Increase of sialyltransferase activity in the serum and liver of inflamed rats

Induction of inflammation by turpentine injection caused 1.5-2-fold increase of both sialyl- and galactosyltransferase activity in liver homogenates. The effect was apparent after 12 h of turpentine treatment. Serum sialyltransferase activity started to increase in the inflamed rats after 18 h, reaching a maximum of 4-fold at 48 h. In contrast, galactosyltransferase activity in serum showed no significant increase. The coordinated and temporal increase of sialyltransferase activity in liver and serum suggest involvement of a specific mechanism for the preferential release of this enzyme into serum.     

Effect of partial hepatectomy and hydrocortisone administration on liver and serum sialyltransferase activities

Partial hepatectomy of rats was followed by a rise in liver sialyltransferase activity. The maximum (2.5-fold increase) was reached on the third day after the operation, after which the level started to decline, returning to normal by day 6. Determination of serum sialyltransferase in these animals showed a parallel pattern. Daily injection of 5 mg hydrocortisone to adrenalectomized rats led to a maximal 3-fold elevation in liver sialyltransferase within 3 days, but failed to elicit any changes in the corresponding enzyme in the serum. Results from these two experiments suggest that the elevations of sialyltrasferase in the tissue and in the circulation are independently regulated.     

Iron content and degree of lipid peroxidation in liver mitochondria isolated from iron-loaded rats

1.The content of non-heme iron and the degree of lipid peroxidation were measured in liver mitochondria isolated from rats injected with either Jectofer (an iron-sorbitol-citric acid complex) or iron-nitrilotriacetate. 2. The sedimentation profiles of the mitochondria from controls and iron-treated rats as revealed by analytical differential centrifugation, indicated single population of mitochondria with $\text{s}{}_{\mathrm{4,B}}$ values of 13200± 560 S and 14200±590 S for controls and iron-loaded animals, respectively. In contrast, the sedimentation profiles of the acid phosphatase activity and the non-heme iron revealed marked polydispersities with at least three populations of particles for both controls and iron-loaded animals. 3. The mitochondria and iron-rich lysosomes were separated by density-gradient centrifugation in an isotonic medium of Percoll and sucrose. With this technique, the amount of non-heme iron in a mitochondrial fraction by differential centrifugation decreased from 69±28 nmol/mg protein to 5.6±1.1 nmol/mg protein and from 19.3±5.6 nmol/mg protein to 3.3±0.6 nmol/mg protein for Jectofer and iron-nitrilotriacetate injected rats, respectively. For control rats the amount of mitochondrial non-heme iron was about 2.7 nmol/mg protein both before and following density gradient centrifugation. The extra amount of non-heme iron still present in the purified mitochondrial fraction from iron-loaded rats, as compared to controls, was further characterized by the reactivity towards bathophenanthroline sulfonate. The results suggest that the extra iron was due to a small amount of either ferritin or hemosiderin still contaminaning the mitochondrial fraction. The amount of mitochondrial heme iron was the same in iron-loaded rats and controls. 4. The degree of lipid peroxidation in the mitochondria was estimated from the amount of malondialdehyde. The thiobarbituric acid method used for the quantitation of malondialdehyde was modified so that it was insensitive to variable amounts of iron present in the samples. No difference in the degree of lipid peroxidation was observed between the mitochondria from iron-loaded rats and controls. 5. In contrast to recent proposals (Hanstein, E.G. et al. (1981) Biochim. Biophys. Acta 678, 293–299), the present study showed that the amounts of non-heme iron and the degrees of lipid peroxidation are the same in mitochondria isolated from iron-loaded and control animals.     

Increase of intracellular pH in secreting frog gastric mucosa

A method of estimation of pH in frog gastric mucosa by measuring the apparent creatine kinase equilibrium was studied. In a resting, in vitro preparation of frog stomach the intracellular pH was found to increase linearly with an increase in the serosal pH. This increase was also accompanied by an increase in the apparent equilibrium constant of the creatine kinase reaction. A similar increase was found when the resting mucosa was stimulated with histamine plus theophylline. During this procedure the total content of adenine nucleotides and creatine plus creatine phosphate remained constant.     

Regulation of glycogen synthase activity in the isolated mouse soleus muscle effect of insulin,epinephrine, glucose and anti-insulin receptor antibodies

The effects of insulin, epinephrine, glucose and anti-insulin receptor antibodies on enzymes involved in the regulation of glycogen synthesis were investigared in the isolated mouse soleus muscle. Insulin maximally increased the percentage of glycogen synthase active form after 15 min in the absence of glucose in the extracellular medium; half-maximal and maximal effects were obtained with 1.5 and 33 nM insulin, respectively. The basal percentage of glycogen phosphorylase active form was not altered by insulin. Antibodies to the insulin receptor had similar effects to those of insulin on both enzymes. The percentage of glycogen synthase active form was maximally decreased and that of phosphorylase maximally increased after a 2 min exposure to epinephrine in the absence of extracellular glucose. Glucose alone had no effect on muscle glycogen synthase. When muscles were incubated with insulin (33 nM) plus glucose (20 mM) for 5–10 min, the increase in the percentage of glycogen synthase active form was greater than with insulin alone. This enhancing effect of glucose on insulin activation of glycogen synthase disappeared after 20 min. The results suggest the existence of two mechanisms whereby insulin activates muscle glycogen synthase. The main effect is operative in the absence of extracellular glucose and occurs at insulin concentrations close to the physiological range. The other effect requires glucose and may result from the stimulation by insulin of glucose transport and/or metabolism.     

Changes in the chondroitin sulfate-rich region of articular cartilage proteoglycans in experimental osteoarthritis

The chondroitin sulfate-rich region was cleaved from cartilage proteoglycans of experimental osteoarthritic canine joints to establish whether changes in this region of the molecule contribute to the well-documented increase in the chondroitin sulfate to keratan sulfate ratio in osteoarthritis. Experimental osteoarthritis was induced in eight dogs by severance of the right anterior cruciate ligament, the left joint serving as a control. Proteoglycans were extracted from the femoral cartilage of both joints, isolated as A1 fractions by associative density gradient centrifugation and cleaved with hydroxylamine. The chondroitin sulfate-rich region was isolated by either gel chromatography or dissociative density gradient centrifugation. The chondroitin sulfate-rich region from the proteoglycans of the experimental osteoarthritic joints was slightly larger in hydrodynamic size and had both a higher uronate/protein weight ratio and galactosamine/glucosamine molar ratio than the corresponding control. We conclude that the chondroitin sulfate-rich region of proteoglycans in articular cartilage of experimental osteoarthritic joints is larger and has more chondroitin sulfate than that of proteoglycans of normal cartilage.     

The effects of potassium and membrane potential on sodium-dependent glutamic acid uptake

The uptake of l-glutamic acid into brush-border membrane vesicles isolated from rat renal proximal tubules is Na⁺-dependent. In contrast to Na⁺-dependent uptake of d-glucose, pre-equilibration of the vesicles with K⁺ stimulates l-glutamic acid uptake. Imposition of a K⁺ gradient ($\text{[}\text{K}{}_{\mathrm{i}}{}^{\mathrm{+}}\text{] > [}\text{K}{}_{\mathrm{o}}{}^{\mathrm{+}}\text{]}$) further enhances Na⁺-dependent l-glutamic acid uptake, but leaves K⁺-dependent glucose transport unchanged. If K⁺ is present only at the outside of the vesicles, transport is inhibited. Intravesicular Rb⁺ and, to a lesser extent, Cs⁺ can replace intravesicular K⁺ to stimulate l-glutamic acid uptake. Changes in membrane potential incurred by the imposition of an H⁺-diffusion potential or anion replacement markedly affect Na⁺-dependent glutamic acid uptake only in the presence of K⁺. Experiments with a potential-sensitive cyanine dye also indicate that, in the presence of intravesicular K⁺ a charge movement is involved in Na⁺-dependent transport of l-glutamic acid.The data indicate that Na⁺-dependent l-glutamic acid transport can be additionally energized by a K⁺ gradient. Furthermore, intravesicular K⁺ renders Na⁺-dependent l-glutamic acid transport sensitive to changes in the transmembrane electrical potential difference.     

Intracellular transport and degradation of 125I-labelled denatured serum albumin in isolated nonparenchymal rat liver cells

The intracellular movement, following uptake of ¹²⁵I-labelled denatured serum albumin into nonparenchymal liver cells, was followed by means of subcellular fractionation. Isolated nonparenchymal rat liver cells were prepared by means of differential centrifugation. The cells were homogenized in a sonifier and the cytoplasmic extract subjected to isopycnic centrifugation in a sucrose gradient. The intracellular movement of the labelled albumin was followed by comparing the distribution profile of radioactivity in the sucrose gradient with those of marker enzymes for plasma membrane and lysosomes. The distribution profiles for radioactivity after the cells had been exposed to the labelled denatured albumin for different time periods indicated that the radioactivity was first associated with subcellular fractions of lower modal densities than the lysosomes. With time of incubation the radioactivity moved towards higher densities. After prolonged incubations in the absence of extracellular labelled denatured albumin the radioactivity peak coincided with that of the lysosomal marker β-acetylglucosaminidase. When the cells were treated with the lysosomal inhibitor leupeptin, degradation of the labelled albumin was decreased, resulting in a massive intracellular accumulation of radioactivity. The radioactivity peak coincided with the peak of activity for the lysosomal marker β-acetylglucosaminidase, suggesting lysosomal degradation.     

Hydrogen oxidation activity in membranes from Rhizobium japonicum

Membranes capable of oxidizing H₂ with O₂ as terminal acceptor were obtained from free-living Rhizobium japonicum. Membranes contained highest H₂-uptake specific activities when isolated in the presence of an H₂ atmosphere, and when the oxygen radical scavenger butylated hydroxytoluene was included in the buffer used for rupturing cells. After breaking cells, all of the O₂-dependent H₂-uptake activity was associated with a particulate membrane-containing fraction, whereas approx. 75% of the methylene blue-dependent H₂-uptake activity was sedimented. The particulate and soluble fractions containing H₂-uptake activity with methylene blue were separated by sucrose gradient centrifugation. The particulate and soluble activities behaved identically with regard to artificial electron acceptor specificity and reversible inhibition by oxygen. The hydrogenase in membranes coupled H₂ uptake with the reduction of many positive potential electron acceptors, but not with negative potential acceptors. The optimal pH for H₂ uptake with O₂ as acceptor in membranes was approx. 7.2. H₂-uptake activity in membranes was associated with an inner (lighter) membrane fraction that also contained succinate oxidase activity. H₂-reduced minus O₂-oxidized difference spectroscopy of membranes indicated the involvement of b and c-type cytochromes in the H₂-oxidation pathway, with an absorption peak at 551.5 nm and a shoulder at 560 nm. The addition of sodium dithionite to H₂-reduced membranes caused additional b-type cytochrome reduction. The methylene blue-dependent H₂-uptake activity in membranes was reversibly inhibited by brief exposure to oxygen. Recovery of full activity after oxygen exposure was achieved only after several minutes of incubation under strict anaerobic conditions.     

Changes in the sequence content of albumin mRNA and in its translational activity in the rat liver with age

To investigate the regulation of age-related changes in albumin synthesis in the rat liver, total postnuclear RNA and polyribosomes, both membrane-bound and free, were prepared from livers of rats of different ages. By the use of a specific complementary DNA probe, the albumin mRNA sequence content was quantitated in these RNA fractions. These studies showed a specific increase in albumin mRNA sequence content in total postnuclear RNA and membrane-bound polyribosomes at between 12 and 24 months of age. Between 24 and 36 months of age, the increase in the amount of albumin mRNA in these two fractions was due only to an increase in liver weight. The increase in albumin mRNA sequence content was not found in the poly(A)⁺ fraction but in the RNA extracted from the void of oligo(dT)-cellulose column chromatography. The isolated polyribosomes were translated in a cell-free system to assess age-related changes in total protein and albumin synthesis due to translational control. No changes with age were found in the translational capacity of membrane-bound and free polyribosomes per RNA unit. Immunoprecipitation of the synthesized albumin in the translation products revealed that albumin synthesis in the cell-free system is not increased proportionally with the elevated albumin mRNA level between 12 and 24 months of age. This indicates that albumin mRNAs present in the livers of old rats are biologically less active than those found in younger animals.     

An investigation into the feasibility of using yeast protoplasts to study the ion transport properties of the plasma membrane

A method for studying ion uptake in enzymatically isolated protoplasts from the yeast, Saccharomyces cerevisiae, is described. The kinetics of K⁺ and Rb⁺ uptake, metabolic proton extrusion and cell electrophoretic mobility bave been determined. Enzymic removal of the cell wall does not significantly alter the above-mentioned properties of the yeast cells. It is concluded that studies of these properties can be performed equally well with intact yeast cells or protoplasts. However, in studies aimed at determining effects of complex organic substances, e.g., antibiotics, on plasma membrane function the use of protoplasts is recommended. The effectiveness of the antibiotic, Dio-9, for example, in reversing the metabolic proton extrusion into a net proton influx is at least 50 times higher after enzymic removal of the yeast cell wall.     

Inhibition of brain ST8SiaIII sialyltransferase leads to impairment of procedural memory in mice

Several glycoproteins in mammalian brains contain α2,8-linked disialic acid residues. We previously showed a constant expression of disialic acid (DiSia) in the hippocampus, olfactory bulb and cortex, and a gradual decrease of expression in the cerebellum from neonatal to senile mice. Previous publications indicate that neurite extension of neuroblastoma-derived Neuro2A cells is inhibited in the presence of DiSia antibody. Based on this, we treated Neuro2A cell cultures with RNA interference for ST8SiaIII mRNA, the enzyme responsible for DiSia formation. We observed that neurite extension was inhibited by this treatment.     

Fusion kinetics of phosphatidylcholine-phosphatidic acid mixed lipid vesicles: A proton nuclear magnetic resonance study

The kinetics of Ca²⁺-induced fusion of phosphatidylcholine-phosphatidic acid vesicles has been studied using the dependence of proton nuclear magnetic resonance linewidths on vesicle size. The linewidth of the lipid acyl chain methylene resonance been shown to be sensitive to changes in vesicle size but insensitive to vesicle aggregation. For vesicle systems with the same lipid composition, the linewidth increases in a linear fashion with vesicle radius over the range 125–300 Å. This dependence has been used to determine quantitatively fusion rates and the dependence of such rates on Ca²⁺ as well as an vesicle concentration. For vesicle concentrations in the range of 3 · 10^?6–10^?5 M and Ca²⁺ concentration at a level approaching 1 : 1 with respect to phosphatidic acid, the initial fusion rates have been found to be fast, with half-times of 1–10 min. An order of reaction of 2.7 with respect to vesicle concentration has been observed. Mechanisms of vesicle fusion are discussed in view of these observations.     

An improved purification method and a further characterization of the 33-kilodalton protein of spinach chloroplasts

The 33-kDa protein was purified in a high yield from thylakoid membranes of spinach chloroplasts. The extinction coefficient and A^1%_1cm value at 276 nm of the protein were 22000 M^?1·cm^?1 and 6.8, respectively. The 33-kDa protein and a polypeptide appearing at 32 kDa in the SDS-polyacrylamide gel electrophoresis of thylakoid membranes were compared by peptide mapping after limited proteolysis. This indicates that the 32-kDa band is entirely due to the 33-kDa protein. The molar ratio of chlorophyll to the 33-kDa protein in the chloroplasts was estimated to be 300. This suggests that one photosynthetic unit possesses one or two molecules of the 33-kDa protein.     

Partial characterization of the glucose transport activity in the golgi-rich fraction of fat cells

The glucose transport activity solubilized from the basal and plus insulin forms of the Golgi-rich fraction of adipocytes was partially characterized, and the results were compared with those of the activity obtained from the plus insulin form of the plasma membrane-rich fraction. The transport activity was determined in a cell-free, reconstituted, system. Prior to reconstitution, the activities in the three preparations were all (a) stable at 0°C for at least 4 h, but not at 37°C or above; (b) most stable at pH 7–9, and (c) less stable in Tes than in Tris buffer. After reconstitution, the three activities were all (d) stable at 0°C, (e) most active at pH 5.5, (f) mildly stimulated by divalent cations, (g) unaffected by insulin or 1 mM of several SH-blocking agents, (h) inhibited by heavy metal ions, 10–100 mM of monovalent salts, organic solvents, several sugar isomers, and specific sugar-transport inhibitors. The rates of d-glucose uptake by the three liposome preparations were all inhibited more strongly by 2-deoxy-d-glucose or $\text{3}\text{-O-}\text{methyl-}\text{d}\text{-glucose}$ than by d-glucose. These data indicate that the general properties of the glucose transport activity in the Golgi-rich fraction are similar to those of the activity in the plasma membrane-rich fraction.     

Sequestration of adenosine in crude extract from mouse liver and other tissues

Adenosine (1 μM) was incubated in the presence of dialyzed crude tissue extract from mouse liver and its degradation determined. At high concentration of tissue extract, a fraction of adenosine was not metabolized. This phenomenon, termed sequestration of adenosine, was shown to be affected in the same way by the same factors (pH, salt, reducing agent and adenine) as those affecting the protection of adenosine against deamination in the presence of the purified cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase from mouse liver (Sæbø, J. and Ueland, P.M. (1979) Biochim. Biophys. Acta 587, 333–340). These data point to a role of this protein in the sequestration of adenosine in crude extract.The sequestration potency in crude extract could be determined by diluting the extract in the presence of a constant amount of adenosine deaminase added to the tissue extract. Under these conditions there was linearity of adenosine not available for degradation versus the concentration of tissue extract, and a total recovery of the sequestration potency of purified binding protein added to the crude extract was observed.The tissue level of the cyclic AMP-adenosine binding protein/S-adenosylhomocysteinase in mouse liver was determined by two independent procedures based on the sequestration of adenosine and the hydrolysis of S-adenosylhomocysteine, respectively. The intracellular concentration was calculated to be 10 μM.The sequestration of adenosine in crude extract from mouse, rat, rabbit and bovine tissues was determined and showed requirements similar to those of the sequestration in mouse liver extract.The ability to sequester adenosine was high in liver and decreased in the following order: liver, kidney, adrenal cortex, brain, uterus, cardiac and skeletal muscle.     

Metabolism of somatostatin and its analogues by the liver

The rate of degradation of ¹²⁵I-labelled [Tyr¹¹]somatostatin by isolated rat hepatocytes was similar to that of unlabelled somatostatin. Reaction was dependent upon cell concentration and temperature, being rapid at 37°C and negligible at 0°C. The apparent K_m for the overall degradation process was approximately the same for degradation by hepatocytes and by partially-purified liver plasma membranes. Extracellular breakdown of somatostatin, by proteases released from cells into the incubation medium, represented less than 10% of the cell-associated degradation. Homogenization of hepatocytes resulted in a 10–20-fold increase in the degrading ability of the cells. After incubation of ¹²⁵I-labelled [Tyr¹¹]somatostatin and ¹²⁵I-labelled [Tyr¹]somatostatin with hepatocytes, ¹²⁵I-labelled tyrosine was the major radioactive product identified in the incubation medium. The rate of release of ¹²⁵I-labelled tyrosine from the labelled [Tyr¹] analogue was approximately 11 times greater than from the labelled [Tyr¹¹] analogue. ¹²⁵I-labelled [Tyr¹¹]somatostatin bound to the cells in a non-saturable manner and approx. 70% of the cell-associated radioactivity could be dissociated by dilute acid. The rate of degradation of somatostatin was unchanged by reagents that inhibit the internalisation and lysosomal degradation of polypeptides by cell suspensions but was reduced by reagents that inhibit sulphydryl-dependent proteases. It is proposed that plasma-membrane associated proteolysis, involving both endo- and exopeptidases may represent the predominant degradative pathway of somatostatin in vivo.     

Comparison of calmodulin binding to brain synaptic and coated vesicles

Several characteristics of calmodulin association with brain synaptic and coated vesicles were analyzed and compared. Radioimmunoassay revealed that both classes of vesicles contain approx. 1 μg of calmodulin per mg of vesicle protein. Discontinuous sucrose gradients revealed that coated and synaptic vesicles preparations were homogeneous and had different sedimentation properties. Binding of ¹²⁵I-labeled calmodulin to synaptic and coated vesicles was Ca²⁺ dependent and displaced by unlabeled calmodulin but not by troponin-C. Scatchard analysis revealed the presence of two binding sites. In both vesicle types there was one high-affinity, low-binding-capacity site ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 1–39}\text{nM and B}{}_{\mathrm{<mtext>max</mtext>}}\text{= 4–16}\text{pmol}\text{/}\text{mg}$) and one low-affinity, high-binding-capacity site ($\text{K}{}_{\mathrm{<mtext>d</mtext>}}\text{= 102–177}\text{nM and B}{}_{\mathrm{<mtext>max</mtext>}}\text{= 151–202}\text{pmol}\text{/}\text{mg}$). (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated in both synaptic and coated vesicles by calmodulin. Thus synaptic and coated vesicles may possess similar calmodulin binding sites.     

Ca2+-stimulated,Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m  0.25 μM, V_max  24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

Studies on the ferrochelatase activity of isolated rat liver mitochondria with special reference to the effect of oxidizable substrates and oxygen concentration

The mitochondrial ferrochelatase activity has been studied in coupled rat liver mitochondria using deuteroporphyrin IX (incorporated into liposomes of lecithin) and Fe(III) or Co(II) as the substrates.

1. 1. It was found that respiring mitochondria catalyze the insertion of Fe(II) and Co(II) into deuteroporphyrin. When Fe(III) was used as the metal donor, the reaction revealed an absolute requirement for a supply of reducing equivalents supported by the respiratory chain.

2. 2. A close correlation was found between the disappearance of porphyrin and the formation of heme which allows an accurate estimate of the extinction coefficient for the porphyrin to heme conversion. The value Δ (mM⁻¹ · cm⁻¹) = 3.5 for the wavelength pair 498 509 nm, is considerably lower than previously reported.

3. 3. The maximal rate of deuteroheme synthesis was found to be approx. 1 nM · min⁻¹ · mg⁻¹ of protein at 37 °C, pH 7.4 and optimal substrate concentrations, i.e. 75 μM Fe(III) and 50 μM deuteroporphyrin.

4. 4. Provided the mitochondria are supplemented with an oxidizable substrate, the presence of oxygen has no effect on the rate of deuteroheme synthesis.