Isolation ofn-ethylmaleimide-labelled phlorizin-sensitived-glucose binding protein of brush border membrane from rat kidney cortex

A glucose receptor with high affinity for phlorizin from isolated brush border of rat kidney was labelled specifically withN-[¹⁴C]ethylmaleimide and then extracted from the membranes.After the solubilization of the brush borders with sodium dodecyl sulphate theN-[¹⁴C]ethylmaleimide-labelled receptor protein was isolated and was found to have a molecular weight of approximately 30 000 as determined by sodium dodecyl sulphate-polyacrylamide gel disc electrophoresis. The receptor protein eluted from the sodium dodecyl sulphate-containing gels migrates as a single band on sodium dodecyl sulphate-free polyacrylamide gels.The receptor protein can also be released from the brush borders with low concentrations of sodium deoxycholate. Under these conditions the molecular weight of theN-[¹⁴C]ethylmaleimide-labelled receptor protein is approximately 60 000 in contrast to the protein component solubilized with sodium dodecyl sulphate. Since this detergent is known to dissociate the brush border membrane into its protein components, our results suggest that the phlorizin- sensitive glucose receptor protein has a molecular weight of about 30 000.     

Rapidly metabolized glycoproteins in a neuroplastoma cell line

The metabolism of neuroblastoma cell glycoproteins was examined using l-[³H]fucose. Incubation of monolayer cultures with [³H]fucose resulted in a rapid uptake of the radioactive precursor and its incorporation into acid-insoluble macromolecules. Less than 3% of the [³H]fucose that was isolated from neuroblastoma cells by trichloroacetic acid precipitation was associated with glycolipids. The metabolism of fucosylated macromolecules was studied in cells which were labelled to a steady state, and then reincubated under conditions which limited reutilization of the radioactive precursor (40 mM unlabelled fucose). During reincubation of the cells, we observed a rapid metabolism (27% by 2 h)_ of the prelabelled macromolecules which stabilized within a cell generation time to give an overall rate of turnover of 9%. This rapid loss of radioactivity from the cells was not due to exocytosis since less than 4% of the [³H]-fucose was lost into the media as macromolecules during a 5 h reincubation period. The presence of 40 mM fucose in the media did not affect cell growth until after 24 h of incubation or cellular synthesis until after 15 h of incubation. When the metabolism of neuroblastoma cell glycoproteins was measured in the presence of 1.8 · 10^?4 M cycloheximide, there appeared to be a less rapid decrease in cell-associated specific activity, and an increased reutilization of [³H]fucose. Although the major proportion of the radioactivity remained as [³H]fucose, extensive incubation of neuroblastoma cells with this radioactive precursor led to increased amounts of tritium associated with other cellular components. However, a rapid rate of glycoprotein metabolism could also be demonstrated with cells incubated with [⁴C]fucose. This eliminated the possibility that the above results were restricted to the tritiated precursor and merely a reflection of hydrogen-tritium exchange.     

HE TURNOVER RATE OF TUBULIN IN RAT BRAIN

The turnover rate of tubulin in rat brain was determined from the decay in specific radioactivity of the protein after pulse-labeling. When precursors were administered by a parenteral route, the shortest half-life, 9.8 days, was obtained with [¹⁴C]NaHCO₃; the longer half-lives obtained with [U-¹⁴C]glucose or [4,5-³H]leucine suggest significant reutilization of label. Furthermore, with leucine as precursor maximal specific radioactivity of tubulin was not obtained until eight days after administration of label. Labeling and decay kinetics obtained with [4,5-³H]leucine were markedly different when the isotope was administered directly into the lateral ventricle. The difference between the turnover rates of the -α and β subunits of tubulin purified by means of high resolution polyacrylamide gel electrophoresis was not statistically significant. A half-life for tubulin of 6.2 days was measured by continuous intravenous infusion of [U-¹⁴C]tyrosine.     

Amo 1618 effects on incorporation of 14C-MVA and 14C-acetate into sterols in Nicotiana and Digitalis seedlings and cell-free preparations from Nicotiana

Incorporation of radioactivity from acetate-[¹⁴C] and MVA-[¹⁴C] into sterols and sterol precursors in tobacco was inhibited by Amo 1618; differing patterns of accumulation were obtained with the two precursors, suggesting more than one point of inhibition. This was borne out with cell-free preparations with which it was demonstrated that both HMG-CoA reductase and squalene-2,3-epoxide cyclase were inhibited, the latter more strongly than the former. GLC analysis of gross sterol and hydrocarbon fractions confirmed previous indications that incorporation of radioactivity into individual sterols was inhibited by Amo 1618. Finally, incorporation of MVA-[¹⁴C] into sterols and sterol precursors of Digitalis was significantly altered by the retardant, thus expanding the generality of the relationship between sterol (particularly 4-desmethylsterol) biosynthesis inhibition and retardant effect.     

Utilization of exogenously supplied primary precursors for essential oil synthesis in cymbopogon species

The relative efficiency of incorporation of the exogenously supplied primary precursors [2-¹⁴C]acetate, [U-¹⁴C]glucose and [U-¹⁴C]sucrose into essential oil was determined in the immature leaves of threeCymbopogon species(C. martinii,C. winterianus andC. flexuosus). Acetate was most efficiently incorporated into essential oil inC. winterianus andC. flexuosus, whereas glucose was the best precursor inC. martinii. The observations are consistent when expressed as radioactivity [Bq] of essential oil per leaf, as percent incorporation or moles of precursors utilized for essential oil synthesis. Thus, there is selectivity in the efficiency of precursor utilization for the oil synthesis inCymbopogon species.     

Effect of pyridoxine deficiency on nucleic acid metabolism in the rat

1. The nucleic acid metabolism in the pyridoxine-deficient rat has been investigated through studies on the incorporation of radioactivity from various isotopically labelled compounds into liver and spleen DNA and RNA. 2. In pyridoxine deficiency, the incorporation of radioactivity from sodium [¹⁴C]formate was apparently increased. The magnitude of this effect on incorporation into liver RNA and DNA and spleen RNA was approximately the same. The incorporation into spleen DNA was enhanced to a much greater degree. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [¹⁴C]formate. 3. In pyridoxine deficiency, the incorporation of radioactivity from dl-[3-¹⁴C]serine, [8-¹⁴C]adenine, [Me-³H]thymidine and [2-¹⁴C]deoxyuridine was decreased. The incorporation of radioactivity from l-[Me-¹⁴C]methionine was not affected. No noteworthy differences in the effect of pyridoxine deficiency on the incorporation of radioactivity from dl-[3-¹⁴C]serine into DNA and RNA were observed, whereas the effect of the deficiency on the incorporation of radioactivity from [8-¹⁴C]adenine into spleen DNA was somewhat greater than that into spleen RNA. Administration of pyridoxine 24hr. before the rats were killed reversed the changes in incorporation of radioactivity from [3-¹⁴C]serine and [8-¹⁴C]adenine. 4. The adverse effects of pyridoxine deficiency on the biosynthesis of nucleic acids and cell multiplication are discussed in relation to the role of pyridoxal phosphate in the production of C₁ units via the serine-hydroxymethylase reaction.     

Intracellular localization of metabolism of lupeol and its palmitate in Calendula officinalis flowers

In the non-fractionated flowers and in the chromoplast and extra-chromoplast fractions obtained from Calendula officinalis flowers the incorporation of radioactivity after incubation with [3-³H]lupeol and [3-³H]lupeyl[¹⁴C]palmitate were determined. It was shown that both precursors were metabolized. Monol esters are the main precursors of diol 3-monoesters, whereas free monols are hydroxylated to the diols and triols. The shape and course of the dynamic curves suggest that the process of hydroxylation of free monols, free diols and monol esters is situated only inside chromoplasts. On the other hand the hydrolysis of monol esters and esterification of free monols proceeds both outside and inside the chromoplasts.     

Axonal transport of phospholipids in rabbit optic pathway

The uptake of different labeled precursors, their incorporation into lipids, and transport along the rabbit optic pathway [ipsilateral retina and optic nerve (ON), and contralateral optic tract (OT), lateral geniculate body (LGB), and superior colliculus (SC)] were investigated. Albino rabbits were used. The following radioactive precursors, either combined or separately, dissolved in 50 l of saline containing 15% BSA, were injected into vitreous body: [2-³H]glycerol (50 Ci), [1-¹⁴C]palmitate (15 Ci), and [1-¹⁴C]linoleate (7.5 Ci). Animals were killed at different time intervals from 1 hr up to 24 days. The radioactivity of total lipids and of different phospholipid classes from total tissue was measured. One hour after the administration of precursors, the radioactivity into the retina was high and the incorporation of [³H]glycerol and [¹⁴C]palmitate increased until 12 hr and 24 hr, respectively. The incorporation of [¹⁴C]linoleate reached a maximum on the second day. The phospholipids of LGB and SC were intensively labeled after 4–8 hr, and their radioactivity increased up to the 10th day after injection, independent of the precursor employed. The results obtained indicate that the labeled hydrophilic and hydrophobic precursors used were actively incorporated into the retina. The phospholipids were later transported at a rapid rate along the optic pathway.A preliminary report of this study has been presented at the Satellite ISN Meeting, Istanbul, September 8–10, 1979.     

Equilibration of [3H]glucosamine and [35S]sulfate with intracellular pools of UDP-N-acetylhexosamine and 3′-phosphoadenosine-5′-phosphosulfate (PAPS) in cultured fibroblasts

Nucleotides and sugar nucleotides were extracted from cultures of human fibroblasts with perchloric acid, separated by isotachophoresis, and quantified by uv absorption analysis at 254 nm. ATP (936 pmol/μg DNA) was, as expected, the dominating nucleotide pool. The energy charge was estimated to 0.9. The UDP-N-acetylhexosamine pool was also a very prominent compound (596 pmol/μg DNA). After incubation of fibroblasts with [³H]glucosamine, more than 95% of the acid-soluble radioactivity was found in the UDP-N-acetylhexosamine pool. Incubation with [³⁵S]sulfate resulted in the incorporation of [³⁵S]sulfate into 3′-phosphoadenosine-5′-phosphosulfate (PAPS). The latter could, however, only be measured as radioactivity, as the amount was too small to be quantified as total mass. Pulse-labeling of fibroblasts with [³⁵S]sulfate and [³H]glucosamine from 5 min to 16 h showed that [³⁵S]PAPS was equilibrated in less than 10 min, while [³H]glucosamine required a longer time, 2–4 h, to attain a steady state with UDP-N-acetylhexosamine. [¹⁴C]Glucose required approximately the same time as [³H]glucosamine to reach steady state with UDP-acetylhexosamine, which suggests that the reason for the long equilibration time is the slow turnover of this pool.     

THE METABOLISM OF LABELLED ETHANOLAMINE IN NEURONAL AND GLIAL CELLS OF THE RABBIT IN VIVO1

Abstract— Adult rabbits were injected intraventricularly with [¹⁴C]ethanolamine and the incorporation of the base into the phosphatidylethanolamine and ethanolamine plasmalogen (and their water-soluble precursors) of isolated neuronal and glial cells was investigated. All the radioactivity was incorporated into the base moiety of the ethanolamine lipids for the time intervals examined in both types of cells. In neurons, maximum labelling of the two ethanolamine lipids occurred at 7 h after administration, whereas the highest specific radioactivity for glial phosphatidylethanolamine and ethanolamine plasmalogen was reached at 20 and 36 h, respectively. The two lipids had a faster turnover in neurons than in glia, and in both populations incorporated the base at a faster rate than did whole brain tissue. The maximum incorporation rates for phosphorylethanolamine and CDP-ethanolamine were reached in both types of cell at about 6 h after administration but the content of radioactivity per unit protein for phosphorylethanolamine was much higher in glial than in neuronal cells. It is concluded that the site of most active synthesis of ethanolamine phospholipids in vivo is the neuronal cell, with a possible transfer of intact lipid molecule to the glial compartment.     

The uptake of cyclic AMP by human erythrocytes and its effect on membrane phosphorylation.

The effects of time and cyclic AMP concentration on cyclic AMP uptake and membrane phosphorylation were studied using intact human erythrocytes. The rate of uptake of cyclic [³H]AMP was nearly linear with respect to cyclic AMP concentration. The amount taken up was small compared to the extracellular cyclic AMP concentration, but was sufficient to significantly increase the intracellular cyclic AMP concentration. Incubation with cyclic AMP resulted in increased incorporation of ³²P_i into several phosphorylated membrane peptides of the intact erythrocytes. Although cyclic AMP altered the distribution of radioactivity among the membrane components, the total amount of incorporation was not increased. The effect of cyclic AMP on phosphorylation of membrane peptides was observed with extracellular cyclic AMP concentrations as low as 1 μm and was most pronounced in incubations of 1 to 4 h. These results indicate that cyclic AMP can enter erythrocytes in sufficient amounts to alter the activity of cyclic AMP-dependent protein kinases, or to alter the rate of turnover of certain phosphorylated membrane peptides.     

The labelling of nucleic acids by radioactive precursors in the blue-green algae Anacystis nidulans and Synechocystis aquatilis Sanv.

Summary The labelling of nucleic acids of growing cells of the blue-green algae Anacystis nidulans and Synechocystis aquatilis by radioactive precursors has been studies. A. nidulans cells most actively incorporate radioactivity from [2-¹⁴C]uracil into both RNA and DNA, while S. aquatilis cells incorporate most effectively [2-¹⁴C]uracil and [2-¹⁴C]thymine.Deoxyadenosine does not affect incorporation of label from [2-¹⁴C]thymidine into DNA, but weakly inhibits [2-¹⁴C]thymine incorporation into both nucleic acids and significantly suppresses the incorporation of [2-¹⁴C]uracil.The radioactivity from [2-¹⁴C]uracil and [2-¹⁴C]thymine is found in RNA uracil and cytosine and DNA thymine and cytosine. The radioactivity of [2-¹⁴C]thymidine is incorporated into DNA thymine and cytosine. These results and data of comparative studies of nucleic acid labelling by [2-¹⁴C]thymine and [5-methyl-¹⁴C]thymine suggest that the incorporation of thymine and thymidine into nucleic acids of A. nidulans and S. aquatilis is accompanied by demethylation of these precursors. In this respect blue-green algae resemble fungi and certain green algae.     

Biosynthesis of securinine,the main alkaloid of Securinega suffruticosa

Biosynthesis of securinine was studied by incorporation experiments in Securinega suffruticosa. Among presumed precursors tested, lysine, cadaverine, and tyrosine showed the highest incorporation into securinine. Degradation experiments revealed that cadaverine-[1,5-¹⁴C] labelled specifically the piperidine ring of securinine and the radioactivity from dl-tyrosine-[2-¹⁴C] was introduced into the C-11 lactone carbonyl. Experiments with L-tyrosine-[U-¹⁴C] and L-tyrosine-[3′,5′-³H; U-¹⁴C] prove that the remaining C₆Sz.sbnd;C₂ moiety is derived from the aromatic ring and the C-2 and C-3 or tyrosine.     

Kinetic study of possible intermediates in the biosynthesis of chlorogenic acid in Cestrum poeppigii

p-Coumaric and 3-O-p-coumarylquinic acid seem to be important precursors of chlorogenic acid in the leaves of Cestrum poeppigii. 3-O-Cinnamylquinic acid, which has a very small metabolic activity, is of little importance in this respect. The kinetics of incorporation of radioactivity from t-cinnamic acid-3-[¹⁴C] into p-coumaric, 3-O-p-coumarylquinic, chlorogenic and 3-O-cinnamylquinic acid showed that the biosynthetic rates for these products decrease in the order shown. For p-coumaric acid, which has a markedly high metabolic activity, a turnover rate of 28 μg/hr and per gram fresh plant leaf, was calculated. Some trapping experiments with caffeic acid, and the acids mentioned above and using either t-cinnamic acid-3-[¹⁴C] or p-coumaric acid-2-[¹⁴C] as precursor, are discussed. A HPLC method for the rapid determination of phenolic acids in plant extracts, is described.     

Preparation, Characterization, and Microbial Degradation of Specifically Radiolabeled [14C]Lignocelluloses from Marine and Freshwater Macrophytes

Specifically radiolabeled [¹⁴C-lignin]lignocelluloses were prepared from the aquatic macrophytes Spartina alterniflora, Juncus roemerianus, Rhizophora mangle, and Carex walteriana by using [¹⁴C]phenylalanine, [¹⁴C]tyrosine, and [¹⁴C]cinnamic acid as precursors. Specifically radiolabeled [¹⁴C-polysaccharide]lignocelluloses were prepared by using [¹⁴C]glucose as precursor. The rates of microbial degradation varied among [¹⁴C-lignin]lignocelluloses labeled with different lignin precursors within the same plant species. To determine the causes of these differential rates, [¹⁴C-lignin]lignocelluloses were thoroughly characterized for the distribution of radioactivity in nonlignin contaminants and within the lignin macromolecule. In herbaceous plants, significant amounts (8 to 24%) of radioactivity from [¹⁴C]phenylalanine and [¹⁴C]tyrosine were found associated with protein, although very little (3%) radioactivity from [¹⁴C]cinnamic acid was associated with protein. Microbial degradation of radiolabeled protein resulted in overestimation of lignin degradation rates in lignocelluloses derived from herbaceous aquatic plants. Other differences in degradation rates among [¹⁴C-lignin]lignocelluloses from the same plant species were attributable to differences in the amount of label being associated with ester-linked subunits of peripheral lignin. After acid hydrolysis of [¹⁴C-polysaccharide]lignocelluloses, radioactivity was detected in several sugars, although most of the radioactivity was distributed between glucose and xylose. After 576 h of incubation with salt marsh sediments, 38% of the polysaccharide component and between 6 and 16% of the lignin component (depending on the precursor) of J. roemerianus lignocellulose was mineralized to ¹⁴CO₂; during the same incubation period, 30% of the polysaccharide component and between 12 and 18% of the lignin component of S. alterniflora lignocellulose was mineralized.     

Coronatine biosynthesis: Incorporation of l-[U-14C]isoleucine and l-[U-14C] threonine into the 1-amido-1-carboxy-2-ethylcyclopropyl moiety

Addition of either l-[U-¹⁴C]threonine or l-[U-¹⁴C]isoleucine to 2.7-day-old shaking liquid cultures of Pseudomonas syringae pv. atropurpurea resulted in incorporation of radioactivity into coronatine, but not into N- coronafacoylvaline, another phytotoxin excreted by P.s. atropurpurea. In contrast, addition ofl-[U-¹⁴C]valine did not lead to incorporation of radioactivity into coronatine, but instead into coronafacoylvaline. Acid hydrolysis of the purified [¹⁴C] coronatine obtained after incorporation of either [¹⁴C]isoleucine or [¹⁴C]threonine demonstrated that > 94% of the radioactivity was present in the 1-amido-1-carboxy-2-ethylcyclopropyl moiety of coronatine, and < 6 % was in the coronafacoyl moiety. These findings are used to propose a biosynthetic pathway for coronatine.     

Sialic acid incorporation into gangliosides and glycoproteins of the fish brain

—The concentration of lipid- and non-lipid-bound sialic acid in the optic nerve tract and tectum and in whole brain of fish was estimated. The incorporation of sialic acid into gangliosides and non-lipid components was studied in fish by intracranial or intraocular application of N-[³H]acetylmannosamine or N-[³H]acetylglucosamine. After intracranial injection of N-[³H]acetylmannosamine autoradiography showed lipid- and non-lipid-bound radioactivity in the tectum opticum evenly distributed over regions of nerve fibres or perikarya indicating an ubiquitous incorporation of label. Sialic acid incorporation into glycoproteins after intracranial injection of N-acetylmannosamine always exceeded that into gangliosides. TCA-precipitable non-lipid material is labelled from intracranially applied N-acetylmannosamine in the sialic acid portion and also in nonsialic acid components, whereby the percentage of label in sialic acid increases reaching 90 per cent of the total radioactivity after 90 min. After intraocular application of N-[³H]acetylmannosamine, sialic acid in gangliosides was generally found to be more highly labelled than in glycoproteins. The ratio of radioactivity in gangliosides and glycoproteins increased with time of incubation and the distance from the eye. TCA-soluble radioactivity was translocated by fast axonal transport. Cycloheximide inhibited incorporation of N-acetylmannosamine-derived radioactivity into gangliosides and proteins but not the transport of TCA-soluble material, which accumulates in the tectum. After intraocular application of N-[³H]acetylglucosamine, TCA-soluble label arrives later in the optic tectum than radioactivity of high molecular weight components. The ratio of lipid to non-lipid-bound radioactivity does not change considerably with the time after injection or the distance from the eye. There was no accumulation of TCA-soluble radioactivity after the inhibition of incorporation into high molecular weight components.     

Incorporation of [14C] ethanolamine and [3H] Methionine into phospholipids of rat brain and liver in vivo and in vitro

Comparative studies were undertaken on the in vivo and in vitro incorporation of [¹⁴C] ethanolamine, [³H] methionine and [¹⁴C] S-adenosyl-methionine into phosphatidylethanolamine (PhE) and phosphatidylcholine (PhC) of rat liver and brain. It was observed that brain can synthesize de novo PhC from PhE via the transmethylation pathway, however synthesis rates were (1) markedly lower than those of liver and (2) decreased significantly with age. In the choline-containing lipids more than 95% of the radioactivity was found in PhC. Studies on the localization of the radioactivity in PhC following the intracranial injection of [³H] methionine or [¹⁴C] ethanolamine revealed that both precursors are incorporated almost exclusively into the choline moiety of this phospholipid. There was significant labeling of PhC only when the precursors were administered intracranially and much less incorporation was observed with the systemic routes. Thus following the intravenous administration of [¹⁴C] ethanolamine, the specific radioactivities of liver PhE and PhC were up to 75 times as high as those of brain and 4 to 5 times as high in the organs of the 20-day old as those of the adult. In contrast, when this precursor was administered intracranially the specific radioactivities of both phospholipids in liver were only twice as high as those of brain. Although the short-and long-term time-course studies on the in vivo incorporation of [¹⁴C] ethanolamine and [³H] methionine into PhC of both organs could suggest a precursor-product relationship between the biosynthesis of this phospholipid in liver and brain, this apparent relationship could also be due to the high turnover of PhE in liver, with half-life of 2.87 hr, and its low turnover in brain, with half-life of 10.7 days. The present findings on the low rate of formation of PhC from PhE in brain coupled with the fact that this conversion declines sharply with age, especially when the isotopes are administered systemically, could explain the observation of previous investigators that the brain cannot synthesize its own choline and thus it must derive its choline from exogenous sources such as lipid-choline. It was concluded that the brain can synthesize its own choline; however it remains also dependent on liver and dietary choline which are probably transported into the brain as free choline.     

Labelling of intestinal brush border membrane proteins in vivo using diazotised [125I]iodosulfanilic acid

Membrane proteins of the intestinal brush border were labelled in vivo by intraluminal injection of diazotised [¹²⁵I]iodosulfanilic acid, a highly polar molecule. Sodium dodecyl sulfate polyacrylamide gel electrophoresis of brush border membranes labelled in this manner showed 20 protein bands, 11 of which contained significant radioactivity. The most heavily labelled proteins had molecular weights greater than 150 000, indicating that they were the most exposed to the intestinal lumen. Little radioactivity was detected in proteins with molecular weights of less than 94 000. The majority of these smaller proteins were likely to have been brush border core proteins. The evidence that diazotised [¹²⁵I]iodosulfanilic acid bound primarily to brush border membrane proteins when administered in this way, was: (a) the specific activity of brush border proteins was up to 3-fold greater than that of total cell particulate proteins (pelleted at 27 000 × $\text{g}$ from mucosal homogenates); (b) principal peaks in the gel radioactivity profile of total cell particulate proteins corresponded to the most heavily labelled proteins of the isolated brush border membrane; and (c) brush border core proteins showed minimal radioactivity in vivo, but considerably higher radioactivity when brush border membranes were labelled in vitro. A small amount of label was absorbed across the intestinal mucosa. However, secondary labelling of brush border proteins by this absorbed label was minimal, since the specific activity of brush border proteins in jejunum adjacent to the labelled loop was only 0.22% of the level for those proteins in the labelled segment. Since this technique did not affect the cellular morphology, enzyme activity or biochemical integrity of the membrane, it should prove useful as a means of accurately studying in vivo turnover rates of brush border membrane proteins.     

Thyroxine-stimulated synthesis of microvillus membrane glycoproteins during culture of chick embryonic duodenum

The effect of thyroxine on biosynthesis of microvillus membrane glycoproteins has been investigated in organ culture of 18-day-old chick embryonic duodenum. Explants incorporate [³H]leucine and [³H]glucosamine continuously, and overall incorporation is enhanced by 10 nM thyroxine during 48 h of labeling; this increase in radioactivity is associated with vesicles released from the microvilli. Light microscope autoradiography, pulse labeling of brush border fragments, and pulse chase experiments reveal that [³H]glucosamine is incorporated into brush border at an increasing rate during culture, and that newly synthesized glycoproteins are discharged into the medium along with brush border enzymes (alkaline phosphatase and maltase). These results suggest that thyroxine stimulates biosynthesis of microvillus membrane glycoproteins, in addition to stimulating vesiculation of the membrane. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of ³H-labeled vesicles and brush border fragments show that [³H]leucine and [³H]glucosamine are incorporated into proteins of high molecular weight. Two protein bands are identified as alkaline phosphatase and maltase. Thyroxine stimulates glycosylation of these enzymes, but does not change protein patterns. Radioactivity assay of alkaline phosphatase- and maltase-active gel slices suggests that thyroxine stimulation of these enzyme activities during culture is not correlated with de novo synthesis of these proteins.