Number Of Phosphorylation Sites Research Articles

The number of phosphorylation sites within the cagA gene of Helicobacter pylori may determine disease outcome in infected patients

The number of phosphorylation sites within the cagA gene of Helicobacter pylori may determine disease outcome in infected patients

Regulation of Xenopus Cdc25 protein

This chapter discusses the regulation of xenopus Cdc25 protein. The Cdc25 protein was first identified on the basis of a mutation in Schizosaccharomyces pombe that blocks the cell cycle at the G2 phase. The biochemical function of Cdc25C as a phosphatase that dephosphorylates both Thr-14 and Tyr-15 of Cdc2, thereby activating the Cdc2 protein kinase at the onset of mitosis, is well established. Cdc25 has a highly conserved catalytic domain in its C-terminal half that contains a critical cysteine residue required for catalytic activity. The N-terminal half is less conserved between different species and contains a number of phosphorylation sites. During mitosis, the N-terminal half becomes extensively phosphorylated. Cdc25 has a highly conserved catalytic domain in its C-terminal half that contains a critical cysteine residue required for catalytic activity. The N-terminal half is less conserved between different species and contains a number of phosphorylation sites.

INMP, a novel intranuclear matrix protein related to the family of intermediate filament-like proteins: molecular cloning and sequence analysis

An important step in understanding nuclear structure and its function in replication and gene regulation is the cloning and characterisation of nuclear matrix proteins. A full-length cDNA-clone, encoding a novel nuclear matrix protein, was isolated from a (λ gt11 cDNA library derived from murine macrophages. The antibody used for the screen was raised against a single polypeptide isolated from two-dimensional gel electrophoresis of nuclear matrix preparations. The cDNA encodes a protein of 446 amino acids named INMP for intranuclear matrix protein. INMP displays several salient features, a coiled-coil domain, a leucine zipper, a number of phosphorylation sites and a putative nuclear localisation signal. Sequence homology comparisons indicate that INMP is a unique protein which is evolutionary related to the gene family of intermediate filament-like proteins, especially the nuclear lamins.

Altered cardiac Na+,K+-ATPase activity in prehypertensive spontaneously hypertensive rat

Na+,K+-ATPase activity, Na+-dependent phosphorylation, and [3H]ouabain binding in sarcolemma prepared from 4 week old spontaneously hypertensive rat(SHR) ventricles were compared to the same parameters in sarcolemma from age matched normotensive Wistar-Kyoto (WKY) rat ventricles to examine whether the reduced myocardial Na+-pump activity in SHR is an inherited enzymatic defect or a second phenomenon due to sustained hypertension. The total body weights, ventricular weights, and blood pressures were the same for SHR and WKY. No significant differences in sarcolemmal protein content and protein recovery were noted between the two groups. Sarcolemma isolated from SHR ventricles showed significantly less Na+, K+-ATPase activity and number of phosphorylation sites when, compared to sarcolemma from the WKY ventricles. Equilibrium binding of [3H]ouabain and the tumover number of myocardial Na+,K+-ATPase, however, were the same for both groups. These results indicate that the low affinity (α, or α1) isoform for ouabain is reduced in SHR compared to WKY but that the high affinity (α+, or α2) α isoform is the same in ventricles of SHR and WKY. The reduced amount of isoform of the Na+,K+-ATPase in, prehypertensive SHR ventricles may play some role in the development of hypertension.

Characterization of a soluble Mr-30,000 catalytic fragment of the neuronal calmodulin-dependent protein kinase II.

Chymotryptic digestion of postsynaptic densities releases a soluble, catalytically active fragment of the alpha (Mr 50,000) subunit of the neuronal cytoskeletal calmodulin-dependent protein kinase II. The purified soluble form of the kinase likewise yields the fragment. Denaturation of the enzyme results in more extensive proteolytic degradation. 125I-Iodopeptide maps of the isolated catalytic portions of both forms of the enzyme are similar and are contained within the map of the isolated alpha subunit. Catalytic fragments of both forms of the enzyme comigrate on two-dimensional SDS-PAGE/isoelectric focusing with pI 6.7-7.2. The fragment phosphorylates microtubule-associated protein (MAP-2) but is not activated by Ca+2/calmodulin nor is it inhibited by trifluoperazine. Km values for MAP-2 and ATP are indistinguishable from those of the holoenzyme, while the Vmax is similar to that of the holoenzyme activated with Ca+2/calmodulin. Overlays of Western blots of fragment with 125I-calmodulin shows a loss of calmodulin binding. Both the number of phosphorylation sites and the ability to autophosphorylate are markedly reduced in the catalytic fragment. Evaluation of the hydrodynamic parameters of the purified fragment yielded Mr value of 25,600 with a frictional ratio (f/f0) of 1.12; the Mr value determined by SDS-PAGE was 30,000. Thus, the catalytic fragment appears to represent an activated form of the kinase with a monomeric, globular structure unlike the native enzyme which exhibits oligomerization and cytoskeletal association. These results are consistent with a tertiary structure for the calmodulin-dependent protein kinase that contains distinct domains responsible for catalytic activity, regulation by calmodulin, cytoskeletal association and the multimeric organization of enzyme subunits.

Kinetic properties of C 12E 8-solubilized (Na ++K +)-ATPase

(1) The properties of the rectal gland (Na + + K +)-ATPase (ATP phosphohydrolase, EC 3.6.1.8) solubilized in octaethyleneglycol dodecylmonoether (C 12E 8) have been investigated. (2) The Kinetic properties of the solubilized enzyme resemble those of the membrane-bound enzyme to a large extent. The main difference is that K m for ATP for the (Na + + K +)-ATPase is about 30 μM for the solubilized enzyme and about 100 μM for the membrane-bound enzyme. (3) The Na +-form (E 1) and the K +-form (E 2) can also be distinguished in the solubilized enzyme, as seen from tryptic digestion, the intrinsic fluorescence and cosin fluorescence responses to Na + and K +. (4) The number of vanadate-binding sites is unchanged upon solubilization, and it is shown that vanadate binding is much more resistant to detergent inactivation than the enzymatic activities. The number of phosphorylation sites on the 95–100% pure supernantant enzyme is about 3.8 nmol/mg, and is equal to the number of vanadate sites. (5) Inactivation of the enzyme by high concentrations of detergent can be shown to be related to the C 12E 8/protein ratio, with a weight ratio of about 4 being a threshold for the onset of inactivation at low ionic strength. At high ionic strength, more C 12E 8 is required both for solubilization and inactivation. (6) It is observed that the commercially available detergent polyoxyethylene 10-lauryl ether is much less deleterious than C 12E 8, and its advantages in the assay of detergent-solubilized (Na + + K +)-ATPase are discussed. (7) The results show that (Na + + K +)-ATPase can be solubilized in C 12E 8 in an active form, and that most of the kinetic and conformational properties of the membrane-bound enzyme are conserved upon solubilization. C 12E 8-solubilized (Na + + K +)-ATPase is therefore a good model system for a solubilized membrane protein.

Decrease in Na+,K+-ATPase activity and [3H]ouabain binding sites in sarcolemma prepared from hearts of spontaneously hypertensive rats.

Na+,K+-ATPase activity, phosphorylation, and [3H]ouabain binding in sarcolemma isolated from spontaneously hypertensive rat (SHR) hearts were compared to the same parameters in sarcolemma from normotensive rat (WKY) hearts. Sarcolemma prepared from SHR heart contained significantly less ouabain-inhibitable ATPase activity than sarcolemma from WKY heart. No significant differences in sarcolemmal protein content or recovery were noted between the two groups. The numbers of phosphorylation sites and ouabain binding sites were lower for SHR hearts than for WKY hearts. The KD values for ouabain binding were the same (0.30 muM) in cardiac sarcolemma of SHR and WKY. The I50 values for inhibition by ouabain of Na+,K+-ATPase were also the same for both groups (SHR = 49 microM; WKY = 44 microM). These data suggest that the decrease of cardiac sarcolemmal Na+,K+-ATPase activity in SHR hearts is due to a decrease in the number of active sites.

Development of insulin responsiveness of the glucose transporter and the (Na+,K+)-adenosine triphosphatase during in vitro adipocyte differentiation.

The development of insulin responsiveness of the transport systems for glucose (2-deoxyglucose) and potassium (Rb+) was compared during in vitro adipocyte differentiation of 3T3-L1 cells. Growing cells exhibited minimal increases in 2-deoxyglucose and Rb+ transport rates in response to insulin. Low levels of insulin stimulation of both transport processes became apparent when cultures attained confluence, and these levels were maintained in uninduced cells which retained their fibroblast-like morphology. Following a 48-h induction treatment with methylisobutylxanthine and dexamethasone, a dramatic and simultaneous increase in insulin sensitivity of both deoxyglucose and Rb+ uptake was observed. In fully differentiated 3T3-L1 adipocytes, insulin caused a 6-10-fold increase above the basal rate for deoxyglucose uptake and a 1.50-1.60-fold increase of Rb+ uptake. The insulin dose response relationships were identical for both deoxyglucose uptake and Rb+ uptake and half-maximal stimulation occurred at insulin concentrations of 2-3 nM in 3T3-L1 fibroblasts, 550 pM in 3T3-L1 adipocytes, and 100 pM in mature rat adipocytes. Basal rates of deoxyglucose and Rb+ uptakes were 2-9-fold higher in growing cells than in confluent cells, and fatty cells exhibited lower transport rates relative to nonfatty cells. The number of active Na+ pumps on the cell surface was determined by quantitation of the covalent phosphorylated intermediate of the (Na+,K+)-ATPase. Plasma membranes of growing cells contained a larger number of (Na+,K+)-ATPase specific phosphorylation sites and higher (Na+,K+)-ATPase activity than those of confluent cells. Although (Na+,K+)-ATPase and Rb+ transport activities were greater in uninduced cells than in induced cells, both cultures exhibited the same number of phosphorylation sites, implying that the Na+ pump in 3T3-L1 adipocytes was operating at a reduced efficiency.

Theoretical calculations on the influence of the inorganic nitrogen source on parameters for aerobic growth of microorganisms.

The amount of ATP required for the formation of microbial cells growing in a minimal medium with various nitrogen sources was calculated. In a glucose-mineral salts medium 28.8 g cells can be formed per mole ATP with ammonia and 23.1 for growth with nitrate. For growth with molecular nitrogen 11.1; 8.7; 7.1 and 6.0 g cells can be formed per mole ATP for ATP/N2 ratios of 12, 18, 24 and 30 respectively. A method is given for the calculation of Ysub, YO2 and Yc02 values for aerobic growth with glucose, succinate or methanol and various nitrogen sources. In this method use is made of the elementary composition of the cells and of mass balance equations. As an assimilation equation: C6H12O7 + 1.4 HNO3 + 6.85 “H2” → C6H10.84N1.4O3.07 + 8.13 H2O is given for growth ofParacoccus denitrificans with gluconate and nitrate. From this equation and the molar growth yield for gluconate the oxygen uptake, carbon dioxide evolution and the YO2 value can be calculated. A very good agreement between the calculated values and the experimental values was obtained. For the calculation of the ATP production it is essential to know the number of phosphorylation sites in the respiratory chain. Calculations are given for 2 (sites I + II) and 3 phosphorylation sites. The molar growth yields for growth with nitrate and nitrogen are much smaller than that for growth with ammonia. The YO2 values for growth with glucose and nitrate are higher (with 2 sites) or somewhat smaller (with 3 sites) than for growth with ammonia. The YO2 values for growth with nitrogen are always very low. The calculations show that especially YO2 is very dependent on the number of phosphorylation sites. For growth with methanol YCO2 is strongly dependent on the nature of the assimilation pathway for methanol and on the nitrogen source. The molar growth yields for growth with glucose, succinate or methanol and nitrogen are about the same as when nitrate is the nitrogen source for organisms with 3 phosphorylation sites. The theoretical efficiency of nitrogen fixation in grow-ing cells is much lower (dependent on the ATP/N2 ratio) than that in nongrowing cells.

Kinase activities in the non-histone chromosomal proteins of resting and proliferating BHK21 C13 cells

When BHK21 C13 cells in saturation density are stimulated to proliferate by a change of medium, a synchronized DNA synthesis occurs with a maximum at about 15 h. After stimulation the nonhistone chromosomal proteins (NHC proteins) phosphorylation in vivo increases and reaches a maximum in late G 1 phase. We show that the endogenous phosphorylation in vitro of NHC proteins is 4-fold larger when the NHC proteins are extracted from cells in late G 1 phase than from resting cells. The electrophoretic patterns of [ 32P]distribution show that the [ 32P]incorporation enhancement in NHC proteins from stimulated cells is more significant for the slowly migrating proteins. The NHC protein kinase activity was fractionated on polyacrylamide gel in 10 fractions, in resting as in stimulated cells. Only some of these kinase activities were increased after cell stimulation. The enhancement of phosphorylation may be due in part to an increase in the number of phosphorylation sites and in part to an increase in kinase activities. The use of phosvitin as exogenous substrate allow the conclusion that the activity of some kinase is enhanced after stimulation. Furthermore, by fractionation on polyacrylamide gel five phosvitin kinase activities were found in low molecular weight NHC protein fractions, in control as in stimulated cells. After cell stimulation, two phosvitin kinase activities only are specifically increased. The results show the multiplicity of kinase activities and support the hypothesis that these various kinase activities correspond to specific differentiated functions.

Enhanced cell aggregation in Dictyostelium discoideum by ATP activation of cylic AMP receptors

Cell aggregation in the cellular slime mold Dictyostelium discoideum is mediated by cyclic AMP. In the presence of ATP the onset of cell aggregation is enhanced and cyclic AMP receptors are activated. The number of phosphorylation sites is species dependent, and two main phosphorylated proteins with MW of, respectively, 20,000 and 15,000 are localized.

Growth phase and the number of phosphorylation sites in the mitochondrial electron transport chain of Acanthamoeba castellanii.

SYNOPSIS. Mitochondria isolated from the soil ameba Acanthamoeba castellanii growing exponentially on complex medium have rotenone‐insensitive oxygen uptake and ADP:O ratios which indicate the presence of only 2 phosphorylation sites in the electron transport chain. Stationary phase amebae yield mitochondria which are sensitive to inhibition by rotenone when respiring NAD+‐Minked substrates and have 3 sites of phosphorylation. The levels of cytochromes (a +a3), b, and c are similar in mitochondria isolated from log or stationary phase amebae, and, with the exception of succinate, the respiratory rates obtained with different substrates do not change significantly from log to stationary growth phase.