⁺ concentrations (above 0.05 – 0.1 mM) after trypsin treatment still inhibit the (Ca²⁺ + Mg²⁺)-ATPase activity. Mg²⁺ activates in the same concentration range ( _Mg = 25 μM) as in the undigested preparation ( _Mg = 27 μM) and retains its competitive behaviour towards Ca²⁺ after trypsin treatment.It is concluded that (1) trypsin treatment unmasks high affinity sites for Ca²⁺ ( _Ca 1 μM) and that, therefore, such sites are not added to the system by calmodulin, and (2) that inhibition by high Ca²⁺-concentrations is not due to Ca - Mg competition at sites located on the calmodulin molecule.     

The amino acid sequence of the fluorescein isothiocyanate reactive site of lamb and rat kidney Na+- and K+-dependent ATPase

Fluorescein 5'-isothiocyanate has been used to label ouabain sensitive and insensitive (Na,K)-ATPases from lamb and rat kidney, respectively. The labeled enzymes were digested with trypsin to generate soluble peptides, which were purified by high performance liquid chromatography and sequenced on a gas phase sequenator. The sequence of the labeled peptide from both species is His-Leu-Leu-Val-Met-Lys-Gly-Ala-Pro-Glu-Arg. Thus, it appears that the primary structure of the fluorescein 5'-isothiocyanate reactive site, and therefore presumably the ATP binding site, is completely conserved in ouabain sensitive and ouabain insensitive (Na,K)-ATPases.     

1-(5-Isoquinolinesulfonyl)-2-methylpiperazine (H-7) is a selective inhibitor of protein kinase C in rabbit platelets

Effects of 1-(5-isoquinolinesulfonyl)-2-methylpeperazine (H-7), a potent inhibitor of protein kinase C in vitro (1), were investigated with regard to stimulus-induced protein phosphorylation of rabbit platelets. While H-7 inhibited the protein kinase C-mediated phosphorylation in 12-0-tetradecanoylphorbol-13-acetate (TPA)-stimulated platelets, this compound did not block the Ca2+-calmodulin-dependent phosphorylation in Ca2+ ionophore A23187-stimulated cells. This selective inhibitor of protein kinase C, in intact cells, will facilitate studies on the biological functions of protein kinase C.     

A central role for intermolecular dityrosine cross-linking of fibrinogen in high molecular weight advanced oxidation protein product (AOPP) formation

Background

Advanced oxidation protein products (AOPPs) are dityrosine cross-linked and carbonyl-containing protein products formed by the reaction of plasma proteins with chlorinated oxidants, such as hypochlorous acid (HOCl). Most studies consider human serum albumin (HSA) as the main protein responsible for AOPP formation, although the molecular composition of AOPPs has not yet been elucidated. Here, we investigated the relative contribution of HSA and fibrinogen to generation of AOPPs.

Methods

AOPP formation was explored by SDS-PAGE, under both reducing and non-reducing conditions, as well as by analytical gel filtration HPLC coupled to fluorescence detection to determine dityrosine and pentosidine formation.

Results

Following exposure to different concentrations of HOCl, HSA resulted to be carbonylated but did not form dityrosine cross-linked high molecular weight aggregates. Differently, incubation of fibrinogen or HSA/fibrinogen mixtures with HOCl at concentrations higher than 150 μM induced the formation of pentosidine and high molecular weight (HMW)-AOPPs (> 200 kDa), resulting from intermolecular dityrosine cross-linking. Dityrosine fluorescence increased in parallel with increasing HMW-AOPP formation and increasing fibrinogen concentration in HSA/fibrinogen mixtures exposed to HOCl. This conclusion is corroborated by experiments where dityrosine fluorescence was measured in HOCl-treated human plasma samples containing physiological or supra-physiological fibrinogen concentrations or selectively depleted of fibrinogen, which highlighted that fibrinogen is responsible for the highest fluorescence from dityrosine.

Conclusions

A central role for intermolecular dityrosine cross-linking of fibrinogen in HMW-AOPP formation is shown.

General significance

These results highlight that oxidized fibrinogen, instead of HSA, is the key protein for intermolecular dityrosine formation in human plasma.     

Studies on actin fragments obtained by digestion with thrombin,BNPS-skatole and nitrothiocyanobenzoic acid

We have isolated fragments of actin prepared by thrombic digestion (residues 40–374 [I], and 114–374 [II]), BNPS-skatole cleavage (residues 87–339 [III]) and nitrothiocyanobenzoic acid treatment (residues 10–216 [IV]) using preparative electrophoretic and chromatographic techniques. Analysis of the filament-forming and tropomyosin-binding properties of demonstrably homogeneous fragments revealed that only fragments I and II oculd form F-actin-like filaments after attempted renaturation, and that only fragment I could bind to tropomyosin. These results in addition to our previous studies on actin fragments and chemically-modified intact actin suggest that residues 1–86 and 340–374 are not required for F-actin filament formation, whereas residues 70–86 and/or 340–374 are essential for tropomyosin-binding activity.     

ATP and magnesium drive conformational changes of the Na/K-ATPase cytoplasmic headpiece

Conformational changes of the Na⁺/K⁺-ATPase isolated large cytoplasmic segment connecting transmembrane helices M4 and M5 (C45) induced by the interaction with enzyme ligands (i.e. Mg²⁺ and/or ATP) were investigated by means of the intrinsic tryptophan fluorescence measurement and molecular dynamic simulations. Our data revealed that this model system consisting of only two domains retained the ability to adopt open or closed conformation, i.e. behavior, which is expected from the crystal structures of relative Ca²⁺-ATPase from sarco(endo)plasmic reticulum for the corresponding part of the entire enzyme. Our data revealed that the C45 is found in the closed conformation in the absence of any ligand, in the presence of Mg²⁺ only, or in the simultaneous presence of Mg²⁺ and ATP. Binding of the ATP alone (i.e. in the absence of Mg²⁺) induced open conformation of the C45. The fact that the transmembrane part of the enzyme was absent in our experiments suggested that the observed conformational changes are consequences only of the interaction with ATP or Mg²⁺ and may not be related to the transported cations binding/release, as generally believed. Our data are consistent with the model, where ATP binding to the low-affinity site induces conformational change of the cytoplasmic part of the enzyme, traditionally attributed to E2 → E1 transition, and subsequent Mg²⁺ binding to the enzyme-ATP complex induces in turn conformational change traditionally attributed to E1 → E2 transition.     

Purification and characterization of trehalose phosphorylase from the commercial mushroom Agaricus bisporus

Trehalose phosphorylase (EC 2.4.1.64) from Agaricus bisporus was purified for the first time from a fungus. This enzyme appears to play a key role in trehalose metabolism in A. bisporus since no trehalase or trehalose synthase activities could be detected in this fungus. Trehalose phosphorylase catalyzes the reversible reaction of degradation (phosphorolysis) and synthesis of trehalose. The native enzyme has a molecular weight of 240 kDa and consists of four identical 61-kDa subunits. The isoelectric point of the enzyme was pH 4.8. The optimum temperature for both enzyme reactions was 30°C. The optimum pH ranges for trehalose degradation and synthesis were 6.0–7.5 and 6.0–7.0, respectively. Trehalose degradation was inhibited by ATP and trehalose analogs, whereas the synthetic activity was inhibited by P_i (K_i=2.0 mM). The enzyme was highly specific towards trehalose, P_i, glucose and α-glucose-1-phosphate. The stoichiometry of the reaction between trehalose, P_i, glucose and α-glucose-1-phosphate was 1:1:1:1 (molar ratio). The K_m values were 61, 4.7, 24 and 6.3 mM for trehalose, P_i, glucose and α-glucose-1-phosphate, respectively. Under physiological conditions, A. bisporus trehalose phosphorylase probably performs both synthesis and degradation of trehalose.