Specific stimulation of α2-6 sialyltransferase activity by a novel cytosolic factor from rat colon

A factor present in the 100 000 g supernatant from the homogenate of rat colon stimulated the activity of purified GaIβ1-4GlcNAc α2,6 sialyltransferase [α2-6ST(N)] from rat liver and α2-6ST(N) from either liver microsomes or Golgi membrane. The stimulation of α2-6ST(N) activity by the colon factor using protein acceptors was about four-fold and highly reproducible when the reaction product of the α2-6ST(N) was assayed by either precipitation or affinity chromatography. In contrast, the colon factor did not stimulate the GaIβ1-4GlcNAc α2,3 sialyltransferase [α2-3ST (N)], from rat jejunum microsomes or purified Galβ1-3GalNAc α2,3 sialyltransferase [α2-3ST (O)] from porcine liver, or purified β1,4 galactosyltransferase (GT) from bovine milk. In addition to rat colon, the 100 000 g supernatant from the homogenates of rat brain and kidney also stimulated the α2-6ST(N) activity. The stimulation of α2-6ST(N) by the colon factor resulted in a decrease in the K_m (by about two-fold) and an increase in V_max (about 2- to 3-fold) for desialylated α₁ acid glycoprotein and CMP-[¹⁴C]N-acetylneuraminic acid. The stimulation of α2-6ST(N) activity by the colon factor was temperature dependent, protease sensitive and was inhibited by CTP, but did not need the presence of either metal ions or detergent. The cytosolic factor was partially purified by ion-exchange chromatography with the retention of the activator activity in the peaks containing low molecular weight proteins, but the activity was lost on attempts to further purification. A specific marked stimulation of the α2-6ST(N) activity by cytosolic factors in certain tissues might suggest a physiological role for these factors in the regulation of α2-6ST(N) activity.     

Reversible effect of calcium-binding protein regucalcin on the Ca2+-induced inhibition of deoxyuridine 5′-triphosphatase activity in rat liver cytosol

The effect of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on deoxyuridine 5′-triphosphatase (dUTPase) in the cytosol of rat liver was investigated. Addition of Ca²⁺ up to 5.0 μM to the enzyme reaction mixture caused a significant decrease of dUTPase activity, while Zn²⁺, Cd²⁺, Co²⁺, Al³⁺, Mn²⁺ and Ni²⁺ (10 μM) did not have an appreciable effect. The Ca²⁺-induced decrease of dUTPase activity was reversed by the presence of regucalcin; the effect was complete at 1.0 μM of the protein. Regucalcin had no effect on the basal activity of the enzyme. Meanwhile, the reversible effect of regucalcin on the Ca²⁺ (10 μM)-induced decrease of dUTPase activity was not altered by the coexistence of Cd²⁺ or Zn²⁺ (10 μM). The present data suggest that liver cytosolic dUTPase is uniquely regulated by Ca²⁺ of various metals, and that the Ca²⁺ effect is reversed by regucalcin.     

Binding of thyroid hormones to human hemoglobin and localization of the binding site

Radiolabeled thyroid hormones were allowed to bind to erythrocyte cytosol and the complex was fractionated by Sephadex G-100 or by high-performance liquid chromatography (HPLC). On Sephadex G-100, four radioactive peaks (P₁P₄) were obtained, whereas HPLC gave only three radioactive peaks (P₁P₃). Chromatographic studies with human adult Hb and non-Hb cytosol protein fractions, which had been reacted with radiolabeled thyroid hormones, and immune precipitation with specific antisera for the hormones, confirmed that the first peak of Sephadex G-100 radioactivity was a mixture of Hb and non-Hb proteins, while the second peak was Hb. The third peak was free¹²⁵I and the fourth peak was unbound¹²⁵I-T₃ or¹²⁵I-T₄. The third peak of HPLC was confirmed to be a mixture of free¹²⁵I and unbound radiolabeled thyroid hormones. Scatchard analysis of the interaction between T₄ and apo-Hb, and the - and -chains of human Hb suggested the presence of the specific binding site(s) for the hormone. Interaction between T₄ and synthesized peptides, which constitute the heme pocket of the -chain of Hb (61–75, 71–85, 81–95), indicated that the T₄ binding site of Hb resides within the heme-binding cavity. It is concluded that human erythrocyte cytosol does not contain receptor for thyroid hormones and cannot be a model for studying functions of cytosol receptor for the hormones; rather, it contains binding protein with large binding capacity, including Hb and non-Hb proteins, which possibly constitute a large reservoir for the hormone in blood.     

Effect of inhibitors on polyphosphate metabolism in the yeast Saccharomyces cerevisiae under hypercompensation conditions

After re-inoculation of the yeast Saccharomyces cerevisiae from phosphate-deficient to complete medium, the total content of polyphosphates increased tenfold during 2 h (hypercompensation), but the content of certain fractions increased differently. The content of acid-soluble polyphosphate increased to the maximal extent. The ratio of the activities of two exopolyphosphatases also changed in the cytosol. Activity of a low molecular weight exopolyphosphatase (40 kD) decreased almost twice, whereas activity of a high molecular weight exopolyphosphatase (830 kD) increased tenfold. Cycloheximide blocks the increase in activity of high molecular weight exopolyphosphatase and hence, under these conditions the latter is synthesized de novo. Inhibitors of energy metabolism and cycloheximide, an inhibitor of protein synthesis, differently influence accumulation of certain polyphosphate fractions under hypercompensation conditions. The effect of iodoacetamide, an inhibitor of glycolysis, on any fraction is negligible, while cycloheximide suppresses accumulation of only polyP4 fraction associated with the cell envelope and bafilomycin A1, an inhibitor of vacuolar H⁺-ATPase, suppresses accumulation of polyP3 fraction. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) to variable extent inhibits accumulation of all the fractions. Analysis of the effect of inhibitors on accumulation of polyphosphates under hypercompensation conditions confirms various localization, heterogeneity, and multiplicity of the routes of biosynthesis of certain fractions of these macroergic phosphorus compounds and also suggests interrelation between their biosynthesis and the gradient of H⁺ electrochemical potential.     

Effect of PPX1 inactivation on the exopolyphosphatase spectra in cytosol and mitochondria of the yeast Saccharomyces cerevisiae

Inactivation of PPX1 encoding exopolyphosphatase PPX1 in Saccharomyces cerevisiae results in a change in the exopolyphosphatase spectrum in the yeast cells. In the PPX1-deficient strain, elimination of an 45 kD exopolyphosphatase is observed in the cytosol, and activity of an exopolyphosphatase with molecular mass of 830 kD increases fivefold. The latter activity differs greatly in properties from the low-molecular-mass enzyme of the parent strain. In the soluble fraction of the mutant mitochondria, exopolyphosphatase of 45 kD characteristic of the soluble mitochondrial fraction in the parent strain is eliminated, and exopolyphosphatase with a molecular mass of 440 to 830 kD is found. On PPX1 inactivation, a membrane-bound form of mitochondrial exopolyphosphatase is unaffected in its activity level and properties. Therefore, the membrane-bound exopolyphosphatase of mitochondria and the high-molecular-mass enzyme of the cytosol of S. cerevisiae are not encoded by the PPX1 gene, unlike the soluble low-molecular-mass exopolyphosphatase of mitochondria, which is probably a product of this gene with a posttranslational modification. In the PPX1 mutant, exopolyphosphatase properties in the cell as a whole undergo modifications including the ability to hydrolyze polyphosphates (polyP) with different polymer degree.     

Lysophospholipase activity in rat brain subcellular fractions

Lysophospholipase activity in brain subcellular fractions was measured by the release of myristic acid from 1-myristoylglycerophosphocholine or through the formation of [³²P]glycerophosphocholine from [³²P]lysophosphatidylcholine. Although the lysophospholipase activity was highest in microsomes, considerable enzyme activity was also found in other subcellular membrane fractions. The pH optimum for the microsomal enzyme was around 7, whereas the synaptosomes and non-synaptic plasma membranes exhibited a pH maximum around 8. Although the enzyme did not require divalent cations for activity, divalent cations (1 mM) such as Hg²⁺, Cu²⁺, and Zn²⁺ inhibited potently the enzyme activity. Enzyme activity was also partially inhibited by both saturated and polyunsaturated fatty acids (25–200 M), and the inhibition seemed to be greater in the membrane than in the cytosolic fractions. Ionic detergents such as deoxycholate and taurocholate inhibited the lysophospholipase. On the other hand, the effect of Triton X-100 was biphasic, i.e., stimulation at concentrations below 100 g/mg protein and inhibition at higher concentrations. Addition of cholesterol (50–250 g/ml), but not cholesteryl esters, also potently inhibited enzyme activity. The presence of active lysophospholipase(s) in brain is probably an important mechanism for preventing unnecessary accumulation of lysophospholipids which may exert a deleterious effect on the membranes because, of their detergent properties.     

Effects of Polyamines on Proline Endopeptidase Activity in Rat Brain

The in vitro effects of polyamines on the activity of proline endopeptidase (PEPase) in rat brain cytosol, which contains an endogenous PEPase inhibitor, have been studied. Of the three amines tested (spermine, spermidine, and putrescine), spermine and spermidine markedly enhanced the enzyme activity in brain cytosol. At 6.25 mM spermine or 25 mM spermidine, a 13- or 14-fold enhancement of the enzyme activity was observed. When Mg2+ was used, an approximately fourfold enhancement of the enzyme activity was observed at 50 mM. The enhancement produced by spermine or spermidine was unaffected by Mg2+ up to 50 mM. The activity of purified PEPase was only slightly affected by each polyamine, but it was inhibited 50% by 50 mM Mg2+. On the other hand, 50% inhibition of the enzyme produced by the purified PEPase inhibitor (Mr 7,000: Ki 0.67 mM) was completely restored by addition of 0.7 mM spermine, 3.5 mM spermidine, or 28 mM putrescine. This restoration of inhibition by polyamines was reversed by increasing the inhibitor concentration. These data suggest that polyamines effectively reverse the inhibition of PEPase by its endogenous inhibitor by the reversible formation of a kinetically significant complex. The possible functions of polyamines in the regulation of PEPase in vivo are discussed.