Evidence for the Existence of a Soybean Resistant Protein That Captures Bile Acid and Stimulates Its Fecal Excretion

Feeding HMF, an insoluble “high-molecular-weight fraction” from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestable protein or peptide that can be called a “resistant protein” in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestable fraction of HMF.     

Enzymatic Modification of Proteins in Foodstuffs

Peptic hydrolyzate of soy protein was submitted to the plastein reaction with α-chymotrypsin under the following condition: substrate concentration, 20%; enzyme-substrate ratio by weight, 1/100; reaction pH, 7.0; and reaction temperature, 37°C. The plastein yield resulting from the plastein reaction for 24 hr was found to depend on the degree of hydrolysis of the substrate (per cent ratio between nitrogen amount in 10% trichloroacetic acid soluble nitrogen and that in whole hydrolyzate); the optimum degree of hydrolysis for the highest plastein yield seemed to lie around 80%. A turbidity appeared in the process of the plastein reaction, whose intensity was correlative to the plastein yield. The peptic hydrolyzate of soy protein per se had bitterness and its magnitude decreased with increasing plastein yield.

As a result of the plastein reaction applied for 24 hr to the hydrolyzate whose degree of hydrolysis was 80%, the average molecular weight estimated by the change in amino nitrogen content increased by approximately three times. The molecular weight distribution pattern obtained by gel filtration supported the above result. The total amount of amino acids liberated from the plastein reaction product by its treatment with either leucine aminopeptidase or carboxypeptidase A was significantly less than that liberated from the original hydrolyzate by its similar treatment. This result also supports the formation of higher-molecular protein-like substances by the plastein reaction. Deuteration study followed by IR spectrometry showed the occurrence of peptide bond formation, i.e. decrease in ionized carboxyl group at 1575 cm^?1 and increase in deuterated amide at 1450 cm^?1, even at the earlier stages of the plastein reaction.     

The incorporation of glycerol and lysine into the lipid fraction of Staphylococcus aureus

1. Incubation of washed cells of Staphylococcus aureus with [1-¹⁴C]glycerol results in the incorporation of glycerol into the lipid fraction of the cells. The rate of incorporation is increased by the presence of glucose and amino acids. The presence of amino acids increases incorporation into the fraction containing O-amino acid esters of phosphatidylglycerol. 2. Glycerol, incorporated into washed cells by incubation with glycerol, glucose and amino acids, is rapidly released from the lipid fraction when cells are incubated at low suspension densities in buffer. 3. Of nine amino acids tested, only lysine is significantly incorporated into the lipid fraction. The incorporation is increased by the presence of glycerol, glucose and other amino acids, especially aspartate and glutamate. 4. The incorporation of lysine is increased by the addition of puromycin at concentrations that inhibit protein synthesis. Chloramphenicol does not increase the incorporation of lysine but abolishes the enhancing effect of puromycin. 5. The enhancing effect of puromycin is accompanied by a similar increase in the incorporation of lysine into the fraction soluble in hot trichloroacetic acid. 6. Lysine is incorporated into the lipid fraction that contains O-amino acid esters of phosphatidylglycerol and corresponds in properties to phosphatidylglyceryl-lysine. 7. Lysine is rapidly released from the lipid of cells incubated in buffer only at low suspension densities. 8. Incubation of cells with the phosphatidylglyceryl-lysine fraction does not lead to the appearance of free lysine or to incorporation into the fraction insoluble in hot trichloroacetic acid.     

The incorporation of glucose into alpha-1,4-glucan proteins of bovine retina membranes.

—Incubation of bovine retina membranes with UDP-[¹⁴C]glucose resulted in the incorporation of [¹⁴C]glucose into endogenous α-1, 4-glucan proteins. The transferring system was concentrated in membranes that floated at 0.94 and 1.10m -sucrose when centrifuged in a discontinuous sucrose density gradient and was almost absent in the rod outer segment (ROS) and the 100, 000 g supernatant fractions. The glucan proteins labelled by incubation with the radioactive sugar nucleotide at micromolar concentrations were distinguished in two fractions by their solubilities in trichloroacetic acid (TCA): glucan protein-I (GP-I), insoluble in TCA, and glucan protein-II (GP-II), soluble in TCA and precipitable by ethanol from the TCA soluble fraction. GP-I and GP-II were precipitated by trichloroacetic acid-phosphotungstic acid (TCA-PTA). A third fraction, glucan protein-III (GP-III) was found when incubations were carried out with UDP-[¹⁴C]glucose at millimolar instead of micromolar concentrations. GP-III was soluble in TCA and in TCA-PTA and precipitable by ethanol from the TCA soluble fraction. GP-II was excluded from a Sephadex G-200 column and showed a greater size than GP-I in a Sepharose 2B column. The radioactive residues obtained from the glucan proteins after digestion with pronase were totally included in a Sephadex G-25 column and were of a greater size than the labelled residues released with salivary α-amylase. Only radioactive maltose was found after a-amylase treatment. When membranes containing labelled GP-I and GP-II were incubated with unlabelled UDP-glucose at millimolar concentrations, GP-I was converted into GP-II and GP-III was formed.     

Effects of Carbon Dioxide on Coccidian Oocysts from 6 Host Species*

SYNOPSIS Activation of sporozoites in oocysts of Eimeria acervulina (chicken), E. intricata (sheep), and E. scabra (swine) occurred after pretreatment in aqueous 0.02 M cysteine hydrochloride under an atmosphere of CO₂, followed by incubation in a trypsin-bile mixture. Sporozoites of E. stiedae (rabbit), E. bilamellata (squirrel), and Isospora canis (dog) became activated when incubated in trypsin and bile with or without prior CO₂-pretreatment of oocysts; however, when CO₂-pretreatment was used, activation of these species in trypsin and bile was greatly enhanced. For E. acervulina, 12% of the oocysts were activated after 4 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 43 C; higher temperatures or longer pretreatment times did not cause greater activation. Eimeria intricata oocysts became activated after 1 hr pretreatment and 10 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively. The highest activation (31%) occurred after 20 hr pretreatment and 10 hr incubation in trypsin and bile at 41 C. Ninety percent of E. scabra oocysts contained active sporozoites after 1 hr CO₂-pretreatment and 10 hr incubation in trypsin and bile at 37 C. At 39 or 41 C, 100% activation occurred with this species after similar pretreatment and treatment periods. With E. bilamellata, 64% activation occurred in nonpretreated oocysts incubated 10 hr in trypsin and bile at 41 C, whereas 100% activation occurred if oocysts were pretreated with CO₂ for 1 hr before treatment with trypsin and bile. Thirty-one, 35, and 36% of CO₂-pretreated E. stiedae oocysts were activated after 1 hr incubation in trypsin and bile at 37, 39 or 41 C, respectively, whereas 1, 2, and 20% activation occurred in nonpretreated oocysts incubated at the same temperatures. Sporozoites in 99-100% of I. canis oocysts were activated after 10 hr treatment in trypsin and bile with or without 1 hr CO₂-pretreatment at 23, 37, 39 or 41 C.     

Changes of Nucleic Acids and Proteins in the Oocysts of Eimeria tenella during Sporulation

SYNOPSIS. The total content of DNA in Eimeria tenella , estimated at 5.8 × 10⁻¹² gm/oocyst, varies little during sporulation. Its buoyant density is 1.682 gm/cm³, reflecting a G + C content of ∼41%. Thymidine is not incorporated into any TCA insoluble fraction of sporulating oocysts, but radioactivity from [³H]uridine and [³H]deoxyuridine are incorporated into RNA at a linear rate during the first 5 hr of sporulation. The labeled RNA, found mainly in the paranuclear bodies of newly formed sporozoites, contains ∼0.15 nmole [³H]uridine/10⁶ oocysts at the completion of sporulation. One nmole of leucine is incorporated into the hot TCA insoluble fraction of 10⁶ oocysts during the first 7 hr of sporulation after an initial lag. The incorporated amino acid is mainly in the cytoplasm of the sporozoites, and an analysis by SDS-gel electrophoresis reveals most of the radioactivity in a narrow band with a molecular weight of ∼50,000 daltons. Incorporation of uridine and leucine, however, can be totally suppressed by respiratory inhibition. Further analysis of the proteins in the oocysts reveals that the total protein content remains relatively unchanged at 2.64 × 10⁻¹⁶ gm/oocyst during sporulation, but there is a shift of 13–14% of total protein from the soluble cytoplasm to the 15,000 g pellets. By polyacrylamide gel electrophoresis, a major protein band. possibly a glycoprotein, is shown in the soluble cytoplasm of unsporulated oocysts. This band disappears during sporulation.     

Non-enzymic Browning of Soy Sauce

Ion exchange resins have been used to separate soy sauce into three fractions of distinctly different composition: a cation fraction, a neutral fraction and an anion fraction. Almost all of the constituents responsible for browning were recovered in these three fractions.

Storage experiments show that when the three fractions were stored separately, only the cation fraction darkened considerably. When they were combined and stored, the color of the mixture increased at nearly the same rate as that of the original soy sauce. Neutral sugars are important constituents of the neutral fraction with respect to browning. The browning rate of a sugar-amino acid mixture (simulated soy sauce), was about 10% of soy sauce. The effect of the anion fraction (mainly caused by organic acids) and the ashed cation fraction on the over-all browning of soy sauce is calculated to be 1O~12% and 20%, respectively.

The sum of the contribution rate of the anion fraction, the neutral fraction, the amino acids and the ashed cation fraction in the browning of soy sauce was concluded to be approximately 40%. Compounds responcible for residual part of 60% should be considered to exist in the cation fraction. It was suggested that such compounds have strong reducing power and 0₂-uptaking ability.     

Evidence for the existence of a soybean resistant protein that captures bile acid and stimulates its fecal excretion

Feeding HMF, an insoluble "high-molecular-weight fraction" from an industrial enzymatic digest of a soy protein isolate, increased the fecal excretion of bile acid concomitant with increased fecal nitrogen. An amino acid analysis revealed that this increased fecal nitrogen could be explained by an increase in the insoluble protein fraction. This suggests the existence of an indigestible protein or peptide that can be called a "resistant protein" in the feces. The presumed resistant protein was rich in hydrophobic amino acids and bound bile acid by hydrophobic interaction. The residual fraction of HMF obtained after in vitro pepsin and pancreatin digestion, showed higher in vitro bile acid-binding capacity and excreted more bile acid in vivo than HMF. Its amino acid composition was similar to that of the feces of rat fed with HMF. These results suggest that the fecal resistant protein with bile acid-binding ability could be derived from the indigestible fraction of HMF.     

A self-inhibitor of protein synthesis in the conidia of Glomerella cingulata

Summary A diffusible self-inhibitor of germination of conidia of Glomerella cingulata appears to act as a regulator of protein synthesis. Both uptake of labeled amino acids and their incorporation into protein are reduced by the inhibitor or by crowding. Compared to conidia incubated without self-inhibitor, conidia incubated with self-inhibitor incorporated no labeled amino acids into protein in the first hour and 80% less in 6h. Thoroughly washed conidia were more permeable to amino acids and incorporated 6 times more precursor into proteins than unwashed conidia. At high density in nutrient medium, conidia of G. cingulata preferentially form secondary conidia instead of germ tubes and a mycelium. This inhibition of germination of conidia and regulation of development is mimicked by exposing them to an auto-inhibitor extracted from used culture medium and conidial washings. Germination of conidia of G. cingulata involves two steps, an initial step of 5 h duration which continues unaffected by crowing (1.7×10⁸/ml) and a subsequent 2 h step which conidia do not take unless they are sufficiently diluted. It is this step for which protein synthesis may be required.Non-Standard Abbreviations CHM cyloheximide - NM Neurospora minimal medium - psi pound per square inch - RPH reconstituted algal protein hydrolysate - TCA trichloroacetic acid     

AMINO ACID INCORPORATION IN VITRO INTO PROTEIN OF NEURONAL AND GLIAL CELL-ENRICHED FRACTIONS

Slices of rabbit cerebral cortex were incubated in the presence of labelled amino acids. Following incubation, neuron- and gliaenriched fractions were obtained by density gradient centrifugation and the TCA-insoluble radioactivity determined. The protein-bound radioactivity was five to six times higher in the neuronal-enriched fraction than in the glial-enriched fraction after incubation with tritiated leucine. The neuronal fraction incorporated also a number of other amino acids to a higher extent than the glial fraction (neuron/glia ratio 2·5-6). A definite dependence of incorporation on the rate of oxygenation was demonstrated. The suppression of amino acid incorporation was more marked for the neuronal fraction than for the glial fraction during incubation in relative hypoxia. An increase of potassium concentration in the incubation medium enhanced the amino acid incorporation in both fractions. Low sodium levels decreased the incorporation. Puromycin inhibited incorporation to approximately 30 per cent of control for both fractions. Addition of cycloheximide and dinitrophenol resulted in greater inhibition of incorporation in the neuronal fraction than in the neuroglial fraction. Actinomycin D did not markedly affect the incorporation in any fraction. These results are discussed in relation to in vivo and in in vitro differences for transport and incorporation of amino acids.     

A novel iron protein from Desulfovibrio gigas

The isolation, purification, and partial characterization of a novel iron-containing protein from the sulfate-reducing anaerobic bacterium, Desulfovibrio gigas, is described. The highly insoluble protein was isolated from the cell debris following osmotic shock of the bacteria. The insoluble fraction consistently contained about 90% of the cell-associated iron. Elemental analysis of a crude protein preparation gave 5.3% iron, 2.9% sulfur and 11.9% nitrogen. An independent colorimetric iron analysis showed 6.4% iron. The iron could be dissociated from the protein by treatment with 5% SDS. The iron-free protein was purified by a combination of organic extraction and DEAE-cellulose chromatography. The purified protein showed only one major band, M_r 14 000, by SDS-polyacrylamide gel electrophoresis. The protein could be reconstituted upon treatment with an appropriate mixture of FeS and β-mercaptoethanol. The reconstituted protein had the same physical and chemical properties as the native protein. The amino acid composition was not unusual except for the high isoleucine content.     

Identification and Quantitation of New Glutamic Acid Derivatives in Soy Sauce by UPLC/MS/MS

Glutamic acid is an abundant amino acid that lends a characteristic umami taste to foods. In fermented foods, glutamic acid can be found as a free amino acid formed by proteolysis or as a non‐proteolytic derivative formed by microorganisms. The aim of the present study was to identify different structures of glutamic acid derivatives in a typical fermented protein‐based food product, soy sauce. An acidic fraction was prepared with anion‐exchange solid‐phase extraction (SPE) and analyzed by UPLC/MS/MS and UPLC/TOF‐MS. α‐Glutamyl, γ‐glutamyl, and pyroglutamyl dipeptides, as well as lactoyl amino acids, were identified in the acidic fraction of soy sauce. They were chemically synthesized for confirmation of their occurrence and quantified in the selected reaction monitoring (SRM) mode. Pyroglutamyl dipeptides accounted for 770 mg/kg of soy sauce, followed by lactoyl amino acids (135 mg/kg) and γ‐glutamyl dipeptides (70 mg/kg). In addition, N‐succinoylglutamic acid was identified for the first time in food as a minor compound in soy sauce (5 mg/kg).     

Effect of temperature on fatty acid composition in each lipid fraction of Spirometra erinaceieuropaei plerocercoids

The effects of temperature and host fatty acids on the fatty acid contents of Spirometra erinaceieuropaei plerocercoids were investigated to clarify their role in sparganosis. After 24 hr incubation at 18 C in host snake serum, omega6 series fatty acids, especially arachidonic acid in the phospholipid fraction of the plerocercoids, increased compared with those of plerocercoids incubated at 37 C. The changes in the ratio of polyunsaturated to saturated fatty acids in the phospholipid fraction of plerocercoids incubated in physiological saline for 6 hr at 10 C were almost the same as the changes at 37 C. The ratio of polyunsaturated to saturated fatty acids of the triglyceride fraction showed almost opposite change versus the phospholipid fraction. The percentage of arachidonic acid in the phospholipid fraction of plerocercoids increased during the first 3 hr of incubation and then decreased, regardless of temperature. At 37 C, the percentage of arachidonic acid in the free fatty acid fraction fell for the first 3 hr of incubation and was significantly elevated at the end of the 6-hr incubation. At 10 C, however, arachidonic acid in the free fatty acid fraction decreased for the first hour of incubation, increased at 3 hr of incubation, then decreased again. These results suggest that fatty acids of the plerocercoids are frequently exchanged between fractions. Plerocercoids can mobilize arachidonic acid to the free fatty acid fraction more quickly at lower temperature than at higher temperature. They may utilize mobilized arachidonic acid early in the infection stage to produce prostaglandins. Alternatively, they can incorporate arachidonic acid into the phospholipid fraction again when arachidonic acid is readily available in the environment.     

Some Nutritional and Physiological Factors Affecting the Urinary Excretion of Acid Soluble Peptides in Rats and Women

Urinary excretion of acid soluble peptide (ASP)-form amino acids was lower in rats deprived of protein than in rats fed on a 20% casein or 20% gluten diet. However, the amino acid pattern of urinary ASP was similar among each of the three dietary groups, suggesting that urinary ASP is mainly endogenous origin under these nutritional conditions.

College women who were given a meat-free protein diet for 3 days after 10 days’ protein deprivation excreted 1.4 times the amount of ASP-form amino acids during protein deprivation.

The rate of urinary excretion of ASP-form amino acids in the state of protein deprivation was proportional to the metabolic body size of organisms as far as rats and women were concerned.

Streptozotocin-induced diabetic rats excreted two times the amount of ASP-form amino acids compared with normal rats. This suggests that endogenous protein catabolism doubled in diabetic rats.

When labelled urinary ASP was injected into rats, approximately 40% of the label was recovered as urinary ASP within 24 hr. This excretion rate was far higher than that after the injection of free leucine.

The rate of urinary excretion of ASP-form amino acids correlated with that of N^τ-methylhistidine in rats.

These results favor the hypothesis that urinary ASP reflects the catabolism of body proteins.     

Chemical Composition of Giant Cells Induced by Tunicamycin and Normal Mycelia of Penicillium citrinum

Growth of Penicillium citrinum was reduced in the presence of tunicamycin. Under this condition, reduction of yield of cell wall was greater than that of cellular protein.

Chitin content in the cell wall was several times higher in giant cells formed from conidia in the presence of tunicamycin than in normal mycelia, while reducing sugar content, presumably reflecting glucan content, did not significantly differ. Galactosamine, which was present in normal mycelia and absent in conidia, could not be detected in giant cells. The amino acid composition of the cell wall and whole cells of giant cells differed distinctly from that of normal mycelia.

Tunicamycin did not significantly inhibit the synthesis of DNA, RNA and protein as judged by incorporation of radioactive precursors, while cell wall synthesis, as judged by incorporation of radioactive N-acetylglucosamine, glucose and alanine into acid insoluble fraction, was inhibited by more than 40% in the presence of 10 μg/ml of tunicamycin. In fungi tunicamycin probably acts primarily as an inhibitor of cell wall glycoprotein synthesis and not of chitin synthesis.

Cyclic nucleotides level also differed distinctly between giant cells and mycelia.     

Effect of sulfite on compositional changes of cell constituents in germinating spores ofAdiantum capillus-veneris L.

Spores ofAdiantum capillus-veneris L., which were preincubated at 25 C for three days in the dark, were suspended in 1 mM potassium phosphate buffer, pH 6.0, and incubated for four days under continuous red light in the presence or absence of 3 mM sulfite. At day 0, 2 and 4 of the incubation, contents of cell constituents were determined. Total lipid content decreased continuously over four days of incubation in the absence of sulfite or in the presence of 3 mM sulfate. In contrast, when sulfite was added to the medium, the decrease stopped after day 2. The content of insoluble glucan increased markedly between day 2 and 4 in the medium without sulfite, whereas it decreased continuously for four days in the medium containing sulfite. The protein content decreased promptly by day 2, but its decrease was delayed when 3 mM sulfite was added to the medium. The content of amino acids also decreased by day 2, but it increased thereafter in the absence of sulfite or in the presence of 3 mM sulfate. In the presence of sulfite, however, the content continued to decrease until day 4. The results indicate that 3 mM sulfite in the incubation medium depressed the utilization of reserve lipid and protein, the synthesis of insoluble glucan and the increase of amino acid pool sizes in fern spores.     

Partial Purification and Some Properties of Aggregation Factor from Pronase-Susceptible Floe Forming Flavobacterium Strain B

Globomycin inhibited the incorporation of [¹⁴C]diaminopimeric acid (Dap) into the cold 5% TCA insoluble fraction of Escherichia coli H2143 at higher concentrations than the minimum inhibitory concentration (MIC).

One-sixth or -seventh molecules of the lipoprotein were found per one molecule of N-acetyl glucosamine (GlucNAc) or Dap in globomycin-treated cells as compared with one-twelfth or -thirteenth molecules in normal cells. Among globomycin-resistant cells isolated, one-tenth were lipoprotein-less mutants and they showed a slightly swollen form and leaked RNase into the medium. It was interesting that spheroplast formation of the mutants in the presence of the antibiotic was not observed even at a high concentration.     

Studies on Cell Wall of Alkalophilic Bacillus

Cell walls of alkalophilic Bacillus No. C-125 and No. A-59 which grew in different pH conditions were prepared and analyzed. In the walls from cells grown at pH 10.3 (pH 10.3-cell wall) and the walls from cells grown at pH 7.5 (pH 7.5-cell wall) of the alkalophilic bacilli, the contents of neutral sugar and phosphorus were low as compared with those of Bacillus subtilis 6160, while uronic acid and amino acids were abundant. The uronic acid content of the pH 10.3-cell walls was higher than that of the pH 7.5-cell walls in both strains. The insoluble fraction (peptidoglycan) of cell walls of Bacillus No. C-125 consisted of muramic acid, glutamic acid, alanine, diaminopimelic acid and glucosamine as in neutrophilic bacilli. In the TCA soluble fraction of pH 10.3-cell walls of Bacillus No. C-125, uronic acid was a polymer of glucuronic acid containing a small amount of hexosamine, and 2/3 of the ninhydrin positive material was glutamic acid which was derived mainly from poly γ-L-glutamic acid.     

Production of high-quality edible protein from Candida yeast grown in continuous culture

Candida utilis NRRL Y-900 was grown in aerobic continuous culture with cane molasses as the source of the growth-limiting carbon. At 1% reducing sugar in the chemostal (10 liter working volume) feed medium, addition of Zn (25μM) to a minimal salts medium resulted in an increase in the biomass productivity of the chemostat from 1.7 to 2.6 g/liter/hr with a growth yield of 0.55 g dry biomass/g reducing sugar utilized at D_max. On the average, the yeast biomass was 50–55% protein. At S_R > 2% sugar, the biomass productivity was limited by the oxygen supply. With O₂-supplemented aeration (at S_R = 4.2%)the maximum biomass productivity Was 7.25 g/liter/hr. Aerobic ethanol production was not observed. A highquality undenatured protein fraction was isolate from the yeast homogenate by isoelectric precipitation at pH 4.5. Contaminating nucleic acid was removed as an insoluble complex by chelation with an organic cation (cetavlon). The final protein product contained about 3% RNA (DWB) and was suitable for use as a food additive.     

Role of carbon dioxide in germination of spores of Streptomyces viridochromogenes

CO₂ in required continuously during germination of Streptomyces viridochromogenes spores. Spores incubated in a defined germination medium in the absence of CO₂ remain phase bright and do not release spore carbon. In the presence of CO₂, the spores initiate germination accompanied by loss of refractility and spore carbon. The CO₂ requirement is replaced by oxaloacetate or a mixture of tricarboxylic acid cycle (TCA) intermediates. Labeled CO₂ is taken up by germinating spores, and is incorporated into protein and RNA. TCA cycle intermediates and related amino acids contain most of the acid-soluble label following short term exposures of germinating spores to ¹⁴CO₂. TCA cycle inhibitors repress germination and ¹⁴CO₂ uptake whereas folic acid antagonists do not. The results indicate that CO₂ is incorporated into oxaloacetate which is converted to biosynthetic intermediates required for germination. Operation of the TCA cycle appears to be essential for spore germination. The conclusion is reached that CO₂ is required during germination in order to maintain the cycle by an anaplerotic reaction.Abbreviations SN sucrose-nitrate medium - TX buffer Trisbuffer pH 7.3 containing-Triton X-100 - DGM defined germination medium - TX salts TX buffer plus Mg and Ca ions - TA trichloroacctic acid - TCA tricarboxylic acid