Fucose Incorporation Research Articles

Long-term memory formation in chicks is blocked by 2-deoxygalactose, a fucose analog

When day-old chicks are trained on a passive avoidance task there is enhanced synthesis of glycoproteins. Bilateral intracerebral injections of 20 mumole of 2-deoxygalactose (2-D-gal), administered just before and just after training on the task, produce amnesia for the avoidance. Amnesia develops slowly over the first hour and persists for at least 24 h subsequently. If 2-D-gal injections are administered 4 h prior to the training or delayed for 3 h after training, no amnesia occurs. Apart from a brief initial suppression of pecking following injection there are no effects of 2-D-gal on other observed behaviors of the birds. Within the first hour this dose of 2-D-gal inhibits [3H]fucose incorporation into acid-insoluble material by 26% (or 68%, calculated relative to free pool fucose). The amnestic effect of 2-D-gal is not shown by galactose, glucose, fucose, or 2-D-glucose. Injecting 40 mumole of galactose simultaneously with the 2-D-gal abolishes the 2-D-gal-induced amnesia; 40 mumole of fucose, however, does not abolish the amnesia. The utility of 2-D-gal as an agent for analyzing the role of glycoproteins in memory formation is discussed.

Inhibition of mannose incorporation into glycoproteins and dolichol-linked intermediates of Sarcoma 180 cells by 6-methylmercaptopurine ribonucleoside.

6-Methylmercaptopurine ribonucleoside (6-MMPR), an inhibitor of purine nucleotide biosynthesis de novo, was used as a model compound to evaluate the relationship between the levels of intracellular guanosine triphosphate (GTP) and the formation of cellular glycoproteins and their dolichol-oligosaccharide precursors in Sarcoma 180 cells. Previous studies using the purine antimetabolite, 6-thioguanine (6-TG), demonstrated a relationship between the drug-induced decrease in GTP levels and the incorporation of radiolabelled mannose and fucose into cellular glycoproteins; estimation of the importance of these cell-surface alterations to the cytotoxicity produced by this agent was complicated by the incorporation of 6-TG nucleotides into cellular DNA and RNA. In this report, evidence is presented to show that the toxicity of 6-MMPR to Sarcoma 180 cells is associated with the effects of this agent on the intracellular pools of purine nucleotides. GTP functions in part in the activation of the sugar mannose, a step necessary for the biosynthesis of glycoproteins from nucleotide sugar precursors. Thus, 6-MMPR, which blocks the de novo pathway of purine nucleotide biosynthesis, caused a pronounced decrease in the intracellular pools of GTP in Sarcoma 180 cells; this phenomenon was accompanied by a marked reduction in the incorporation of radiolabelled mannose into cellular glycoproteins and their dolichol-linked oligosaccharide precursors. In contrast, the incorporation of glucosamine, a sugar not metabolically activated by GTP, into glycoproteins, and of leucine into protein, were depressed only after prolonged incubation with either 6-MMPR or 6-TG. Adenine restored purine nucleotide pools depleted by 6-MMPR and partially prevented both the reduction in mannose incorporation into glycoprotein and the cytotoxic effects of this antimetabolite. Guanosine partially reversed the effects of 6-MMPR on intracellular GTP pools and mannose incorporation but not the depression of ATP pools produced by this anti-metabolite. However, guanosine did not reverse the cytotoxicity of 6-MMPR but instead enhanced its toxicity. The findings are consistent with the possibility of membrane changes being involved in the cytotoxicity of 6-MMPR, but clearly other factors are involved as well.

Incorporation of [ 3H]fucose in rat hippocampal structures after conditioning by perforanth path stimulation and after LTP-producing tetanization

The contribution of glycoprotein synthesis to functional synaptic changes and to the formation of memory traces was investigated by autoradiographic determination of the incorporation of [ 3H]fucose into the hippocampal structures of rats. In the first experiment, the fucose incorporation was measured after induction of post-tetanic long-term potentiation (LTP) in granular cell synapses by repeated tetanization (200 cps) of the perforant path, and after stimulation of this hippocampal input by the same number of impulses with very low frequency (0.2 cps) not producing LTP. In the second experiment, the incorporation of fucose was determined after an active avoidance training using the stimulation of the perforant path by impulse trains of 15 cps as conditioning stimuli, and after a session of corresponding unpaired stimulations of the perforant path. Unstimulated animals were used in both experiments to measure the basal glycosylation. LTP-producing tetanization resulted only in a slight increase of incorporation into the ipsilateral hippocampal structures without significant differences to similar changes after the corresponding control stimulation with single impulses. After a session of unpaired stimulation of the perforant path with impulse trains of 15 cps only slight and inconsistent changes of incorporation occurred in the hippocampus too. However, after conditioning by the corresponding perforant path stimulation as conditioned stimulus, considerable increases of incorporation were observed in all structures of the ipsilateral hippocampus, when compared to the unpaired control stimulation. An enhanced labeling occurred also in some structures of the contralateral hippocampus mainly receiving commissural inputs. The results suggest again, that the activation of one single hippocampal afferent, even if producing LTP, would not be sufficient to induce an increased glycosylation of neuronal proteins. The increase of glycoprotein formation seems to require the convergence of several inputs, which can be assumed to occur during learning. Therefore, LTP of a single synaptic population seems not to represent the complete long-lasting memory trace, but only one of its components, or a preceding transient storage mechanism.

Increased fucosylation of chick brain proteins following training: effects of cycloheximide.

When chicks are trained to avoid pecking a bead coated with methylanthranilate in a one-trial passive avoidance task there is an increase in fucose incorporation in vivo and in vitro in the right forebrain base of methylanthranilate (M)-trained compared to water (W)-trained chicks. The relation of this increase to de novo protein synthesis in vivo and in vitro has been examined. Cycloheximide (Cx), 1 mM, inhibited in vitro fucosylation of chick brain slices by 60% after 3 h. However, the training-related increase in in vitro fucosylation still persisted. When Cx was injected intraventricularly 10 min before training, the subsequent increase in in vitro fucosylation due to training was still apparent. When Cx was injected and [14C]leucine and [3H]fucose incorporation studied in vivo in M-trained and W-trained chicks, there was no increase in fucosylation due to training in the Cx-treated M-trained over the W-trained chicks. These results are taken to indicate that in vitro fucosylation and its increase subsequent to training is not protein synthesis-dependent, but that both in vivo and in vitro there are interactions between Cx and fucosylation steps that are independent of Cx's effects on protein synthesis.

Components of increased labelling with putrescine and fucose during healing of skin wounds.

To study the glycoproteins and transglutaminase substrates involved in healing, wounds were made in the skin of anesthetized rats with a biopsy punch. Explants made 1-5 days later were incubated with [3H]-labelled putrescine, fucose or proline. As compared with unwounded skin there was an increased incorporation of label which was greatest at 3 days. Incubation for various times suggests that the incorporation of fucose and proline is dependent on protein synthesis, whereas putrescine is incorporated into preformed proteins. Putrescine and fucose label polypeptides with an Mr of about 45,000 before and 14,000 after reduction. These correspond in size with the aminopropeptide of type III collagen. Other labelled material of higher molecular weight is partly degraded to similar polypeptides on collagenase digestion. Much of the [3H]putrescine in the polypeptides is in the form of gamma-glutamyl putrescine. It is hypothesized that isopeptide linkage of the aminopropeptide III occurs in wound healing.

Abnormal glycosylation of myelin-associated glycoprotein in quaking mouse brain

Brain slices from actively myelinating (26-28 days) quaking and normal littermates were dual-labeled with radioactive mannose and fucose for 2 h. Following the incubation myelin was isolated by sucrose density gradient centrifugation and the incorporation of sugars into the major myelinassociated glycoprotein (MAG) determined. The incorporation of mannose (an internal monosaccharide) and fucose (a terminal monosaccharide) was impaired in quaking by approximately 70 and 83% respectively as compared to control. The mannose/fucose ratio in quaking myelin was approximately 70% higher than in control. The results indicate an abnormal processing of the N-linked oligosaccharide moiety of MAG in quaking oligodendrocytes.

Complex glycosphingolipids of the pheochromocytoma cell line PC12: Enhanced fucosylglycolipid synthesis following nerve growth factor treatment

The effect of nerve growth factor treatment on the expression of neutral glycosphingolipids and gangliosides was examined in PC12 pheochromocytoma cells grown in spinner culture. These studies show that PC12 cells contain an unusual group of fucose containing neutral glycolipids and gangliosides. The fucose containing neutral glycolipids, which appear to be derivatives of globoside, are very minor components. However, nearly all of the gangliosides in PC12 cells contain fucose. Two minor neutral glycolipids were isolated from these cells and were identified as fucosylgloboside and galactosylgloboside. Two major gangliosides were also isolated and identified as fucosyl GM2 and fucosyl GD2. Studies using [ 3H]fucose to examine the synthesis of fucosylglycolipids indicate that nerve growth factor enhances the incorporation of fucose into glycolipids and gangliosides by as much as 80%. These studies show that all of the gangliosides in PC12 cells appear to increase in concentration when the cells are treated with nerve growth factor in spinner culture. However, only the complex fucose containing neutral glycolipids increase in concentration during this same treatment, while the non-fucosylated precursors do not.

Subcellular Localization of Fucose Incorporation into Mouse Thyrotropin and Freeα-Subunits: Studies Employing Subcellular Fractionation and Inhibitors of the Intracellular Translocation of Proteins*

To determine the subcellular sites of fucose incorporation into TSH subunits, pituitaries from hypothyroid mice were incubated with [3H]fucose and fractionated by sucrose gradient centrifugation. To assess potential molecular cross-contamination between subcellular fractions enriched in rough endoplasmic reticulum (RER) or Golgi elements, trace amounts of exogenous [35S]methionine-labeled proteins or [125I]rat TSH were added before tissue homogenization. Particulate contamination of fractions was monitored by electron microscopy. TSH subunits were immunoprecipitated from fractions and analyzed by gel electrophoresis. After both a 2-h pulse incubation and a 2-h pulse, 3-h chase incubation, about half (range, 44-71%) of the [3H]fucose-labeled TSH subunit precursors present in microsomes were in the RER (amounts in excess of estimated contamination by nonspecific readsorption of molecules to vesicles or the presence of Golgi vesicles in the RER fractions); [3H]fucose-labeled free alpha-subunits were also detected in RER as well as in Golgi fractions. During chase incubations, both monensin and carboxyl cyanide m-chlorophenylhydrazone inhibited the appearance of [35S]methionine- or [3H]fucose-labeled TSH subunits in medium in a dose-dependent manner, suggesting that [3H] fucose was added to subunits, in part, early in the secretory pathway. Free alpha-subunits were more fucosylated than was TSH; in TSH heterodimers, beta-subunits were richer in fucose than were alpha-subunits. Thus, the fucosylation of TSH and free alpha-subunits in pituitaries of hypothyroid mice appears to begin at an unusually early stage of intracellular transport and may represent an adaptation to special posttranslational processing requirements.

Isolation of the dorsal, ventral and intracellular domains of HeLa cell plasma membranes following adhesion to a gelatin substrate

The plasma membrane is a complex organelle responsible for many cellular functions. In addition to mediating the exchange of components with the extracellular fluid, the plasma membrane is involved in cell adhesion to matrix proteins in vivo and in vitro. In vitro, adherent cells have three distinct plasma membrane domains to carry out these functions: one attached to the substrate (ventral); another exposed to the media (dorsal); and an intracellular domain involved in endocytosis and secretion. A technique has been developed for the rapid isolation of these specific domains from HeLa cells immediately following adhesion to a gelatin substrate. The isolation procedure utilizes the tight binding of cationic colloidal silica to the dorsal plasma membrane domain of attached cells. Following silica binding and cell lysis, the silica-coated dorsal plasma membrane domain is readily separated from intracellular plasma membrane components by virtue of the high density of the silica pellicle, and the intact ventral plasma membrane domain remains attached to the gelatin substrate. Fluorescence and electron microscopy and biochemical studies using 125I-lactoperoxidase labeling, 125I-labeled wheat germ agglutinin binding, and [ 3H]-fucose incorporation into plasma membrane glycoproteins confirmed the separation of these three topologically distinct plasma membrane domains. The fractions isolated by the technique contained essentially all of the plasma membrane components present in intact cells. This unique membrane-isolation procedure is now being used to analyze membrane flow during plasma membrane domain formation accompanying cell adhesion to an extracellular matrix.

Human spreading factor: synthesis and response by HepG2 hepatoma cells in culture.

Human serum spreading factor (SF) is a cell adhesion and spreading-promoting glycoprotein purified from serum or plasma that mediates effects in a wide variety of animal cell culture systems. HepG2 human hepatoma cells were found to synthesize and secrete SF into culture medium. Quantitative immunoassay of the protein indicated a concentration of about 1 microgram/ml in 48 hr-conditioned medium from confluent cultures. Although fibronectin also was synthesized and secreted into the culture medium, HepG2 cell spreading was observed in response to human serum SF, but not in response to human plasma fibronectin. Immunoprecipitation of SF from culture medium of cells metabolically-labeled with leucine, fucose or glucosamine identified a single form of the molecule of approximately 70,000 daltons. Treatment of cultures with tunicamycin inhibited incorporation of fucose and glucosamine into immunoprecipitated SF, but did not prevent synthesis and secretion of the protein. Electrophoretic analysis and cell spreading assays showed that SF secreted by tunicamycin-treated HepG2 cells was of molecular weight (mw) approximately 60,000, and was biologically active.

Glycoprotein biosynthesis during inflammation in normal and streptozotocin-induced diabetic rats.

Extracellular glycoproteins play an important role in wound healing; yet little is known about glycoprotein biosynthesis and its regulation by insulin in inflammation. Using [1-14C] fucose as a marker, glycoprotein biosynthesis was studied in carrageenan-induced granuloma from diabetic and control rats. Fucose incorporation into glycoproteins was followed for 24 h after an intraperitoneal injection of the label. Radioactivity in trichloroacetic acid precipitable serum glycoproteins and saline-soluble and insoluble glycoproteins was assessed in five-, seven-, and ten-day-old granuloma tissues. Fucose incorporation was higher in soluble glycoproteins (P less than 0.01) at all points in controls than in diabetic granulomas, and peak incorporation was reached in both groups on the seventh day. Incorporation of fucose into insoluble glycoproteins was higher in normals on the seventh day than in diabetics. Liver-, kidney-, and intestine-soluble glycoproteins showed a maximum incorporation on the seventh day, but no difference was noted between diabetic and normal rats. Incorporation of fucose in insoluble glycoproteins showed a gradual decline with the age of granuloma in all tissues from both groups, with the exception of the kidney. In the kidney, fucosylation of insoluble glycoproteins was decreased (P less than 0.01) in diabetics compared to controls. These results indicate an active phase of biosynthesis, with an increase in glycosylation during inflammation that is probably insulin dependent.

Effect of hormone administration on the sialylation and fucosylation of intestinal microvillus membranes of suckling rats.

Cortisone, thyroxine, epidermal growth factor, or insulin were administered to 8-day-old rats for 4 days. In comparison to saline-injected controls, cortisone treatment: 1) lowered the sialic acid and raised the fucose content of the intestinal microvillus membranes, 2) increased [3H]fucose incorporation into these membranes, and 3) decreased the membrane binding of 125I-wheat germ agglutinin, while increasing the binding of 125I-ulex europeus agglutinin I and 125I-peanut agglutinin. Thyroxine treatment had similar effects on fucose content and 125I-ulex europeus agglutinin I binding, but did not alter [3H] fucose incorporation or sialic acid content. At the doses used, epidermal growth factor and insulin had no significant effects. The effect of cortisone treatment on sialic acid and fucose was commensurate with a 5- to 6-day acceleration of postnatal intestinal maturation. The changes in lectin binding, however, suggested qualitative differences between developmental and cortisone-induced membrane glycosylation. In addition, this study demonstrates significant quantitative and qualitative differences in the response of intestinal glycosylation to pharmacologic doses of the four hormones.

Biosynthesis of Lewis antigenic glycolipid by cell-free extracts of human intestinal tumor cells cultured in serum-free medium

Extracts of the human intestinal tumor cell line SW1116 were able to stimulate the incorporation of (14C) fucose from GDP-(14C) fucose into organically soluble glycolipid. The reaction required a purified glycolipid preparation from human meconium as lipid acceptor. The active glycolipid co-migrated with standard globoside on high performance thin-layer chromatography (HPTLC) and had molecular species (M + H) under fast-atom bombardment mass spectrometry of 1199, 1245 and 1269. Globoside itself was inactive and asialo GM1 had low activity. The radiactive products co-purified with Lewisa and Lewisb and comigrated principally (60–90%) with Lewisb monoclonal antibody binding cellular glycolipids on HPTLC. Analysis of fucosidase digests suggested the presence of two different fucosyl-hexose linkages one of which was suceptible to cleavage. We conclude that the data are consistent with fucosylation of lactotetraosyl ceramide to Lewisa and Lewisb antigenic glycolipids.

Passive avoidance training increases fucose incorporation into glycoproteins in chick forebrain slices in vitro.

When day old chicks are trained to avoid pecking at a bright bead coated with methyl anthranilate, many neurochemical changes, both transient and longer lasting, have been found. These include an increased fucose incorporation in vivo into particulate glycoproteins, which persists for at least 24 hrs after training. We have now developed an in vitro method for studying fucose incorporation and have been able to replicate effects of training found in vivo. Chick forebrain slices incubated at 42 degrees in a glucose containing-medium incorporate L-[U14C]fucose linearly for up to 3 hrs at rates of 30-35 nmol/g prot/hr. Incorporation was only 60% inhibited by cycloheximide indicating that some fucosylation is occurring on pre-existing proteins. Fucose incorporation was compared in slices from trained and control chicks and, as in vivo, a 16% increase in incorporation into the right forebrain base of trained birds was found. This increase was confined to the microsomal fraction. When cycloheximide was added to the incubation medium, the enhanced fucose incorporation in slices from trained birds was still observed.

Glycoprotein composition and turnover in subcellular fractions from the cerebral cortex of normal and reeler mutant mice.

Twenty-day-old reeler and normal mice were either injected intraventricularly with radiolabelled fucose before subcellular fractionation of the cerebral cortex followed by SDS-PAGE, or gels of such fractions were overlaid with [125I]concanavalin A (ConA). While there were no differences in polypeptide profiles of normal and reeler subcellular fractions there were marked differences in the abundance of particular ConA-binding glycoproteins and in fucose incorporation into particular glycoproteins, especially in the synaptic plasma membrane (SPM) and 100,000 g soluble fractions. In addition there was a significantly lower binding (greater than 50%) of quinuclidinyl benzilate to reeler microsomal and SPM fractions as compared with normal. The differences in glycoprotein expression may be pertinent to anatomical observations of abnormal interactions between neurons and glial fibres during development of the reeler cerebrum.

Fucosylation of Membrane Proteins in Soybean Cultured Cells

Cultures of soybean cells incorporate [5,6-(3)H]-l-fucose into various cellular components including lipids and proteins. The membrane glyco-proteins were digested with pronase to produce glycopeptides, and the glycopeptides were isolated on columns of Biogel P-4. The major fucoselabeled glycopeptide sized as a Hexose(15-17)-N-acetylglucosamine(2) (GlcNAc(2)) on columns of Biogel P-4. Fucose incorporation was also examined in the presence of the processing inhibitor swainsonine, and the glycosylation inhibitor tunicamycin. In the presence of swainsonine, the incorporation of fucose was not reduced but the glycopeptides were smaller in size and migrated like Hexose(12-13)-GlcNAc(2) structures. On the other hand, tunicamycin inhibited the incorporation of fucose into the glycopeptides by 70 to 80%, indicating that the l-fucose was present in N-linked oligosaccharides.The membrane glycoproteins were doubly-labeled by incubating soybean cells in [(3)H]fucose and [(14)C]mannose. By repeated separation on Biogel P-4, six glycopeptides were purified that ranged in size from Hexose(8)GlcNAc(2) to Hexose(15-17)-GlcNAc(2). The three larger glycopeptides (I, II, III) were highly labeled with [(3)H]fucose and also contained [(14)C] mannose. Evidence that both isotopes were in the same glycopeptide was obtained by the finding that the mannose-labeled glycopeptides were shifted to smaller-sized structures when the [(3)H]fucose was removed by mild acid hydrolysis. Glycopeptide IV also contained [(3)H]fucose and [(14)C] mannose but only part of the [(3)H]fucose was released by mild hydrolysis. Glycopeptides V and VI contained only small amounts of tritium, but were labeled with [(14)C]mannose. None of the six glycopeptides was susceptible to the action of endo-beta-N-acetylglucosaminidase H, and none of these glycopeptides was bound to columns of concanavalin A-sepharose. The smaller glycopeptides (IV, V, VI) were partially susceptible to alpha-mannosidase digestion, but this enzyme did not release any radioactive mannose from the larger-sized glycopeptides. These data indicate that the fucosylated glycopeptides are N-linked structures containing fucose, mannose, GlcNAc, and probably other sugars, and that the mannose units are blocked by other sugars. Thus, these results indicate that plant membrane glycoproteins contain a significant amount of modified oligosaccharide side chains.

Regional and cellular differences in fucosylation of glycomacromolecules in the mouse brain. A biochemical and autoradiographic study of early postnatal and adolescent animals

Mice of CBA strain of both sex were injected with [ 3H]fucose at age 2, 6, 12 or 30 days and the incorporation was determined biochemically 45, 90 and 180 min later. Biochemical measurements of the whole brain (at 90 min postinjection interval) revealed a stepwise age decrease in the amount of incorporated isotope (per mg protein). The amount of [ 3H]fucose available in the brain has however been found to decrease with age while the degree of its utilization increases. Thus, after correction of the data on [ 3H]fucose incorporation for the pool of the free isotope, a transient increase in fucosylation rate appeared at postnatal day 12 while the differences among 2-, 6- and 30-day-old animals became negligible. Further, the synthesis of fucosylated soluble glycomacromolecules appeared relatively higher at postnatal day 2 than in older age groups. Examination of different brain regions revealed that the rate of fucosylation is relatively highest in the olfactory bulbs; this prevalence starts appearing with age and becomes most evident in 30-day-old mice. Autoradiography carried out with 6- and 12-day-old animals revealed that the incorporation of [ 3H]fucose into meningovascular apparatus and the choroid plexus is a few times higher than into brain parenchyma. The regional differences appeared to be similar to those indicated by the biochemical data. Cellular analysis has shown that the incorporation is relatively higher in the cytoplasm of large projecting neurons of the cerebellum, hippocampus and the olfactory bulbs; in the latter region high amounts of macromolecule-bound [ 3H]fucose also appeared in the synaptic glomeruli. The olfactory bulbs have also been shown to be relatively more rich in newly synthetized fucosylated soluble glycoproteins than other brain regions.

Ammonium chloride, methylamine and chloroquine reversibly inhibit antibody secretion by plasma cells.

The effects of the lysosomotropic weak bases, NH4Cl, methylamine and chloroquine, on the secretory process of antibody-synthesizing cells were studied. Popliteal lymph node cells taken from rats immunized against horseradish peroxidase (HRP) were incubated with the lysosomotropic agents. The rate of secretion of anti-HRP antibodies was measured using an indirect enzyme-linked immunosorbent assay. These agents induced an inhibition of antibody release within 5 min, and for all four concentrations tested, maximal inhibition was reached after 15 min. A 50% inhibition was obtained with 20 mM NH4Cl, 21.7 mM methylamine and 8.8 X 10(-4) M chloroquine. This effect was rapidly and entirely reversible, regardless of the weak base used, and it increased as the pH of the extracellular media was raised. Under these conditions, intracellular ATP contents remained normal, and protein synthesis did not undergo marked changes except with chloroquine. Inhibition of secretion was accompanied by an intracellular accumulation of antibodies which was equal to the degree of inhibition of antibody release. Immunocytochemical studies of the weak base-treated cells performed by light and electron microscopy showed that this accumulation probably occurred within certain dilated Golgi saccules. In addition, reduced incorporation of fucose into immunoglobulins as well as partial inhibition of the secretion of fucosylated immunoglobulins were observed in the presence of weak bases. These results are consistent with the hypothesis that weak bases inhibit antibody secretion by acting within saccules of the Golgi apparatus. These saccules could maintain an acidic pH important for the migration and/or sorting of immunoglobulins.

Synthesis and renal activity of rat atrial granules depend on extracellular volume.

Extracellular fluid volume (by 22Na) and extent of 4-h [3H]fucose incorporation into atrial-specific granules were measured in deoxycorticosterone acetate (DOCA)/salt-loaded or Na-deficient rats. The natriuretic potency of extracts from their atria was also measured in assay rats. DOCA/salt-treated animals had a significantly greater extracellular volume, a significantly greater degree of fucose uptake, and a significantly more potent diuretic and natriuretic effect than did Na-deficient rats. These observations, together with the known decrease in atrial granularity with DOCA treatment, suggest that a chronic increase in extracellular fluid volume is associated with increased synthesis and metabolism of atrial natriuretic factor. They also confirm the finding reported by others that granularity and natriuretic potency are not always directly related. It may be that visible granules represent a peptide storage form that requires further processing to become natriuretic.

Effect of thyroid hormone on carbohydrate content of Na+-K+-adenosine triphosphatase

The effect of L-3,5,3'-triiodothyronine (T3) (50 micrograms/100 body wt) on the incorporation of labeled glucosamine and fucose into the subunits of Na+-K+-ATPase was examined by gel electrophoresis in sodium dodecyl sulfate. T3 augmented the incorporation of glucosamine into the alpha- and beta-subunits by 51 and 58%, respectively, in the 22-h chase experiments. Similarly T3 augmented the incorporation of fucose into the alpha- and beta-subunits by 58 and 43%, respectively. Reverse T3 did not alter the incorporation of labeled fucose in either subunit. The effect of T3 on the rate constant of degradation of renal cortical Na+-K+-ATPase was assessed. The rate constant of degradation (Kd) of the [3H]fucose labeled alpha- and beta-subunits for the hypothyroid rats were both 0.20, and for T3-treated rats, the Kd of the alpha- and beta-subunits were 0.23 and 0.18, respectively, suggesting that T3 enhanced fucose incorporation into the subunits of Na+-K+-ATPase rather than retarding the degradation of this enzyme.