Structural and immunological properties of arabinogalactan polysaccharides from pollen of timothy grass (Phleum pratense L.)

Extracts from pollen of timothy grass (Phleum pratense L.) contain up to 20% arabinogalactan proteins (AGPs). Separation of the AGP polysaccharide moieties by tryptic digestion, size exclusion chromatography (GPC), and reverse phase HPLC yielded arabinogalactan fractions AG-1 and AG-2 with molecular weights of approximately 15,000 and approximately 60,000Da, respectively. The backbones of both polysaccharides are composed of (1-->6)-linked beta-D-galactopyranosides with beta-D-GlcUAp or 4-O-Me-beta-D-GlcUAp at their terminal ends as revealed by chemical analysis, FT-IR, MALDI-MS, and NMR spectroscopy. AG-1 contains a small number of beta-l-Araf side chains while AG-2 possesses a variety of (1-->3)-linked units, which consist of beta-l-Araf-(1-->, alpha-l-Araf-(1-->3)-beta-l-Araf-(1-->, and alpha-l-Araf-(1-->5)-beta-l-Araf-(1--> as well as a small number of longer arabinogalactan side chains. In contrast to crude pollen extracts, the immunological properties of the arabinogalactan mixture reveal an IgG4 reactivity instead of IgE reactivity. Structural properties of timothy pollen arabinogalactan might thus influence the immune response.     

Arabinogalactans in a purified allergen preparation from pollen of timothy (Phleum pratense)

The purified allergen preparation representing a certain fraction of an aqueous timothy pollen extractcontained ca. 20% carbohydrate, mainly as arabinose (7%) and galactose (13%). The protein content was 63%. Fractionation on DEAE-Sephadex and Sephadex G-100 gave one neutral and two acidic fractions, all containing protein, arabinose and galactose. The structure of the carbohydrate moiety was investigated by methylation analysis, periodate oxidation and enzyme incubation. The acidic fraction contained (1→6)-linked galactose residues, some being substituted on O-3 with arabinose. The neutral fraction consisted of a more extensively branched arabinogalactan with longer side chains of (1→3)- and (1→5)-linked arabinose. The arabinose was present mainly as α-l-arabinofuranosyl residues. Alkaline degradation and subsequent fractionation indicated the presence of a covalent linkage between hydroxyproline and arabinose. Periodate oxidation or incubation with α-l-arabinofuranosidase did not affect the allergenic activity of the extract.     

Precursors of Asparagine in Lupins

Fumaric acid-1,4-¹⁴C, L-aspartic acid-4-¹⁴C, and glycine-2-¹⁴Cwere supplied for 1 to 3 h to etiolated lupin shoots that wererapidly synthesizing asparagine. The distribution of ¹⁴C inaspartate and asparagine isolated from this material was determined.With aspartic acid-4-¹⁴C adminstration radioactivity was mostlyin carbons 1 and 4 with carbon 4 having the greater amount;with fumaric acid 1,4-¹⁴C administration, radioactivity wasagain mostly in carbons 1 and 4. Aspartate labelled by fumaratecontained relatively more radioactivity in carbons 2 and 3.Little radioactivity from glycine-2-¹⁴C entered either aspartateor asparagine and this was mainly in carbon 2 and 3. It is concludedthat asparagine synthesis form aspartate is a major pathwayof asparagine biosynthesis in lupins.     

Effect of exo-β-D-(1→3)-galactanase digestion on complement activating activity of neutral arabinogalactan unit in a pectic arabinogalactan from roots of Angelica acutiloba Kitagawa

A complement activating pectic arabinogalactan (AGIIb-1) has been isolated from roots of Angelica acutiloba Kitagawa, and a neutral arabinogalactan (N-I) unit, which was liberated from a side chain in AGIIb-1 by mild acid hydrolysis, has found to show the most potent complement activating activity among the arabinogalactan side chains of AGIIb-1. In order to clarify the essential carbohydrate side chains in N-I for the activity, neutral arabinogalactan (AF-N-I) unit, which was obtained from exo--L-arabinofuranosidase-digested AGIIb-1, was digested with exo-β-d-(1 → 3)-galactanase from Irpex lacteus. The galactanase digestion of the AF-N-I unit completely stopped its reactivity with β-d-glucosyl-Yariv antigen, and significantly reduced its complement activating activity. The digestion products gave high molecular weight fragments (GN-1A and -1B) and intermediate size fragments in addition to β-d-(1 → 6)-galactosyl mono to pentasaccharides as the side chains of the AF-N-I unit. Among the fragments, GN-1A, GN-1B and intermediate size fragments showed relatively potent complement activating activity, suggesting that these side chains in the N-I unit might be responsible for expression of the activity of the N-I unit.     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells II. Partial Purification and Some Physical Characterization of an Intracellular Precursor of Cell Wall Glycoproteins

Arabinose-containing macromolecules accumulate in the Golgiapparatus prior to their secretion into the cell wall. Theseintracellular macromolecules of suspension-cultured tobaccocells were labelled with ¹⁴C-arabinose, extracted and partiallycharacterized. The major component is a glycoprotein, in whicharabinose is linked to hydroxyproline residues as oligosaccharides,among them mainly as disaccharides. A pulse-chase experimentshowed the major glycoprotein to be a precursor of cell-wallmaterials. The glycoprotein has a density of 1.65 g/cm³, indicatinga high content of carbohydrate (90%), of which arabinose, galactoseand uronates are the major components. The glycoprotein is highlyacidic (pI1.3), probably due to the presence of uronate. Thelarge stokes' radius, equivalent to a 5 ? 10⁵-dalton protein,and a small S value (6.5 S) indicate a swollen structure forthe molecule. These data indicate a close similarity of theglycoprotein to an extracellular arabinogalactan protein secretedinto the culture medium. Present address: Institute for Plant Virus Research, TsukubaScience City, Yatabe, Ibaraki 305, Japan. (Received April 12, 1982; Accepted October 20, 1982)     

Isolation of β-galactosidase fractions from Japanese pear: Activity against native cell wall polysaccharides

β-Galactosidase (EC 3.2.1.23) was purified from the cell wall of the fruit of Japanese pear ( Pyrus serotina Rehder var. culta Rehder cv. Hosui) and characterized. Five peaks of β-galactosidase activity, designated as Gal I to V, were separated by hydrophobic chromatography on butyl toyopearl and ion exchange chromatography on Mono S. These isolated β-galactosidases were investigated with regard to their abilities to release monomeric galactose from the fractionated polymers of native cell wall (cyclo-hexane-trans-1,2-diamine tetraacetic acid-, Na₂CO₃-, guanidine thiocyanate- and KOH-soluble fractions) and arabinogalactan (from larch wood). All the β-galactosidase fractions were active against native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall polysaccharides although to varying degrees. Gal I reacted to all fractions of native cell wall and arabinogalactan. Gal II released much galactose only from KOH-soluble polymers and arabinogalactan. Gal III released the most galactose. from cyclohexane- trans -1,2-diamine tetraacetic acid-, Na₂CO₃- and guanidine thiocyanate-soluble cell wall polymers which probably contained galactosyl side chains of pectic polymers, although it did not react much to arabinogalactan. In addition, the activity of Gal Ill dramatically increased as ripening proceeded. Furthermore, Gal III was purified to homogeneity by gel filtration on TSKgel 3000SW and the size of a polypeptide was 80 kDa on SDS-PAGE.     

Polysaccharide and oligosaccharide changes in germinating lupin cotyledons

In germinating lupin cotyledons, there was a rapid depletion of raffinose series oligosaccharides, a temporary increase in sucrose and constant low levels of reducing monosaccharides. The major polysaccharide fraction was extracted with hot NH₄ oxalate—EDTA solution and had the constitution of intercellular/cell wall polysaccharide. GLC examination of component sugars showed that as cotyledons expanded this fraction was depleted and that there was selective hydrolysis of arabinose and galactose, so that the uronic acid proportion increased. Gel and DEAE-cellulose chromatography showed that this fraction became more heterogeneous. The neutral and acidic fractions were separated and the component sugars, viscosities, gel chromatographic behaviour and sedimentation constants of these determined. The results indicated that in the later phase of plant cell wall expansion in germinating lupin cotyledons the arabinogalactan side chains of the pectic polysaccharide fraction are selectively hydrolysed leaving a primary wall with a high uronic acid content.     

An arabinogalactan from the fibres of cotton (Gossypium arboreum L.)

An acidic arabinogalactan has been isolated from fibres of the cotton plant (Gossypium arboreum L.) at the stage of intensive secondary-wall formation. The polysaccharide contains arabinose, galactose, rhamnose, and glucuronic acid residues in the molar ratios 1:1.2:0.1:0.2. Periodate oxidation and methylation studies showed that there is a main chain of (1→3)-linked galactopyranosyl residues to which side chains are attached at O-6. The side chains consist of (1→6)-linked galactopyranosyl residues substituted at O-3 by (1→5)-linked arabinofuranosyl chains. Terminal galactopyranosyl, rhamnopyranosyl, and glucopyranuronosyl groups are also present. Enzymic hydrolysis showed that the configurations of the galactose and arabinose residues are d and l, respectively.     

Metabolic relationships of the isolated fractions of the pectic substances of actively growing sycamore cells

1. d-Glucose and l-arabinose serve as precursors of the pectic polysaccharides of sycamore suspension-callus tissue. 2. The rates and characteristics of the incorporation of radioactive sucrose, glucose and mesoinositol by sycamore callus tissue have been compared and shown to be different. 3. The time-course of the incorporation of radioactive glucose into the major fractions within the cells has been determined. Approx. 7-10% of the radioactivity incorporated is present in the whole pectin of the cells. 4. A study of the continuous incorporation of radioactive glucose showed that the neutral arabinan-galactan fraction of the pectin quickly became saturated with the radioactive label. During the incorporation of radioactivity from a pulse of radioactive glucose the neutral fraction became progressively less labelled, with a corresponding increase in the radioactivity of the weakly acidic pectinic acid, which is known to contain neutral sugars. 5. When the cells were exposed to a pulse of radioactive l-arabinose, the label accumulated first in the neutral fraction and then after 4hr. it passed to the weakly acidic pectinic acid with a corresponding decrease in the radioactivity of the neutral fraction. 6. The product that was initially labelled during the first hour of exposure of the cells in the stationary phase to radioactive glucose was identified as an incompletely methylated galacturonan in which the radioactivity was present in the anhydrogalacturonide residues. This polysaccharide probably acts as the precursor of the polyuronide portions of both the strongly acidic and weakly acidic pectinic acids. 7. The observations are discussed in relation to the structure of the pectic substances and their function in cell growth and development. A tentative model for their metabolic relationship is put forward.     

The occurrence of internal (1 --> 5)-linked arabinofuranose and arabinopyranose residues in arabinogalactan side chains from soybean pectic substances

CDTA-extractable soybean pectic substances were subjected to enzymatic digestion with arabinogalactan degrading enzymes yielding a resistant polymeric pectic backbone and arabino-, galacto-, and arabinogalacto-oligomers. The complex digest was fractionated using size-exclusion chromatography. Monosaccharide composition analysis, HPAEC fractionation and MALDI-TOF MS analysis of the resulting fractions showed that each contained a mixture of oligosaccharides of essentially the same degree of polymerisation, composed of only arabinose and galactose. MALDI-TOF MS analysis was used for molecular mass screening of oligosaccharides in underivatised HPAEC fractions. The monosaccharide sequence and the branching pattern of oligosaccharides (degree of polymerisation from 4 to 8) were determined using linkage analysis and ES-CID tandem MS analysis of the per-O-methylated oligosaccharides in each of the HPAEC fractions. These analyses indicated the presence of common linear (1 --> 4)-linked galacto-oligosaccharides, and both linear and branched arabino-oligosaccharides. In addition, the results unambiguously showed the presence of oligosaccharides containing (1 --> 4)-linked galactose residues bearing an arabinopyranose residue as the non-reducing terminal residue, and a mixture of linear oligosaccharides constructed of (1 --> 4)-linked galactose residues interspersed with an internal (1 --> 5)-linked arabinofuranose residue. The consequences of these two new structural features of pectic arabinogalactan side chains are discussed.     

The Development of a High Performance Liquid Chromatograph with a Sensitive On-stream Radioactivity Monitor for the Analysis of 3H- and 14C-labelled Gibberellins

The development of a high performance liquid chromatograph forthe separation of gibberellins is described. The system combineshigh efficiency, peak capacity, and sample capacity with rapidspeed of analysis. In addition, the constructional details ofa sensitive on-stream radioactivity monitor are outlined. Theoverall versatility of the chromatograph is demonstrated bythe separation of a range of ³H- and ¹⁴C-labelled Gibberellinsand gibberellin precursors. The system also has considerablepotential for the analysis of abscisic acid and acidic and neutralindoles.     

Carbohydrate structural analysis of wheat flour arabinogalactan protein

The water-extractable arabinogalactan protein (AGP) was isolated from bread wheat flour (Triticum aestivum L. variety Cadenza) and the structure of the arabinogalactan (AG) carbohydrate component was studied. Oligosaccharides, released by hydrolysis of the AG with a range of AGP-specific enzymes, were characterised by Matrix Assisted Laser Desorption Ionisation (MALDI)-Time of Flight (ToF)-Mass Spectrometry (MS), MALDI-ToF/ToF high energy collision induced dissociation (CID) and Polysaccharide Analysis by Carbohydrate gel Electrophoresis (PACE). The AG is composed of a β-(1→3)-d-galactan backbone with β-(1→6)-d-galactan side chains. These side chains are highly variable in length, from one to at least 20 Gal residues and are highly substituted with α-l-Araf. Single GlcA residues are also present at the non-reducing termini of some short β-(1→6)-galactan side chains. In addition, the β-(1→6)-galactan side chains are also substituted with β-l-Arap. We propose a polysaccharide structure of the wheat flour AGP that is substantially revised from earlier models.     

Cell Wall Metabolism in Developing Strawberry Fruits

Cell wall metabolism was studied in strawberry receptacles (Fragariaananassa, Duchesne) of known age in relation to petal fall (PF).Polysaccharide and protein composition, incorporation of [¹⁴C]glucoseand [¹⁴C]proline by excised tissue, and the fate of ¹⁴CO₂ fixedby young, attached fruits were followed in relation to celldivision, cell expansion, fine structure, and ethylene synthesis. Cell division continued for about 7 d after PF although vacuolationof cells was already beginning at PF and the subsequent cellexpansion was logarithmic. There was an associated logarithmicincrease in sugar content per cell and a decreasing rate ofethylene production per unit fresh weight. During cell expansion radioactivity from [¹⁴C]glucose was incorporatedinto fractions identified as starch and soluble polyuronideand into glucose and galactose residues in the cell wall. Radioactivityfrom [¹⁴C]proline was also incorporated into the cell wall,but only 10 per cent of this activity was found in hydroxyproline.Correspondingly wall protein contained a low proportion of hydroxyprolineresidues. The proportion of radioactivity from ¹⁴CO₂ fixed byfruitlets remained constant in most sugar residues in the cellwall. The proportion of radioactivity in galactose fell, indicatingturnover of these residues. Between 21 and 28 d after PF receptacles became red and softenedbut there was no change in the rate of ethylene production.Cell expansion continued for at least 28 d. Tubular proliferationof the tonoplast and hydration of middle lamella and wall matrixmaterial had begun 7–14 d after PF but became extremeduring ripening. Associated with the hydration of the wall,over 70 per cent of the polyuronide in the wall became freelysoluble, and arabinose and galactose residues lost from thewall appeared in soluble fractions. There was no increase intotal polysaccharide during ripening and incorporation of [¹⁴C]glucoseinto polysaccharides ceased, although protein increased andincorporation of [¹⁴C]proline into wall protein continued.     

Influence of mitochondria on phospholipid synthesis in preparations from rat liver

1. The addition of mitochondria to an incubation system containing the soluble and microsomal fractions of rat liver enhances severalfold the incorporation of each of ethanolamine, phosphorylethanolamine and CDP-ethanolamine into phosphatidylethanolamine. 2. In the presence of microsomal, mitochondrial and soluble fractions, CDP-ethanolamine exhibits the greatest initial rate of incorporation (approx. 6nmol/h per mg of protein), being slightly faster than that of phosphorylethanolamine (approx. 5nmol/h per mg of protein). Incorporation of ethanolamine proceeds very slowly for the first 20min and only after 30min gives rates approaching those of the other two precursors. 3. By using a substrate ;dilution' technique it was shown that in the reconstituted system the affinity of each of the enzymes for their respective substrates is very high: 10mum for ethanolamine, 25mum for phosphorylethanolamine and 5mum for CDP-ethanolamine. 4. Isolation of the mitochondrial and microsomal fractions from the medium after incubation together with phosphorylethanolamine showed that about 70% of the total radioactivity was present in the microsomal fraction and about 30% in the mitochondria after only 20min. Similar experiments with ethanolamine as precursor revealed that after 20min only about 15% of the total radioactivity was present in the mitochondria but that after 40min about 30% was present in this fraction. 5. Heating and phospholipase treatment of mitochondria, but not freeze-thawing, eliminated the stimulatory effect of mitochondria on phospholipid synthesis. 6. The reconstituted system exhibits an absolute requirement for Mg(2+) (2mm gave maximal rates) and is inhibited by very low concentrations of Ca(2+) (100mum-Ca(2+) produced half-maximal inhibition with 3mm-Mg(2+)). Further addition of Mg(2+) overcame the Ca(2+) inhibition, suggesting that the inhibitory effect is readily reversible. 7. The concept that modification of the Mg(2+)/Ca(2+) ratio is a means of controlling the rate of cellular phospholipid synthesis is introduced.     

Proline-14C metabolism in barley seedlings during vernalization

The metabolism of proline-¹⁴C was examined in vernalized (LT)and unvernalized (HT) barley shoots. The content of prolinein cytoplasmic and cell wall protein fractions was a littlehigher in HT, against the accumulation of free proline in LT.Proline-¹⁴C was more heavily incorporated into HT and the cytoplasmicprotein had a higher radioactivity than the cell wall protein.In LT, the activity in the cytoplasmic protein was lower thanthat in the cell wall. The time course of incorporated proline-¹⁴Cshowed no distinct changes in HT. but decreased remarkably inthe LT cell wall. The distribution of proline-¹⁴C in hydroxyproline and otheramino acids in the two proteins was examined. In the cytoplasmicproteins, the conversion pattern of proline-¹⁴C was very similarin both treatments. The highest hydroxyproline activity wasfound at the beginning of incubation and was maintained at acomparatively high level in the HT cell wall. The LT cell wallshowed a gradual increase in radioactivity due to hydroxyprolineand reached maximum conversion at the last incubation period.Distribution of radioactivity due to incorporated proline-¹⁴Cwas examined by separating barley shoot tissues into three sections. (Received December 5, 1972; )     

Polysaccharide breakdown by mixed populations of human faecal bacteria

Measurements of polysaccharide-degrading activity in different fractions of human faeces showed that bacterial polysaccharidases and glycosidases were primarily associated with the washed bacterial fractions. Amylase, pectinase and xylanase were the major polysaccharide-hydrolysing enzymes detected, whilst α-L-arabinofuranosidase, β-D-xylosidase, β-D-galactosidase and β-D-glucosidase were the most active glycosidases. Starch and 3 non-starch polysaccharides (NSP; pectin, xylan and arabinogalactan) were fermented by mixed populations of human faecal bacteria in batch culture. Detailed carbohydrate analysis demonstrated that starch and pectin were the most rapidly degraded substrates and that arabinogalactan and the relatively insoluble polysaccharide xylan were broken down more slowly. Free sugars and oligosaccharides did not accumulate in culture media with any polysaccharide tested. Time-course measurements of polysaccharide remaining in the batch culture fermentations showed that the arabinose side chains of pectin, xylan and arabinogalactan were co-utilised with the backbone sugars. In these cultures, polysaccharide-degrading activity was mainly cell-associated, but extracellular polysaccharidase activity increased as the fermentations progressed. Molar ratios of acetate, propionate and butyrate produced in these experiments were dependent upon the polysaccharide substrate tested. Molar ratios of acetate, propionate and butyrate in the starch, arabinogalactan, xylan and pectin fermentations were 50:22:29, 50:42:8, 82:15:3, and 84:14:2, respectively. The presence of starch did not inhibit the breakdown of arabinogalactan, xylan or pectin by faecal bacterial, providing evidence that multicomponent substrate utilisation occurs when complex populations of faecal bacteria are provided with mixed polysaccharide substrates.     

Tissue distribution of retinol and its metabolites after administration of double-labelled retinol.

The tissue concentrations and distribution of radioactivity present in retinol and its metabolites were investigated in vitamin A-deficient rats 24h after injection of physiological doses (10mug) of [6, 7-14C2, 11,12-3H2] retinol. The highest concentration of radioactivity was observed in the adrenals, followed by kidney, spleen, liver, intestine and blood. The total radioactivity was greatest in urine, followed in descending order by liver, kidney, blood and intestine. The 14C/3H ratios of crude light-petroleum extracts in the liver, intestines, lungs, heart and faeces were similar to the ratio of the injected retinol dispersion. However, the 14C/3H ratios in the adrenals, kidney, spleen, blood, brain and urine were quite different from that of injected retinol. Alumina chromatography of the kidney and intestinal extracts demonstrated that retinol and retinyl palmitate are the principal forms of vitamin A present. However, alumina chromatography of the liver extract did not reveal the presence of retinol but yielded a major compound with a low 14C/3H ratio. That this compound was not retinol was shown by its inability to react with ethanolic HC1 to yield anhydroretinol. The distribution of radioactivity in ether-soluble, acidic and water-soluble fractions of urine indicated that most of the radioactivity was present in the acidic and water-soluble fractions. The 14C/3H ratios in ether-soluble and acidic fractions were higher than that of injected retinol, whereas in the water-soluble fraction the ratio was similar to the injected material.     

14C2H4: Distribution of 14C-labeled tissue metabolites in pea seedlings

The ¹⁴C-metabolite distribution pattern following ¹⁴C₂H₄ metabolismin intact pea seedlings (Pisum sativum L.) was determined undervarious conditions. After a 24 hr exposure to ¹⁴C₂H₄, the majorityof ¹⁴C-metabolites were water-soluble (60–70%) with lesseramounts in the protein (10–15%), lipid (1%), and insoluble(1–2%) fractions. Ion exchange chromatography of the water-solublecomponents into basic, neutral, and acidic fractions revealeda 50 : 40 : 10 distribution, respectively. Chromatography ofthe neutral fraction revealed two regions of radioactivity (Rf=0.38)and 0.63 which did not cochromatograph with twenty-two knownsugars or neutral metabolites. Chromatograms of the basic fractioncontained 3 regions of radioactivity. Similar distribution patternswere noted when ¹⁴C₂H₄ exposure was followed by a 6 hr air chaseor when 5% CO₂, an antagonist of ethylene action, was presentduring the exposure. Marked differences in the ¹⁴C-metabolite distribution patternswere obtained when ¹⁴CO₂ was substituted for ¹⁴C₂H₄. These resultsindicate that the metabolic pathway involved in ethylene metabolismis different from that involved in intermediary carbon metabolism. ¹ Contribution No. 2338 from Central Research and DevelopmentDepartment, Experimental Station, E. I. du Pont de Nemours andCompany, Wilmington, Delaware. (Received June 28, 1976; )     

Patterns of hemoglobin assembly in reticulocytes of sickle cell trait individuals.

Venous blood was obtained from five sickle cell trait donors with relatively high hemoglobin S concentrations (40% of total hemoglobin) and five donors with unusually low hemoglobin S concentrations (25 to 30%). A fraction of cells with 15 to 20% reticulocytes was isolated from the blood and incubated with [3H]leucine in a medium supporting protein synthesis for various times from 1.25 to 60 min. Previous studies showed an imbalance in globin chain synthesis in reticulocytes of "low hemoglobin S" donors which suggested the presence of an alpha-thalassemia gene; reticulocytes of "high hemoglobin S" donors had balanced globin chain synthesis (DeSimone, J., Kleve, L., Longley, M.A., and Shaeffer, J. (1974) Biochem. Biophys. Res. Commun. 59, 564-569). In the present study the soluble phase of the 3H-labeled reticulocytes was examined by electrophoresis on strips of cellulose acetate. The tetramer hemoglobins A and S were separated from each other and from a small pool of free, newly synthesized alpha and beta chains. Kinetics of labeling studies showed that the free alpha and beta chains were intermediates in tetramer hemoglobin assembly. The distribution of radioactivity between the alpha and beta chains of each of the electrophoretically isolated components were determined by separation of their globin chains on CM-cellulose columns. After 5 min of 3H-labeling of the reticulocytes from donors with 40% hemoglobin S the ratio of newly synthesized alpha chains to beta chains in the tetramer hemoglobins A and S ranged from 0.37 to 0.58. This ratio increased with longer labeling times. Almost all of the radioactivity of the free chain intermediates was in the alpha chain. These results confirmed the presence of a significant pool of newly synthesized alpha chains and a normal pattern of hemoglobin assembly in which initially unlabeled alpha chains combined with labeled beta chains when the cells were exposed to [3H]leucine. Conversely, in the reticulocytes of donors with 25 to 30% hemoglobin S the ratio of newly synthesized alpha chains to beta chains in the completed hemoglobins A and S ranged from 0.96 to 1.37 and remained unchanged throughout the 3H-labelling period. The radioactivity of the free alpha chain pool was substantially less that the total radioactivity of the betaA and betaS chain pools. These results confirmed the existence of a decreased pool size of soluble alpha chain intermediates and a pattern of hemoglobin assembly consistent with the presence of the alpha-thalassemia gene.     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells I. Intracellular Site of Synthesis and Transport

The distribution of arabinose-containing macromolecules in suspension-culturedtobacco cells was examined using sucrose density gradients.Exogenously applied ¹⁴Carabinose was scarcely converted intoother sugars, and concentrated in the Golgi-rich fraction (1.15g/cm³) and then secreted to the cell wall. ¹⁴C-Arabinose wasalso incorporated in a lower sucrose density fraction (1.11g/cm³), which contains small vesicles presumably originatedfrom the Golgi apparatus. The arabinose-containing macromoleculesin this fraction was more easily solubilized in water than thosein the Golgi-rich fraction. Alkaline hydrolysis of the macromoleculesindicated that cell-wall glycoprotein is a major component ofthe macromolecules and that the degree of glycosylation is slightlygreater in the lower density fractions than in the Golgi-richfraction. Based on these results, a scheme is suggested in whichthe glycoproteins and polysaccharides are glycosylated in theGolgi apparatus and secreted to the cell wall via secretionvesicles in the low density fraction. The possibility of ¹⁴C-arabinose-containingmacromolecules, in the early phase of synthesis, being a markerof the plant Golgi apparatus is also proposed. (Received September 21, 1980; Accepted January 27, 1981)