The Incorporation of 14C-Proline into the Proteins of Growing Cells: II. A NOTE ON EVIDENCE FROM CULTURED CARROT EXPLANTS BY ACRYLAMIDE GEL ELECTROPHORESIS

The method of acrylamide gel electrophoresis has been appliedto the separation of the proteins of growing carrot explantswhich are soluble in a buffer solution at pH 8.3. At least ninedistinct bands were detectable in this way. When carrot tissuehad absorbed ¹⁴C-proline it entered into the composition ofall these bands and, in all except one, it was converted tohydroxyproline to different degrees which characterized theband in question. Thus, in the soluble protein the ¹⁴C-hydroxyproline:¹⁴C-proline ratio varied from 0 to 0.53. The bulk of the proteinin the tissue was insoluble in buffer at pH 8.3; it containedthe bulk of the radioactivity absorbed from proline (84.8 percent.); and its average ¹⁴C-hydroxyproline : ¹⁴C-proline ratiowas the highest of all (1.28). A particulate protein preparation,separated at 25,000 g. from the soluble protein, had an intermediateratio (0.643) of ¹⁴C-hydroxyproline to ¹⁴C-proline. Therefore,there are in cultured carrot explants many distinct proteinmoieties which incorporate ¹⁴C-proline, and they convert itto ¹⁴C-hydroxyproline to very different degrees. The evidenceis consistent with the incorporation of the proline, first intothe various soluble proteins which are electrophoretically separableand subsequently, with progressively greater hydroxylation,into the more insoluble protein that constitutes the bulk ofthe protein of the cell and its organelles. It is, therefore,quite incorrect to say (as some have done) that all of the hydroxyproline-containingprotein is in very close association with the cell wall, forpart of it is present in the cell in soluble and electrophoreticallyseparable forms.     

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.     

Isolation of 1-O-trans-p-Coumaroyl-r{beta}-D-glucopyranose from Sweet Potato Roots and Examination of Its Role in Chlorogenic Acid Biosynthesis

1-O-trans-p-Coumaroyl-rß-D-glucopyranose (p-coumaroyl-D-glucose)was isolated from slices of sweet potato root which had beenincubated with trans-cinnamic acid. Pre-loaded trans-cinnamicacid efficiently trapped the radioactivity from L-[U-¹⁴C]-phenylalanineand reduced its incorporation into chlorogenic acid by 75% ofcontrol values in disks of sweet potato root. In the root diskssupplied with trans-[3-¹⁴C]-cinnamic acid, the radioactivitywas transferred first to trans-cinnamoyl- D-glucose, then top-coumaroyl-D-glucose, and subsequently to chlorogenic acidand isochlorogenic acid. These results support our earlier propositionthat p-coumaroyl-D-glucose is involved in the biosynthesis ofchlorogenic acid as an intermediate adjacent in the pathwayto trans-cinnamoyl-D-glucose in sweet potato roots. (Received April 11, 1988; Accepted August 9, 1988)     

In Vitro Phosphorylation of Proteins in IAA-Treated Mesocotyls in Zea mays

Five-mm sections of elongation zones of Zea mesocotyls wereincubated for designated periods with various concentrationsof IAA. In vitro protein phosphorylation in the soluble fraction(85,000 x g supernatant) prepared from the sections was analyzedby sodium dodecyl sulfate-polyacrylamide gel electrophoresis.The phosphorylation of proteins in the soluble fraction thathad been prepared from sections incubated for 20 min in thepresence of 10{small tilde}^s M IAA was greater than that inthe sections incubated for 20 min without IAA. The amount ofphosphorylation of proteins per protein became higher when higherconcentrations increased (10{small tilde}⁸—10{small tilde}⁵M).The growth of sections incubated in the presence of 10{smalltilde}⁸ M IAA or higher concentrations was greater than thatof sections incubated in the absence of IAA. The promotion ofgrowth by IAA was greater at higher concentrations of IAA. Proteinsin the soluble fraction, prepared from sections incubated for20 min in the presence of 10{small tilde}⁵ M IAA, were phosphorylatedin the presence of either 10 fM cAMP, 10 µM cGMP, 100µM W-7, 100 µM W-5, 20 µM H-7 or 20 µMHA1004. The calmodulin antagonist, W-7, and the inhibitor ofprotein kinase C, H-7, inhibited the phosphorylation of proteinsstimulated by incubation with IAA. These results suggest thatIAA promotes cell elongation via protein phosphorylation thatdepends on calmodulin-dependent protein kinase and protein kinaseC. (Received November 29, 1995; Accepted May 20, 1996)     

Increased sensitivity of scleroderma fibroblasts in culture to stimulation of protein and collagen synthesis by serum

Serum effects on ¹⁴C-proline incorporation and ¹⁴C-hydroxyproline synthesis by normal and scleroderma fibroblasts in culture were studied. Serum resulted in 97% and 212% increases in ¹⁴C-proline incorporation in two lines of scleroderma fibroblasts while the increase in normal fibroblasts was only 53%. Effects on collagen synthesis were more pronounced. Addition of serum resulted in 124% and 445% increments in ¹⁴C-hydroxyproline synthesis in the scleroderma fibroblasts but only a 43% increment in the normal fibroblasts. The results indicate that cultured scleroderma fibroblasts have increased sensitivity to biosynthetic stimulation by serum and this mechanism may be of pathogenetic importance in the excessive collagen accumulation characteristic of the disease.     

Variation in 14C Partitioning in the Tips of Growing Stolons of Potato (Solanum tuberosum L.)

¹⁴C partitioning was examined in growing stolons of field-grownpotato (Solanum tuberosum L.) cv. Maris Piper. Considerablevariation was evident on single plants and on a fresh weightbasis many stolon tips, which showed no signs of sub-apicalswelling, had higher specific activities (cpm g^–1 f. wt)of ¹⁴C in both ethanol soluble and insoluble forms than larger,visibly tuberized stolons. Furthermore, many tips of low freshweight had a higher insoluble to soluble ¹⁴C ratio than visiblytuberized stolons suggesting greater efficiency of conversionof soluble ¹⁴C to insoluble ¹⁴C in the smaller stolons. Theresults suggest that the onset of visible ‘tuberization’,namely the sub-apical swelling of the stolon, is preceded byincreased soluble carbon accumulation at the stolon tip togetherwith an increase in the conversion of soluble to insoluble formsof carbon. Tuberization, ¹⁴C, stolon tip     

Protein Modification and Utilization of Starch in Soybean (Glycine max L. Merr.) Seed Maturation

Seeds of soybean (Glycine max L. Merr.) harvested at variousstages of development and allowed to dry in intact pods undergoa maturation process and are viable. Defatted powders of seedharvested 24–66 d after flowering were extracted to yieldbuffer-soluble and alkali-soluble proteins. Imposition of amaturation process increased the level of buffer-soluble proteinsbut had no effect on the disulflde content of these proteins.After undergoing maturation, seeds showed an accumulation ofbuffer-soluble polypeptides in the molecular weight range of43–94 kD. Maturation may be associated with the synthesisof specific polypeptides having a molecular weight of approximately85 kD. Alkali-soluble proteins, which represents the storageproteins, did not show any responses to maturation. Their quantityincreased substantially during seed development and the disulfidelevel was only half that of buffer-soluble proteins, attaininga maximum value of 10.9 mol S per 10⁵ g protein. Matured seedat all harvest dates had a final starch content close to thatof normal seed, 10–20 mg g^–1, and soluble sugarswere maintained at quite high levels, 51–83 mg g^–1.The metabolic program for synthesis and degradation of starchseems quite rigidly followed and is independent of harvest dateor of attachment to the parent plant. Soybean seeds retain considerablesoluble proteins and soluble sugars throughout maturation, andthese collectively may be important in maintaining a desiccationresistant structure.     

Long-term Metabolism of [14C]Sugars in Stored Potatoes

[¹⁴C]Sucrose, [¹⁴C]glucose and [¹⁴C]fructose were introducedinto potato tubers held at 10 °C and the redistributionof label chased over a 65 d period in storage. Respiratory losseswere identical in all treatments, as was the partitioning of¹⁴C between soluble and insoluble forms. Sucrose was the predominantlabelled sugar in the tubers after 20 h, regardless of the original[¹⁴C]sugar introduced, and was loaded and distributed throughoutthe tubers by the internal phloem system. After 20 h the proportionsof labelled sugars bore no relationship to those of the unlabelledendogenous sugars. However, with time the percentage of ¹⁴Cin sucrose fell while that in glucose increased and by 65 dthe proportions of the labelled sugars more closely resembledthe endogenous pools. Fructose represented a consistently lowproportion of both the labelled and unlabelled sugars. By 21d a considerable proportion of the soluble ¹⁴C had been convertedto starch (approx. 25% of the total tuber 14C), this value remainingrelatively constant for the remainder of the storage period.Sprouts which formed on the tubers contained up to 6% of thetotal tuber ¹⁴C but less than 0.2% of the tuber dry matter.It is suggested that the bulk of the translocated [¹⁴C]sucroseentered the symplast and exchanged slowly with the bulk of thesugars in the storage cell vacuoles. [¹⁴C]sugars, phloem loading, starch, potato tuber, Solunum tuberosum, cold storage     

Variations in the Contents and Kinetic Properties of Ribulose-1,5-bisphosphate Carboxylases among Rice Species

The K_m(CO₂) ancl V_max of ribulose 1,5-bisphosphate (RuBP) carboxylaseand its protein ratio to total soluble protein from Oryza speciesincluding cultivars (25 varieties) and wild types (11 species,21 strains) were surveyed. Their variabilities among cultivarsof O. sativa were very small. The averages of the K_m(CO₂) andV_max values and the ratio of carboxylase to soluble protein,and their standard errors were 10.2?1.0µM, 1.72?0.13units.mg^–1(pH 8.0 and 25?C) and 52?2%, respectively. However, some differencesseemed to exist based on genome constitution in the Oryza genus.RuBP carboxylases from the species with the A^gA^g genome, O.graberrima and O. breviligulate, exhibited low K_m(CO₂) values(8.0?0.8 µM). High V_max was associated with the CC genome,O. eichingeri and O. officinalis (2.08?0.15 units.mg^–1).A higher ratio of RuBP carboxylase protein to soluble proteinwas found for the AA genome, O. sativa and O. perennis. (Received September 24, 1986; Accepted April 15, 1987)     

Protein synthesis in Xanthium leaf development

Young, expanding Xanthium leaves had many soluble proteins;older leaves had progressively fewer. The leaves that grew themost rapidly incorporated the most ¹⁴CO₂ into their proteins.The relative intensity of ¹⁴CO₂ incorporation into the differentsoluble proteins changed with leaf development. (Received November 17, 1969; )     

The Partitioning of Photosynthetically Assimilated 14C into Amino Acids in Wheat: 2. Distribution in Amino Acids of Wheat Grain Fractions

Changes in the distribution of ¹⁴C between free and bound aminoacids in wheat grains (Triticum aestivum L. cv. Arkas) at 10and 20 d post-anthesis are described. After ¹⁴CO₂, labellingof the flag leaf, ¹⁴C was initially more rapidly transferredto the grains of 20 d post-anthesis plants than for 10 d post-anthesisplants. However, after a 460 min chase period in the light theamount of ¹⁴C in the grains of the younger and older plantswere similar. In the younger, more rapidly growing grains, agreater proportion of the ¹⁴C was incorporated into structuraltissue and starch. ¹⁴C accumulation in the grains continuedduring the dark in the younger grains but not in the older grains. Although the overall ¹⁴C distribution between the free aminoacid and protein pools of the grain was similar for both treatments,the distribution within the albumin, prolamin and globulin fractionsand between the individual non-bound amino acids differed. Ofthe protein fractions, the albumins were initially the mostheavily labelled but after 460 min chase the prolamins containedmore ¹⁴C. The majority of the ¹⁴C in the albumin and globulinfractions after 280 min chase was in hydrolysable, non-aminoacid compounds. In both tissues, the free amino acid pools lostradioactivity in the dark but the solid residues and proteinscontinued to function as ¹⁴C sinks. Daily fluctuations in the radioactivity in free and bound alanineare consistent with the role of free alanine as a diurnal metabolicnitrogen pool. Wheat, Triticum aestivum¹⁴CO₂, amino acids, proteins, carbon metabolism     

Metabolism of Inorganic Carbon Taken Up by Roots in Salix Plants

The metabolic products of inorganic carbon taken up throughthe roots from nutrient solution were studied in willow plants.Willow cuttings (Salix cv. Aquatica gigantea) were suppliedwith unlabelled or ¹⁴C-labelled NaHC0₃ for 1, 5, 10, and 24h in light or in darkness. After feeding, the plants were dividedinto six samples (upper and lower leaves and corresponding stems,cuttings and roots), which were frozen in liquid N₂. Freeze-driedground samples were extracted into water-soluble, chloroform-solubleand insoluble fractions. The water-soluble fraction was furtherseparated into basic, acidic, and neutral fractions by ion-exchangechromatography. In the light experiment pronase treatment wasused to separate the insoluble fraction into proteins and insolublecarbohydrates. After I h feeding time, most of the ¹⁴C was fixed into organicacids and amino acids both in light and in darkness in all partsof the plants. In the roots a large part of the ^l4C-carbon wasincorporated into the protein and insoluble fractions alreadyduring short feeding times, and the amounts incorporated increasedwith time. In the leaves, after 1 and 5 h the main labelledcompounds were the organic acids and amino acids, but after10 h about half of the total ¹⁴C was in protein and in the insolublefraction. A further analysis of amino acids and organic acidswith HPLC showed that C-4 acids were labelled initially andthat over time the proportion of different acids changed. These results indicate that the metabolism of carbon in rootsmight take place via ß-carboxylation of PEP. Partof the fixed ¹⁴C is transported from the roots, probably asamino acids and organic acids, to the shoot. In roots the C-4acids are metabolized further into structural compounds (proteinsand insoluble carbohydrates). Key words: DIC, Salix, roots, metabolism, HPLC     

Effect of colchicine on atherosclerosis. II. Biochemical studies on skin biopsies from patients treated perorally with colchicine

The biosynthesis of proteins and glycosaminoglycans (GAGs) was determined in skin biopsies from atherosclerotic patients treated perorally for 3 months with 1 mg/day colchicine. The biopsies were incubated with 3H-glucosamine and 14C-proline for 5 h and subsequently digested with pronase. In an aliquot of the pronase digest, the specific radioactivity of 14C-proline and 14C-hydroxyproline were determined. The 3H-glucosamine-labeled GAGs were identified by specific enzymic assay and quantified after electrophoretic separation. 3 months treatment with colchicine did not modify the total amounts of proline and hydroxyproline in skin proteins, but diminished the amount of the GAGs as expressed by uronic acid content. Colchicine treatment decreased also the specific radioactivity of proline and hydroxyproline, which reflects a decrease of total protein and collagen synthesis. The incorporation of 3H-glucosamine in the 3H-GAGs was also decreased, mainly in hyaluronic acid. These results suggest that peroral administration of colchicine modifies the synthesis of extracellular matrix proteins and polysaccharides by skin fibroblasts.     

Metabolism of C1 unit precursors in Neurospora: Effects of media supplements on labelling of nucleic acids and protein

Mycclia of Neurospora crassa wild type (FE SC no. 853), harvestedduring the exponential phase of growth on defined minimal mediaincorporated glycine-2-¹⁴C, serine-3-¹⁴C and formate-¹⁴C intoproteins, DNA and RNA. Supplementing the growth medium with1 mM glycine increased the flow of glycine and formate carboninto these products. In contrast, this supplement decreasedthe incorporation of serine-¹⁴C. When such cultures were preincubatedfor 30 min with adenine, formaldehyde, formate or L-methionine,labelling of the nucleic acids and protein fractions by glycine-2-¹⁴Cwas altered. It is concluded that glycine increases the turnoverof C₁ units in Neurospora, resulting in greater contributionsof the C-2 in nucleic acid and protein synthesis. (Received May 14, 1977; )     

Quantification of mRNA in Chloroplasts of Chlamydomonas reinhardii: Equal Distribution of mRNA for a Soluble and a Membrane Polypeptide in Stroma and Thylakoids

The relative contents of the mRNAs were analyzed for the 32kDa herbicide-binding protein and for the large subunit of ribulose-l,5-bisphosphatecarboxylase in the membrane fraction and in the soluble fractionof chloroplasts from Chlamydomonas reinhardii. The presenceof mRNA for the two proteins in both subchloroplast fractionswas demonstrated by in vitro translation of isolated RNA inthe reticulocyte lysate. The relative amounts of the two mRNAswere measured by hybridizations with cloned chloroplast DNAprobes at two stages of the cell cycle. Both mRNAs were distributedin the same ratio between membrane and soluble fractions, about75% of both mRNAs being in the membrane and 25% in the solublefraction. Therefore, in chloroplasts the accumulation of mRNAson thylakoid membranes does not reflect the final localizationof soluble and membrane proteins. ¹Present address: Department of Biology, Ben Gurion University,Beer-Sheva, Israel. (Received April 28, 1987; Accepted September 29, 1987)     

Effects of Temperature and Photoperiod on Assimilate Partitioning in Potato Plants

The effects of temperature and photoperiod on d. wt partitioningand ¹⁴C translocation were studied in three potato varieties.High temperatures and long days enhanced plant growth in termsof plant height and number of leaves, and also affected d. wtpartitioning between the plant organs. However, no temperatureeffect was noted on total plant d. wt, nor on the export of¹⁴C from the source leaf. Translocation of ¹⁴C to the vegetativeorgans (leaves and stems) was greater at higher temperatures,while translocation to the tubers was less under these conditions.We suggest that, under the temperature regimes studied, themain effect of high temperature is on assimilate partitioningand not on total plant productivity. Differences in responseto high temperatures were observed among varieties, with Norchipshowing the least and Up-to-Date showing the most sensitivity. High temperature, partitioning of assimilates, ¹⁴C-translocation, potato, Solanum tuberosum var. Desirèe, Solanum tuberosum var. Norchip, Solanum tuberosum var. Up-to-Date     

Purification and Characterization of Soluble and Membrane-Associated DNA Polymerases from a Virus PBCV-1 Infected Green Alga Chlorella NC64A

DNA polymerases were purified several hundred-fold from the10 000 x g soluble (polymerase I) and particulate (polymeraseIII) fractions prepared from virus PBCV-1 infected ChlorellaNC64A extracts. Both DNA polymerases exhibited optimal activitywith activated calf thymus DNA at pH 8.5. DNA polymerase I required3.0 mol m^–3 MgSO₄ and 150 to 250 mol m^–3 KCl foroptimum activity whereas, DNA polymerase III required 2.0 molm^–3 MgSO₄ and 150 mol m^–3 KCl. Both enzymes wereinhibited by pyrophosphate, actinomycin D, ethidium bromide,dideoxythymidine triphosphate, and N-ethylmaleimide but wererelatively insensitive to aphidicolin. DNA polymerase I differedfrom DNA polymerase III in its response to cations (particularlyNH₄Cl), elution from a DEAE cellulose column, and molecularweight. Key words: Algal virus, DNA polymerase, Chlorella     

Studies on factors in sweet potato root which induce spore germination of Ceratocystis fimbriata

Spore germination of Ceratocystis fimbriata was studied in termsof host-parasite specificity. The sweet potato, coffee and cacaostrains of Ceratocystis fimbriata germinated well in a fractionof sweet potato root water extract which had been passed througha column of cation exchange resin. The results showed that germinationof these strains was independent of exogenous cations. On theother hand, the prune, oak, taro and almond strains requiredfor germination both the absorbed and unabsorbed fractions ofsweet potato root water extract which were separated from eachother with a cation exchange resin column. Divalent cationssuch as Ca²⁺ Mg²⁺, Mn²⁺ and Zn²⁺ were identified as the activeprinciples in the absorbed fraction and Ca²⁺ showed the highestinductive activity for spore germination in the presence ofthe unabsorbed fraction. The active principle(s) in the unabsorbedfraction has not yet been identified. There was no relationshipbetween the Ca²⁺ and Mg²⁺ contents of the spores and the requirementof exogenous Ca²⁺ for germination. Ca²⁺ appeared to functionas a trigger of spore germination, not as a normal nutrient.These results suggest that the divalent cations such as Ca²⁺and Mg²⁺ in sweet potato contribute to the establishment ofhost-parasite specificity of this system. (Received August 10, 1977; )     

The Use of C14-Proline by Growing Cell: Its Conversion to Protein and Hydroxyproline

C¹⁴-proline is readily absorbed by growing tissue cultures ofcarrot root phloem and of potato tuber in experiments carriedout under aseptic conditions. The C¹⁴-proline rapidly entersinto the protein of the tissue, appearing there in as shorta period as 15 minutes, and, thereafter, the amount incorporatedinto the protein bears a linear relation to time. Virtuallyall the C¹⁴ appears in the protein hydrolysate in the form ofproline and hydroxyproline. It is shown that the conversionfrom proline to hydroxyproline occurs after the C¹⁴-prolineis combined into the protein and that this conversion proceedsprogressively with time. The ratio of C¹⁴ as proline to C¹⁴as hydroxyproline declined progressively from a value of 4.0after 30 minutes of contact and seemed to become stabilizedeventually at 0.7. C¹⁴-hydroxyproline, which can be absorbedby the tissue, seems not to be incorporated into the proteinas such. The protein moiety which contains the C¹⁴-hydroxyprolinefrom C¹⁴-proline represents a stable protein which is not metabolizedand whose carbon does not ‘turn over’. This inertprotein seems to be characteristic of cells which are in rapiddivision under the influence of coconut milk or are synthesizingprotein in response to other stimuli such as the events at acut tissue surface. The protein in question seems to be presentmainly in the cytoplasm rather than in its paniculate inclusions.These results are compatible with earlier views which requirethat part of the protein in the cell ‘turns over’its carbon, whereas another part does not do so.     

The Translocation of 3,5-Dichlorophenoxyacetic Acid in Relation to its Effect on Potato Common Scab

The translocation of 3,5-dichlorophenoxyacetic acid (3,5-D)in the potato plant was studied to clarify the mode of actionagainst potato common scab. 3,5-D was translocated to the tuberfaster and metabolised less than 2,4-D. Translocation to tuberswas proportional to growth and a concentration of 10–25nmol 3,5-D g^–1 fr. wt was attained 8 d after a foliarapplication. Previous evidence indicates this concentrationwas insufficient to be directly toxic to the pathogen Streptomycesscabies and the results suggest that 3,5-D acts in the tuberby altering the host's response to infection.