Assimilation of glucose carbon in subcellular rat brain particles in vivo and the problems of axoplasmic flow

1. Rats were injected with [U-¹⁴C]glucose and the content of ¹⁴C in proteins and lipids of the cerebral P₁ (`nuclear'), P<sub>2</sub> (`mitochondrial'), P₃ (`microsomal') and high-speed supernatant fractions was measured 7, 22 and 93hr. after injection of labelled glucose. 2. The crude brain mitochondrial fractions (P<sub>2</sub>) were subfractionated on continuous sucrose gradients (0·32–1·8m-sucrose) and the <sup>14</sup>C content of the proteins and lipids of about 20 subfractions was measured. 3. About 40–50% of the <sup>14</sup>C assimilated by brain proteins was found in the P<sub>2</sub> (`mitochondrial') fraction. About 68–70% of the ¹⁴C assimilated by brain lipids was also recovered from the lipids of the P₂ fraction. 4. Between 22 and 93hr. after injection of [U-¹⁴C]glucose both the amount of ¹⁴C in the protein of the P₂ (`mitochondrial') fraction and the specific activity of this protein increased. The specific activity of the protein of all other particulate fractions (P₁, P₂ and P₃) and subfractions (obtained from sucrose-density-gradient subfractionation of fraction P₂) when related to the specific activity of the high-speed supernatant protein also increased during 93hr. after injection of [U-¹⁴C]glucose. The amount of ¹⁴C in the protein of the high-speed supernatant and the specific activity of this protein decreased during the same period. 5. The distribution of ¹⁴C in the lipids of all subcellular particulate fractions remained unchanged during the period 22–93hr. after injection of [U-¹⁴C]glucose. 6. It was concluded that a diffusion occurs of some supernatant proteins into subcellular particulate matter of the cerebrum and no significant preference for any subcellular particulate matter was observed. The lipids occur in the cerebrum mainly in a non-diffusible state, which is consistent with the view that they form almost entirely a part of the structure of the cerebrum. 7. The data obtained do not lend further support to the concept of axoplasmic flow within the cerebrum or the concept of a one-directional flow of mitochondria or other subcellular particles within the cerebrum.     

Formation and Action of Lignin-Modifying Enzymes in Cultures of Phlebia radiata Supplemented with Veratric Acid

Transformation of veratric (3,4-dimethoxybenzoic) acid by the white rot fungus Phlebia radiata was studied to elucidate the role of ligninolytic, reductive, and demeth(ox)ylating enzymes. Under both air and a 100% O₂ atmosphere, with nitrogen limitation and glucose as a carbon source, reducing activity resulted in the accumulation of veratryl alcohol in the medium. When the fungus was cultivated under air, veratric acid caused a rapid increase in laccase (benzenediol:oxygen oxidoreductase; EC 1.10.3.2) production, which indicated that veratric acid was first demethylated, thus providing phenolic compounds for laccase. After a rapid decline in laccase activity, elevated lignin peroxidase (ligninase) activity and manganese-dependent peroxidase production were detected simultaneously with extracellular release of methanol. This indicated apparent demethoxylation. When the fungus was cultivated under a continuous 100% O₂ flow and in the presence of veratric acid, laccase production was markedly repressed, whereas production of lignin peroxidase and degradation of veratryl compounds were clearly enhanced. In all cultures, the increases in lignin peroxidase titers were directly related to veratryl alcohol accumulation. Evolution of ¹⁴CO₂ from 3-O¹⁴CH₃-and 4-O¹⁴CH₃-labeled veratric acids showed that the position of the methoxyl substituent in the aromatic ring only slightly affected demeth(ox)ylation activity. In both cases, more than 60% of the total ¹⁴C was converted to ¹⁴CO₂ under air in 4 weeks, and oxygen flux increased the degradation rate of the ¹⁴C-labeled veratric acids just as it did with unlabeled cultures.     

Partial purification and characterization of endoproteinases from senescing barley leaves

Two major endoproteinases were purified from senescing primary barley leaves. The major enzyme (EP₁) appeared to be a thiol proteinase and accounted for about 85% of the total proteolytic activity measured in vitro. This proteinase was purified 5,800-fold and had a molecular weight of 28,300. It was highly unstable in the absence of dithiothreitol or at a pH greater than 7.5. Leupeptin, at a concentration of 10 micromolar, inhibited this enzyme 100%. A second proteinase (EP₂) was purified approximately 50-fold and had a molecular weight of 67,000. It was inhibited 20% by 1 millimolar dithiothreitol and 50% by 1 millimolar phenylmethyl sulfonylfluoride. EP₂ contributed about 15% of the total proteolytic activity measured in vitro. Both proteinases hydrolyzed a variety of artificial and protein substrates, and both had pH optima of 5.5 to 5.7 when either azocasein or [¹⁴C]ribulose-1,5-bisphosphate carboxylase ([¹⁴C]RuBPCase) was the substrate. The thiol endoproteinase hydrolyzed azocasein linearly but hydrolyzed [¹⁴C]RuBPCase biphasically. A third endoproteinase (EP₃), not detected by standard proteolytic assays, was observed when [¹⁴C]RuBPCase was the substrate.     

Katabolismus von 4′,6-dihydroxyauron in pflanzlichen zellsuspensionskulturen

Cell suspension cultures of Glycine max, Phaseolus aureus, Cicer arietinum and Petroselinum hortense were shown to catabolize (α - ¹⁴C) - 4′,6-dihydroxyaurone as measured by ¹⁴CO₂ production and isolation of (¹⁴C)-p-hydroxybenzoic acid. Aurone catabolism in plants is thus comparable with the degradation of chalcones flavanones and flavonols because in all cases the B-ring is liberated as a substituted benzoic acid.     

Transport of dicarboxylic acids in castor bean mitochondria

Mitochondria from castor bean (Ricinus communis cv Hale) endosperm, purified on sucrose gradients, were used to investigate transport of dicarboxylic acids. The isolated mitochondria oxidized malate and succinate with respiratory control ratios greater than 2 and ADP/O ratios of 2.6 and 1.7, respectively. Net accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate into the mitochondrial matrix during substrate oxidation was examined by the silicone oil centrifugation technique. In the presence of ATP, there was an appreciable increase in the accumulation of ¹⁴C from [¹⁴C]malate or [¹⁴C]succinate accompanied by an increased oxidation rate of the respective dicarboxylate. The net accumulation of dicarboxylate in the presence of ATP was saturable with apparent K_m values of 2 to 2.5 millimolar. The ATP-stimulated accumulation of dicarboxylate was unaffected by oligomycin but inhibited by uncouplers (2,4-dinitrophenol and carbonyl cyanide m-chlorophenylhydrazone) and inhibitors of the electron transport chain (antimycin A, KCN). Dicarboxylate accumulation was also inhibited by butylmalonate, benzylmalonate, phenylsuccinate, mersalyl and N-ethylmaleimide. The optimal ATP concentration for stimulation of dicarboxylate accumulation was 1 millimolar. CTP was as effective as ATP in stimulating dicarboxylate accumulation, and other nucleotide triphosphates showed intermediate or no effect on dicarboxylate accumulation. Dicarboxylate accumulation was phosphate dependent but, inasmuch as ATP did not increase phosphate uptake, the ATP stimulation of dicarboxylate accumulation was apparently not due to increased availability of exchangeable phosphate.

The maximum rate of succinate accumulation (14.5 nanomoles per minute per milligram protein) was only a fraction of the measured rate of oxidation (100-200 nanomoles per minute per milligram protein). Efflux of malate from the mitochondria was shown to occur at high rates (150 nanomoles per minute per milligram protein) when succinate was provided, suggesting dicarboxylate exchange. The uptake of [¹⁴C]succinate into malate or malonate preloaded mitochondria was therefore determined. In the absence of phosphate, uptake of [¹⁴C]succinate into mitochondria preloaded with malate was rapid (27 nanomoles per 15 seconds per milligram protein at 4°C) and inhibited by butylmalonate, benzylmalonate, and phenylsuccinate. Uptake of [¹⁴C]succinate into mitochondria preloaded with malonate showed saturation kinetics with an apparent K_m of 2.5 millimolar and V_max of 250 nanomoles per minute per milligram protein at 4°C. The measured rates of dicarboxylate-dicarboxylate exchange in castor bean mitochondria are sufficient to account for the observed rates of substrate oxidation.

     

In Vivo Measurement of Indole-3-acetic Acid Decarboxylation in Aging Coleus Petiole Sections

The concentration of indoleacetic acid (IAA) in plant tissues is regulated, in part, by its rate of decarboxylation. However, the commonly used in vitro assays for IAA oxidase may not accurately reflect total in vivo decarboxylation rates. A method for measuring in vivo decarboxylation was utilized in which ¹⁴CO₂ is collected following uptake of [1-¹⁴C]IAA by excised tissue sections. After a 30-minute equilibration period, the evolution of ¹⁴CO₂ was found to follow an approximately linear course with respect to both time and tissue weight.

Decarboxylation rates were measured by this method in petiole sections of the Princeton clone of Coleus blumei Benth. Both the ¹⁴CO₂ evolved per milligram tissue and the percent of [1-¹⁴C]IAA uptake decarboxylated were highest in sections from the youngest petioles tested, and declined in the older tissue. Thin layer chromatography of acetonitrile extracts from the [1-¹⁴C]IAA-treated petioles showed a decreasing amount of free IAA and an increase at the retardation factor of indoleacetylaspartate in the older sections. The decreased decarboxylation rates in the older petioles may be attributable to a generally lower metabolic rate and increased protection of the IAA by conjugation.

     

Effects of Chronic Internal beta-Radiation from Photoassimilated CO(2) on the Retention and Distribution of C in Young White Pine Plants

Eastern white pines (Pinus strobus L.) in their 3rd year of growth photoassimilated 35, 110, 220, or 400 microcuries of ¹⁴CO₂ on a single occasion in the spring when needle expansion was occurring. Once assimilated into organic products and translocated to various sinks, the retained ¹⁴C represented an internal chronic source of ionizing radiation to the plant. About 2.5 months later, the pines were harvested, and the distribution of ¹⁴C activity was determined. In addition, new needle lengths, fresh weights, and rates of processes involving CO₂ exchange were measured.     

Metabolism of Proline, Glutamate, and Ornithine in Proline Mutant Root Tips of Zea mays (L.)

In excised pro_1-1 mutant and corresponding normal type roots of Zea mays L. the uptake and interconversion of [¹⁴C]proline, [¹⁴C]glutamic acid, [¹⁴C]glutamine, and [¹⁴C]ornithine and their utilization for protein synthesis was measured with the intention of finding an explanation for the proline requirement of the mutant. Uptake of these four amino acids, with the exception of proline, was the same in mutant and normal roots, but utilization differed. Higher than normal utilization rates for proline and glutamic acid were noted in mutant roots leading to increased CO₂ production, free amino acid interconversion, and protein synthesis. Proline was synthesized from either glutamic acid (or glutamine) or ornithine in both mutant and normal roots; it did not accumulate but rather was used for protein synthesis. Ornithine was not a good precursor for proline in either system, but was preferentially converted to arginine and glutamine, particularly in mutant roots. The pro_1-1 mutant was thus not deficient in its ability to make proline. Based on these findings, and on the fact that ornithine, arginine, glutamic acid and aspartic acid are elevated as free amino acids in mutant roots, it is suggested that in the pro_1-1 mutant proline catabolism prevails over proline synthesis.     

Effect of CO(2) Concentration on Glycine and Serine Formation during Photorespiration

Amount and products of photosynthesis during 10 minutes were measured at different ¹⁴CO₂ concentrations in air. With tobacco (Nicotiana tabacum L. cv. Maryland Mammoth) leaves the percentage of ¹⁴C in glycine plus serine was highest (42%) at 0.005% CO₂, and decreased with increasing CO₂ concentration to 7% of the total at 1% CO₂ in air. However, above 0.03% CO₂ the total amount of ¹⁴C incorporated into the glycine and serine pool was about constant. At 0.005% or 0.03% CO₂ the percentage and amount of ¹⁴C in sucrose was small but increased greatly at higher CO₂ levels as sucrose accumulated as an end product. Relatively similar data were obtained with sugar beet (Beta vulgaris L. cv. US H20) leaves. The results suggest that photorespiration at high CO₂ concentration is not inhibited but that CO₂ loss from it becomes less significant.     

Preparation of [U-14C]-labelled glycogen, maltosaccharides, maltose, and D-glucose by photoassimilation of 14CO2 in Anacystis nidulans and selective enzymic degradation.

A procedure is described for the isolation from the phototrophic procaryole Anacystis nidulans of [U-¹⁴C]-labelled glycogen, with high specific radioactivity,formed when NaH¹⁴CO₃ was added to non-dividing cells that continued to photoassimilate CO₂. [U-¹⁴C]-Labelled glycogen was then treated with isoamylase (EC 3.2.1.68), isoamylase plus beta-amylase (EC 3.2.1.2), or glucoamylase (EC 3.2.1.3) to give [U-¹⁴C]-labelled maltosaccharides, maltose-U-¹⁴C, or d-glucose-U-¹⁴C, respectively.     

Silk—A new substrate for UDP-d-xylose: Proteoglycan core protein β-d-xylosyltransferase

The formation of most connective tissue polysaccharides is initiated by transfer of d-xylose from UDP-d-xylose to specific serine residues in the core proteins of the putative proteoglycans. The substrate specificity of the xylosyltransferase catalyzing this reaction has not yet been examined in detail, but it appears that a -Ser-Gly- pair is an essential part of the substrate structure. Since the preparation of the known acceptors (e.g., Smith-degraded or HF-treated cartilage proteoglycan) involves a substantial effort, we have searched for readily available proteins with the -Ser-Gly-sequence, which might serve as alternative substrates. In the present work, it was found that silk fibroin from Bombyx mori, which consists, in large part, of the repeating hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly, is an excellent substrate for the xylosyltransferase from embryonic chick cartilage. Pieces of silk were used directly in the reaction mixtures, and [¹⁴C]xylose transferred from UDP-d-[¹⁴C]xylose was measured by liquid scintillation spectrometry after rinsing the silk in 1 m NaCl and water. Substantially greater incorporation was observed with preparations of silk or fibroin which had been dissolved in 60% LiSCN and subsequently dialyzed exhaustively or diluted appropriately. Under standard reaction conditions, the V_max for fibroin was 531 pmol/h/mg enzyme protein, as compared to 223 pmol/h/mg for Smith-degraded proteoglycan. K_m values were 182 mg/liter (fibroin) and 143 mg/liter (Smith-degraded proteoglycan). The product of [¹⁴C]xylose transfer to silk was alkali labile, and [¹⁴C]xylitol was formed when [¹⁴C]xylosylsilk was treated with borohydride in alkali. Proteolytic digestion with papain, Pronase, leucine aminopeptidase, and carboxypeptidase A yielded a radioactive product which was identified as [¹⁴C]xylosylserine by electrophoresis and chromatography. The identity of the isolated [¹⁴C]xylosylserine was further supported by its resistance to treatment with alkali (0.5 m KOH: 100°C; 8h) and by acid hydrolysis which yielded [¹⁴C]xylose. Tryptic and chymotryptic fragments from fibroin were also good xylose acceptors and had V_max values 60–70% of that observed for the intact protein. Substantial acceptor activity was displayed also by the sericin fraction of silk and by the silk sequence hexapeptide, Ser-Gly-Ala-Gly-Ala-Gly; the latter had a V_max value close to 20% of that of intact fibroin.     

Substrate-binding region of cytochrome P-450scc (P-450 XIA1). Identification and primary structure of the cholesterol binding region in cytochrome P-450scc

Cytochrome P-450_scc (P-450 XIA1) from bovine adrenocortical mitochondria was investigated using a suicide substrate: [¹⁴C]methoxychlor. [¹⁴C]Methoxychlor irreversibly abolished the activity of the side-chain cleavage enzyme for cholesterol (P-450_scc) and the inactivation was prevented in the presence of cholesterol. The binding of [¹⁴C]methoxychlor and cytochrome P-450_scc occurred in a molar ratio of 1:1 and the cholesterol-induced difference spectrum of cytochrome P-450_scc was similar with the methoxychlor-induced difference spectrum. [¹⁴C]Methoxychlor-binding peptides were purified from tryptic-digested cytochrome P-450_scc modified with [¹⁴C]methoxychlor. Determination of the sequence of the amino-acid residues of a [¹⁴C]methoxychlor-binding peptide allowed identification of the peptide comprising the amino-terminal amino-acid residues 8 to 28.     

Cyclic variations in nitrogen uptake rate in soybean plants

Uptake of NO₃⁻ by nonnodulated soybean plants (Glycine max L. Merr. cv Ransom) growing in flowing hydroponic culture at 22 and 14°C root temperatures was measured daily during a 31-day growth period. Ion chromatography was used to determine removal of NO₃⁻ from solution during each 24-hour period. At both root-zone temperatures, rate of NO₃⁻ uptake per plant oscillated with a periodicity of 3 to 5 days. The rate of NO₃⁻ uptake per plant was consistently lower at 14°C than 22°C. The lower rate of NO₃⁻ uptake at 14°C during the initial 5 to 10 days was caused by reduced uptake rates per gram root dry weight, but with time uptake rates per gram root became equal at 14 and 22°C. Thereafter, the continued reduction in rate of NO₃⁻ uptake per plant at 14°C was attributable to slower root growth.     

Removal of the acetate-moiety of 2,4-dichlorophenoxyacetic acid in Ribes sativum

Ten minutes after uptake of 2,4-dichlorophenoxyacetic acid-1-¹⁴C(2,4-D-1-¹⁴C) by excised Ribes sativum leaves, 37·8 % of the radioactivity in water-soluble metabolites was in glyoxylic acid. When 2,4-D- 2-¹⁴C was supplied under the same conditions, 23·0 % of the radioactivity of the water-soluble rnetabolites was in glyoxylic acid. Radioactive glycine and glyoxylic acid, isolated from Ribes sativum 6 hr after uptake of 2,4-D-1-¹⁴C, contained essentially all of the ¹⁴C in the carboxyl-carbon atoms. When 2,4-D-2-¹⁴C was the precursor, the glycine isolated contained 64·8 % of its radioactivity in C₂, while 60·0 % of the radioactivity in glyoxylic acid was in C₂. The side-chain label of 2,4-D-2-¹⁴C-4-³⁶Cl was more efficiently incorporated into ethanol-insoluble plant residue than the ring-label. The metabolism of glyoxylic acid-1-¹⁴C and 2,4-D-1-¹⁴C in excised Ribes sativum leaves were compared. The data suggest a cleavage of the acetate-moiety of 2,4-D resulting in a C₂ compound, perhaps glyoxylate.     

Biosynthesis of C(20) and C(22) Fatty Acids by Developing Seeds of Limnanthes alba: CHAIN ELONGATION AND Delta5 DESATURATION

The storage triacylglycerols of meadowfoam (Limnanthes alba) seeds are composed essentially of C₂₀ and C₂₂ fatty acids, which contain an unusual Δ5 double bond. When [1-¹⁴C]acetate was incubated with developing seed slices, ¹⁴C-labeled fatty acids were synthesized with a distribution similar to the endogenous fatty acid profile. The major labeled product was cis-5-eicosenoate, with smaller amounts of palmitate, stearate, oleate, cis-5-octadecenoate, eicosanoate, cis-11-eicosenoate, docosanoate, cis-5-docosenoate, cis-13-docosenoate, and cis-5,cis-13-docosadienoate. The label from [¹⁴C]acetate and [¹⁴C]malonate was used preferentially for the elongation of endogenous oleate to produce cis-[¹⁴C]11-eicosenoate, cis-13-[¹⁴C]docosenoate, and cis-5,cis-13-[¹⁴C]docosadienoate and for the elongation of endogenous palmitate to produce the remaining C₂₀ and C₂₂ acyl species. The Δ5 desaturation of the preformed acyl chain and chain elongation of oleate and palmitate were demonstrated in vivo by incubation of the appropriate 1-¹⁴C-labeled free fatty acids. Using [1-¹⁴C]acyl-CoA thioesters as substrates, these enzyme activities were also demonstrated in vitro with a cell-free homogenate.     

Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid.

Radioactive gangliosides, N-[¹⁴C]-acetylneuraminylgalactosylglucosylceramide ([¹⁴C]G_M3) and N- [¹⁴C]-acetylneuraminylgalactosyl-N-acetylgalactosaminyl- [N-acetylneuraminyl]-galactosylglucosylceramide ([¹⁴C]G_D1a), were synthesized from CMP-[¹⁴C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G_M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [¹⁴C]G_M3 and [¹⁴C]G_D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K_m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [¹⁴C]G_M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.     

Metabolism of Radiolabeled β-Guaiacyl Ether-Linked Lignin Dimeric Compounds by Phanerochaete chrysosporium

Phanerochaete chrysosporium metabolized the radiolabeled lignin model compounds [γ-¹⁴C]guaiacylglycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether (VI) to ¹⁴CO₂ in stationary and in shaking cultures. ¹⁴CO₂ evolution was greater in stationary culture. ¹⁴CO₂ evolution from [γ-¹⁴C]guaiacyl-glycerol-β-guaiacyl ether and [4-methoxy-¹⁴C]veratrylglycerol-β-guaiacyl ether in stationary cultures was two- to threefold greater when 100% O₂ rather than air (21% O₂) was the gas phase above the cultures. ¹⁴CO₂ evolution from the metabolism of the substrates occurred only as the culture entered the stationary phase of growth. The presence of substrate levels of nitrogen in the medium suppressed ¹⁴CO₂ evolution from both substrates in stationary cultures. [¹⁴C]veratryl alcohol and 4-ethoxy-3-methoxybenzyl alcohol were formed as products of the metabolism of VI and 4-ethoxy-3-methoxyphenylglycerol-β-guaiacyl ether, respectively.     

Increased arginase activity during lymphocyte mitogenesis

A sensitive assay for arginase activity was developed using [guanidino-¹⁴C]arginine as substrate and measuring the production of ¹⁴CO₂ from [¹⁴C]urea in the presence of urease. Arginase activity was measured in bovine lymphocytes after activation by Concanavalin A. The specific enzymatic activity of arginase doubled in 6 hours and increased nearly 4-fold by 24 hours after stimulation. It is suggested that the role of arginase in these cells is to provide ornithine as substrate for the synthesis of putrescine, precursor of the polyamines spermidine and spermine.     

Dark metabolism of carbon monoxide in lettuce leaf discs

In the dark, leaf tissue of crisphead lettuce (Lactuca sativa L.) metabolized ¹⁴CO to ¹⁴CO₂ and acid-stable products. Tissue incubated at 2.5°C for 3.5 hours and 48 hours converted about 1% and 17%, respectively, of the applied ¹⁴CO to ¹⁴CO₂, and incorporated about 0.04% and 0.6% of the ¹⁴C in acid-stable products. Examination of soluble acid-stable products from ¹⁴CO and ¹⁴CO₂-treated leaf tissue revealed that the labeling patterns of both treatments were identical during the 3.5-hour and the 48-hour incubation periods. Malate, citrate, and aspartate together comprised 70% or more of the soluble radioactivity from both treatments. Incorporation of radioactivity from CO into soluble acid-stable products during a 3-hour incubation period at 2.5°C was inhibited 90% by adding 3% nonradioactive CO₂. These results indicate that in head lettuce in the dark ¹⁴CO is metabolized primarily to ¹⁴CO₂ which is the precursor of acid-stable products. In leaf discs at 2.5°C, the apparent K_m for CO oxidation to CO₂ was 5.3 microliters per liter and the V_max was 9.7 nanoliters per gram per hour. The mitochondrial fraction of the leaf homogenate was the most active fraction to oxidize CO to CO₂, and this activity was heat-labile and cyanide-sensitive.     

Photosynthetic and Photorespiratory Carbon Metabolism in Mesophyll Protoplasts and Chloroplasts Isolated from Isogenic Diploid and Tetraploid Cultivars of Ryegrass (Lolium perenne L.)

Photosynthetic ¹⁴CO₂ fixation, [¹⁴C]glycolate formation, and the decarboxylation of [1-¹⁴C]glycolate and [1-¹⁴C]glycine by leaf mesophyll protoplasts isolated from isogenic diploid and tetraploid cultivars of ryegrass (Lolium perenne L.) were examined. The per cent O₂ inhibition of photosynthesis in protoplasts from the tetraploid cultivar was less than that of the diploid line at both 21 and 49% O₂. Kinetic studies revealed that the K_m (CO₂) for photosynthesis by the diploid protoplasts was about twice that of the tetraploid line. In contrast, the K_i (O₂) for protoplast photosynthesis was similar in both cultivars, as was the potential for oxidizing glycolate and glycine to CO₂ via the photorespiratory carbon oxidation cycle. Although the maximal rates of glycolate accumulation by the isolated protoplasts in the presence of 21% O₂ and a glycolate oxidase inhibitor were similar in the two cultivars, the percentage of total fixed ¹⁴C entering the [¹⁴C]glycolate pool and the ratio of the rate of [¹⁴C]glycolate formation to ¹⁴CO₂ fixation at 21% O₂ and low pCO₂ were about two times greater in protoplasts and intact chloroplasts isolated from the diploid line compared to the tetraploid. These results fully support the recent observation that a doubling of ploidy in various ryegrass cultivars reduced the K_m (CO₂) of purified ribulose bisphosphate carboxylase-oxygenase by about one-half without affecting the K_i (O₂) (Garrett 1978 Nature 274: 913-915).