Dynamics of the distribution of labeled etimizol in brain cell structures

The time course of 14C-etimizol distribution in cell structures of the rat cerebral cortex was studied. Two minutes after intraperitoneal injection etimizol penetrates brain cells. At this time the greater portion of the drug is found in cytosol. In five minutes the maximal part of the label gets bound with cell nuclei and microsomal protein fraction extracted with 0.14 M NaCl. These two cell fractions show the highest radioactivity throughout the entire observation period (up to 7 days). Since these fractions are reported to be capable of calcium accumulation, a suggestion is made that metabolic effects of etimizol are likely to be related to calcium metabolism.     

The uptake and subcellular localization of the peptide antibiotic edeine a in HeLa cells in suspension culture

The uptake of [¹⁴C]thymidine, [¹⁴C]uridine and [¹⁴C]leucine by HeLa cells incubated in the presence of 1.52 μg/ml edeine A is inhibited by 7.5, 0 and 4%, respectively. Though edeine A has no gross cytopathic effect on HeLa cells, the peptide antibiotic enters the cells and h after addition to cell cultures is found in the nuclei. After 6 h of incubation, the highest intracellular concentration of edeine is located in the nuclear fraction, but, after 12 h, a higher proportion is in the post-mitochondrial supernatant fraction where it is associated with protein components in the range of molecular weights of 20 000 and 9 500 D. In the nucleus most of the [¹⁴C]edeine is bound to the chromatin fraction after 2 h of incubation. Exhaustive deoxyribonuclease digestion of the chromatin fraction releases all the radioactivity into one ultraviolet absorbing peak, which sediments to a density of 20% sucrose. Exhaustive ribonuclease digestion of the chromatin fraction releases all the radioactivity into two ultraviolet absorbing peaks which sediment to a density of 20 and 40%, respectively; subsequent proteolytic digestion of the RNAse-treated chromatin fraction frees about 70% of the edeine A from the ultraviolet absorbing peaks. This suggests that intranuclear edeine A associates with proteins in the chromatin. The radioactivity was recovered from the enzymatically digested chromatin fractions and characterized as biologically active edeine A.     

A chemical and autoradiographic study of cellulose synthesis during the encystment of Acanthamoeba castellanii

When cells of Acanthamoeba castellanii are placed in a non-nutrient medium, they differentiate into cysts which possess cellulosic walls. In the present study, the source of the glucosyl unit for cyst wall cellulose was investigated by following the encystment of trophozoites grown in the presence of ¹⁴C-labeled fatty acids (uniformly labeled palmitate or oleate) or [3-³H]glucose. Cells were fractionated at the beginning and after 30 hr of encystment using a modified Schmidt-Tannhauser procedure. In cells grown on fatty acids, 90% of the labeled material was in the lipid fractions both before and after encystment with the total amount of label/cell changing very little. Both partial and complete acid hydrolysis of the glycogen of the acidsoluble fraction and the alkali-insoluble residue of the cyst wall indicated that the glucose of both fractions was not radioactive, although Acanthamoeba is known to have a functional glyoxylate pathway.Fractionation data of cells grown on [³H]glucose indicated a sevenfold increase in radioactivity in the wall insoluble fraction and a fivefold decrease in the acid-soluble fraction with the cpm/cell of the other fractions changing very little after 30 hr of encystment. Approximately 70% of the ³H-labeled material was recovered as glucose from the 30-hr wall insoluble fraction following complete acid hydrolysis. The specific radioactivity of glucose in the cyst wall insoluble fraction was the same as that of glycogen glucose isolated from the acid soluble fraction of trophozoites. Electron microscopic autoradiography showed that the majority of nonlipid radioactivity was due to glycogen in trophozoites. Autoradiograms failed to reveal Golgi bodies or any particular region of the cell as being the specialized site of cellulose synthesis. The results of the fractionation and autoradiographic studies are consistent with the concept that glycogen is a precursor of cyst wall cellulose, and that glucosyl units of glycogen and/or other glucose derivatives are converted to cellulose without significant dilution under the experimental conditions used.     

Evidence for a secretion of thyroxine by isolated hog thyroid cells

Isolated thyroid cells prepared from hog thyroid glands by tryptic dispersion were incubated with ¹³¹I⁻ for 1–6 h. Free [¹³¹I]thyroxine was identified in the incubation medium by three chromatographic methods. Neither [¹³¹I]iodotyurosines nor [¹³¹I]triiodothyronine were detected. The [¹³¹I]thyroxine released in the medium by 100 μl of cells (packed cell volume) after a 6-h incubation period amounted to 1.16% (S.E. = ± 0.39) of the total radioactivity. The medium [¹³¹I]thyroxine represented 15–25% of the total [¹³¹I]thyroxine synthesized during the 6 h of incubation. Thyrotropin, 1–60 munits/ml, increased the medium [¹³¹I]thyroxine content 2–4 fold. Dibutyryl cyclic AMP mimicked the effect of thyrotropin. The amount of medium [¹³¹I]thyroxine was strictly related to the amount of incubated cells but was independent of the volume of the incubation medium. When prelabeled cells were incubated in the presence of methimazole the increase in medium [¹³¹I]thyroxine was quantitatively related to a decrease in the intracellular [¹³¹I]thyroxine. Addition of dinitrotyrosine, an inhibitor of the deiodinase activity, induced the release of iodotyrosines in the incubation medium. That the incubation supernatant of isolated thyroid cells did contain free thyroxine but no iodotyrosines suggests that the normal mechanisms of proteolysis of thyroglobulin and deiodination of iodotyrosines inside the cells are preserved. From these data, it was concluded that the thyroxine release by isolated cells represents a real secretion.     

Resistance of Escherichia coli to tetracyclines

1. A strain of Escherichia coli highly resistant to chlortetracycline and partially cross-resistant to tetracycline has been isolated. 2. The nitro-reductase system of the resistant cells was inhibited to a smaller extent by chlortetracycline than was the corresponding enzyme of sensitive cells. 3. The incorporation of leucine in vitro into the ribosomal protein of cell-free preparations from sensitive and resistant cells was equally inhibited by chlortetracycline. 4. Resistant cells accumulated much less chlortetracycline and tetracycline than did sensitive cells when both were cultured in the presence of these drugs. 5. The uptake of tetracycline by both sensitive and resistant E. coli was dependent on the presence of glucose in the medium. 6. Fractionation of cells cultured in medium containing [¹⁴C]chlortetracycline indicated that the largest proportion of radioactivity in sensitive cells was in the fraction consisting mainly of cell-wall material. There was no concentration of radioactivity in any one fraction of the resistant cells. 7. No evidence could be obtained for a specific tetracycline-excretion system in the resistant cells. 8. The significance of these results in relation to current theories of the antibiotic action of and resistance to the tetracycline drugs is discussed.     

Studies on the Induced Synthesis of Maleate cis-trans Isomerase by Malonate

Malonate added to peptone-meat extract medium has been shown to induce maleate cis-trans isomerase in Alcaligenes faecalis IB-l4. This enzyme played an indispensable role in the enzymatic production of L-aspartic acid from maleic acid and ammonia.

Though malonate in the medium inhibited the growth of A. faecalis IB-14 to some extent, cells grown in the medium containing 10^-1 M of malonate showed the highest level of the enzyme activity. Specific activity of the enzyme of malonate-grown cells was approximately ten times as strong as that of maleate-grown cells, while, in basal mediumgrown cells, cis-trans isomerization of maleate did not occur at all. Maleate was utilized not only as carbon source of the cell growth but also as inducer for the formation of maleate cis-trans isomerase. On the other hand, malonate was not utilized as carbon source and the metabolism of it within the cell was rather restricted within narrow limits. Thus it was concluded that malonate was a gratuitous inducer for the formation of the enzyme, while maleate, which was considerably metabolized, was normal inducer.

It was demonstrated that most of radioactivity of l-C¹⁴-malonate taken up by cells was localized in particle fraction which sedimented at 105,000×g for 120 minutes, whereas radioactivity of 1-4-C¹⁴-maleate was uniformly distributed in both particulate and soluble fractions. Maleate cis-trans isomerase activity, in turn, was detected exclusively in soluble fraction in both malonate and maleate induced cells.     

The Uptake and Fractional Distribution of Differentially Labeled Indoleacetic Acid in Light Grown Stems

The uptake of exogenously applied indoleacetic acid (IAA) by light grown stems of bean (Phaseolus vulgaris L. cv. Red kidney) and pea (Pisum sativum L. cv. Alaska) was examined. The IAA was labeled in the 1 and 2 side chain positions with ¹⁴C and the 5 ring position with ³H. The distribution of label in the sections was analyzed by recording the elution into water, ethanol and 1.0 N NaOH, and the amount in the insoluble residue also recorded. Total uptake consisted of a rapid uptake for about 1 h followed by continued uptake at a slower rate for 24 h to give a radioactive concentration in the tissues four to five times, that of the external solution. Most of the radioactivity was initially extractable by water, later by ethanol. With IAA-2-¹⁴C there was a slow increase in radioactivity in NaOH and residue fractions but with IAA-1-¹⁴C most of the radioactivity was present in insoluble residue at times longer than 3 h. From the different residue patterns estimates of the extent of decarboxylation of the IAA were made. The radioactivity in the tissues was largely IAA after 1 h and the content increased until 6 h but there after there was little further increase. The water fraction initially contained the most IAA but by 24 h most IAA was found in the NaOH fraction in bean and the ethanol fraction in pea. The NaOH fraction was the only fraction in which the IAA content continually increased.     

Incorporation of Tritium from Thymidine-methyl-H3 into DNA,RNA, Lipids and Protein in Logarithmic and Stationary Phase Tetrahymena pyriformis,Strain W*

SYNOPSIS. Numerous reports may be found in the literature on cytoplasmic and non-DNA utilization of tritium from H³-thymidine. Such reports underscore the need to clarify the metabolic fate of H³-thymidine. This investigation outlines the fate of thymidinemethyl-H³ (TMH³) in logarithmic phase and stationary phase Tetrahymena pyriformis, strain W. Isotope identification by liquid scintillation spectrometry in chemically derived fractions of log phase cultures grown thruout the initial 48 hours of population growth with TMH³ revealed the majority of the radioactivity (90% of intracellular recovery) to be in the DNA fraction. The remainder of the intracellular label was recovered in the acid soluble fraction, lipid fraction, and a small amount in the RNA and cell residue. On chromatographs, tritium appeared only in the thymine moiety of the nucleic acid derivatives. Hence in dividing cells, thymidine-methyl-H³ is “essentially” specific for DNA at the dosage used although some incorporation into other compounds was detected. Fractionation of the lipid extract from the above experiment on a florisil column localized most of the label to the triglyceride and phospholipid fractions with some recovery in the cholesterol-esters. Similar scintillation counting of the various fractions of early stationary phase cells incubated for the last 48 hours of culture with TMH³ revealed limited tritium distribution in all fractions.     

ACETYLCHOLINE METABOLISM AND CHOLINE UPTAKE IN CORTICAL SLICES

Abstract— The uptake of [¹⁴C]choline was studied in cortical slices from rat brain after their incubation in a Krebs-Henseleit medium containing either 4.7 m m -KCl (low K), 25 m m -KCl (high K) or 25 m m -KCl without calcium (Ca free, high K). With 0.84 μ m -[¹⁴C]choline in the medium the uptake per gram of tissue was 0.62 nmol after incubation in low K medium, 1.13 nmol after incubation in high K medium and 0.78 nmol after incubation in a Ca free, high K medium. The differences caused by potassium were greater in fraction P₂ than in fractions P₁ and S₂. With 17 and 50 μ m -[¹⁴C]choline in the medium greater amounts of [¹⁴C]choline were taken up, but the effect of potassium on the uptake almost disappeared. The amount of radioactive material in fraction P₂ followed Michaelis-Menten kinetics with K _m values of 2.1 and 2.3 μ m after incubation in low and high K medium, respectively. Hemicholinium-3 only slightly inhibited choline uptake from a medium with 0.84 μ m -[¹⁴C]choline, but it abolished the extra-uptake induced by high K medium. The radioactivity in the slices consisted mainly of unchanged choline and little ACh was formed after incubation in low K medium, but after incubation in high K medium 50% of the choline taken up was converted into ACh. The hemicholinium-3 sensitive uptake of choline, the conversion of choline into ACh and the synthesis of total ACh, were stimulated about 7–8-fold by potassium. It is concluded that in cortical slices from rat brain all choline used for the synthesis of ACh is supplied by the high-affinity uptake system, of which the activity is geared to the rate of ACh synthesis.     

Metabolism of DL-Tryptophan-3-14C by the Fruitlets of Citrus aurantifolia

Developing lime fruit [Citrus aurantifolia (Christm.) Swingle] was supplied with dl-tryptophan-3-¹⁴C in a special medium. An incubation period of six hours was sufficient for the radioactivity to reach an equilibrium between the fruit tissue and the incubation medium. Analyses of the fruit tissue and the medium for acidic and neutral metabolites of tryptophan indicated the existence of indolic products. The auxin indole-3-acetic acid (IAA) was identified among the products by dry column chromatography and biological assay. Other acidic metabolites included indolepyruvic acid and an unidentified material. Neutral metabolites included indolealdehyde, indoleacetaldehyde, and two unidentified compounds. Biological activity in the Avena curvature test was obtained from extracted compounds which corresponded to IAA and indolepyruvic acid in the acidic fraction and indoleacetaldehyde in the neutral fraction. Radioactive tryptophan was also found in both the acidic and the neutral fractions due to its amphoteric nature. The experiment demonstrated the conversion of tryptophan to its indolic metabolites, including indole-3-acetic acid, in this Citrus tissue.     

Role of calcium ions in rapid effects of L-thyroxine on phosphoinositide metabolism in rat liver cells

The role of calcium ions in the L-thyroxine-induced initiation of hydrolysis of phosphatidylinositol 4,5-bisphosphate (PtdInsP₂) and also the course of releasing individual fractions of inositol phosphates and diacylglycerides (DAG) were studied in liver cells during early stages of the hormone effect. L-Thyroxine stimulated a rapid hydrolysis in hepatocytes of PtdInsP₂ labeled with [¹⁴C]linoleic acid and [³H]inositol mediated by phosphoinositide-specific phospholipase C. This was associated with accumulation of [¹⁴C]DAG, total inositol phosphates, [³H]inositol 1,4,5-trisphosphate (Ins1,4,5P₃) and [³H]inositol 1,4-bisphosphate (Ins1,4P₂). Elimination of calcium ions from the incubation medium of hepatocytes did not abolish the effect of thyroxine on the accumulation of [¹⁴C]DAG and total [³H]inositol phosphates. Preincubation of liver cells with TMB-8 increased the stimulatory effect of L-thyroxine on the accumulation of [¹⁴C]DAG. During the incubation of hepatocytes in the presence of the hormone the content of ¹⁴C-labeled fatty acids did not change. The L-thyroxineinduced accumulation of [³H]Ins1,4,5P₃ and [³H]Ins1,4P₂ did not depend on the presence of calcium ions in the incubation medium of the cells.     

Thein vitro activity of the microsomal fraction isolated from the spleen and the lymph nodes after immunization of the donor

The microsomal fraction from the spleen (after perfusion) of immunized rabbits incubated for 20 min at 37° C under usual conditions in the presence of energy sources incorporates¹⁴C-labelled amino acids both into the solubilized (by adding deoxycholate), and into the nonsolubilized part (15%). The cell supernatant incorporates under these conditions the¹⁴C-labelled amino acids into total proteins in the absence of microsomes but in a lower degree. The cell supernatant contains gamma globulin detectable by immunoelectrophoresis. Gamma globulin obtained by specific precipitation of the solubilized microsomal fraction with antigamma-globulin serum had an measurable radioactivity. The precipitate of gamma globulin obtained from the supernatant of the incubation medium in the same manner (after removing the microsomes) had a specific activity twice as high. On separating the microsomal fraction extract and the incubation medium supernatant on DEAE cellulose most fractions show on extinction maximum at 260 nm in the first case and at 280 nm in the second case. The microsomal fraction isolated from the spleen and lymph nodes of immunized pigs-48 and 72 h after revaccination, when incubatedin vitro, incorporated¹⁴C-labelled amino acids into total protein. After ultrasonic disintegration in 0.14m NaCl and filtration through a Sephadex G 25 column it is specifically precipitated with the antigammaglobulin serum. Gamma globulin isolated after incubation of the microsomal fraction had a measurable radioactivity. AntiHSA antibodies determined by adsorption on immunosorbent did not possess significant radioactivity. Only the concentrated supernatant of the incubation medium showed minute radioactivity of 75–94 counts/min /ml. The problem of investigating the formation of nascent specific antibodies on a subcellular levelin vitro during the early period of secondary response to the antigen is discussed, in particular the problem of their detection. An erratum to this article is available at .     

Efficient radiolabeling of mammalian cells using 111In-tagged liposomes

When indium-111 oxine labeled neutral liposomes were incubated with Chinese hamster V79 cells in the presence of 100 mM calcium, the cell-associated radioactivity increased approximately 75-fold over that observed in the absence of calcium. This is considerably higher (~20 times) than the cellular uptake obtained when these cells are incubated in the presence of ¹¹¹In-oxine alone. The highest uptake of radioactivity occurred when no bovine serum albumin was present in the medium, while as little as 0.001% of the protein greatly reduced the cell-liposome association. These efficient cell labeling conditions were not found to affect the survival of the cells.     

Control of fatty acid incorporation in membrane phospholipids. X-ray-induced changes in fatty acid uptake by tumor cells

Lymphosarcoma cells isolated from the spleens of tumor-bearing mice were used to study the effect of a low dose of X-rays (5 Gy) on the incorporation of [³H]palmitate and [¹⁴C]arachidonate into the lipids of the tumor cells. Palmitate and arachidonate were rapidly incorporated especially into the phospholipids of the cells. Between one and three hours after the start of the incubation with radiactive palmitate 80–90% of the label of the total lipids was found in the phospholipid fraction. Already after a few minutes of incubation with radioactive arachidonate, about 95% of the label was incorporated in the phospholipids. Irradiation caused a small but significant increase in the rate of fatty acid incorporation for both fatty acids. Concomitantly, a significantly increased amount of fatty acid was removed from the medium by the cells as a result of the irradiation, and the specific radioactivity of the free fatty acids in the cells was found to be enhanced. The radiation effect on the tumor cells could be mimicked by a hypotonic treatment. The magnitude of the radiation-induced stimulation of the fatty acid incorporation was similar to that of the hypotonically induced effect. Cells which had received a hypotonic treatment before the irradiation, did not show an additional radiation-induced enhancement of fatty acid incorporation into the cellular lipids. When the cells were incubated with serum albumin loaded with a relatively large (non-physiological) amount of complexed fatty acids (fatty acid: albumin molar ratio, $\text{ν}\text{= 3.7}$), no radiation effect on the fatty acid incorporation could be detected. It is concluded that hypotonic treatment, irradiation, and increased supply of exogenous fatty acids all lead to an enhanced flux of fatty acids into the cells. These results confirm our previous suggestion that the uptake of fatty acids through the plasma membrane is the rate-limiting step in the fatty acid incorporation into the phospholipids and that ionizing radiation is one of the means to enhance fatty acid uptake through the plasma membrane leading to an increased incorporation into the phospholipids.     

LOCALIZATION OF MACROMOLECULES IN ESCHERICHIA COLI : II. RNA and its Site of Synthesis

The distribution of RNA in cells of E. coli 15 T^-U^- labeled with uridine-H³ was studied by methods involving the analysis of radioautographic grain counts over random thin cross-sections and serial sections of the cells. The results were correlated with electron microscope morphological data. Fractionation and enzyme digestion studies showed that a large proportion of the label was found in RNA uracil and cytosine, the rest being incorporated as DNA cytosine. In fully labeled cells the distribution of label was found to be uniform throughout the cell. The situation remained unchanged when labeled cells were subsequently treated with chloramphenicol. When short pulses of label were employed a localization of a large proportion of the radioactivity became apparent. The nuclear region was identified as the site of concentration. Similar results were obtained when cells were exposed to much longer pulses of uridine-H³ in the presence of chloramphenicol. If cells were subjected to a short pulse of cytidine-H³, then allowed to grow for a while in unlabeled medium, the label, originally concentrated to some extent in the nuclear region, was found dispersed throughout the cell. The simplest hypothesis which accounts for these results is that a large fraction of the cell RNA is synthesized in a region in or near the nucleus and subsequently transferred to the cytoplasm.     

The migration of labeled phosphatidylcholine from the nuclear-associated endoplasmic reticulum to plasma membranes in L-929 cells

Summary The nuclear-associated endoplasmic reticulum of L-929 cells was found to contain the highest amount of labeled phosphatidylcholine after a 60 min incubation with¹⁴C-choline. Radioactivity was otherwise distributed relatively evenly among other membrane-containing organelles (nuclei, mitochondria, plasma membranes and endoplasmic reticulum membranes). During a 120 min chase following removal of isotope and addition of cold choline chloride, there was a considerable reduction in labeled phosphatidylcholine in the NER and nuclei. The decrease in radioactivity in these fractions was matched by an almost identical increase in the fraction containing mitochondria and plasma membranes. Separation of mitochondria and plasma membranes by centrifugation on discontinuous gradients showed that¹⁴C-choline labeled phosphatidylcholine appeared most rapidly in the plasma membranes. The results indicate that phospholipid molecules migrate within a short period of time from their site of synthesis in the NER to plasma membranes.     

Studies on the Secretion of Maize Root Cap Slime: IV. Evidence for the Involvement of Dictyosomes

The involvement of dictyosomes and their vesicles in secretion of slime by maize root cap cells is demonstrated by kinetic and organelle fractionation experiments using l-fucose as a specific marker for the secreted slime. Pulse-chase experiments show that l-[1-(3)H]fucose is incorporated into two distinct fractions of root cap cells. Incorporation into a water-soluble, ethyl alcohol-insoluble fraction of the homogenate has a peak at 20 minutes of chasing followed by rapid loss of label. Seventy per cent of the radioactivity in this fraction is secreted from the tissue during a 2-hour chase period. Incorporation of label from [(3)H]fucose into a water-insoluble fraction is kinetically different suggesting that in situ incorporation of label is occurring into the cell wall. Labeling of the water-soluble, ethyl alcohol-insoluble fraction with an (14)C-amino acid mixture differs from that of [(3)H]fucose. Thus, while release of the [(3)H]fucose-containing polymer begins after 10 to 15 minutes of chasing, the release of the (14)C-amino acid polymer is delayed an additional 5 to 10 minutes and occurs at a lower rate. Cesium chloride density gradient centrifugation of secreted material labeled with radioactivity from [(3)H]fucose indicates the presence of only one major component having a buoyant density similar to that of purified root cap slime (1.63 g cm(-3)). Sucrose density gradient centrifugation of homogenates of [(3)H]fucose-labeled root cap tissue shows that radioactivity in nondialyzable material occurs as a broad band between densities 1.12 and 1.18 g cm(-3) with a peak at density 1.15 g cm(-3), the same density at which dictyosomes were localized by electron microscopy. Autoradiography of organelle fractions shows that radioactivity was associated almost exclusively with dictyosomes.     

Protein synthesis in neuronal perikarya isolated from cerebral cortex of the immature rat

Abstract— Homogenates of neuronal perikarya isolated from the cerebral cortex of the 8-day-old rat were incubated with [³H]leucine, and the characteristics of the protein synthetic process were studied. Incorporation of leucine into protein was linear up to 90 min, proceeded optimally at pH 7.6 and was stimulated by K⁺ and NH₄⁺, unaffected by Li⁺ and inhibited by Na⁺. Puromycin, cycloheximide, RNAse, sulphhydryl blocking agents and phospholipase A exerted a pronounced inhibition, whereas chloramphenicol and phospholipase C had no effect. About 42 per cent of the total radioactive protein formed in the optimally fortified in uitro system was recovered in non-sedimentable form. Incorporation into the subcellular fractions of the neuronal perikarya increased steadily with increasing time of incubation. The microsomal fraction acquired the highest specific radioactivity (d.p.m./mg of protein), followed by the mitochondrial and the nuclear + cell debris fractions. The high-speed soluble fraction exhibited the lowest specific radioactivity. Although the addition of L-methionine to a suitably fortified incubation medium inhibited neuronal protein synthesis by about 80 per cent, the addition of D-methionhe, α-methyl-DL-methionine or L-tryptophan was relatively ineffective by comparison.     

On the role of protein phosphorylation in the ATP-dependent permeabilization of transformed cells

Incubation of transformed mouse fibroblasts with external ATP in alkaline medium low in divalent cations causes an increase in the permeability of the plasma membrane to nucleotides and other small molecules. Previous suggestions that the phosphorylation of a 44,000 dalton membrane protein is involved in this permeabilization process have been pursued. Fractionation of cells that had been incubated with [γ-³²P] ATP revealed that the labeled 44K phosphoprotein was found in both the membrane and mitochondrial fractions. Incubation of fractions isolated from unlabeled cells with [γ-³²P] ATP resulted in substantial formation of ³²P-44K in the mitochondrial fraction and less incorporation in the membrane fraction. The 44,000 dalton protein was identified as the α-subunit of mitochondrial pyruvate dehydrogenase by partial proteolytic mapping and immunological cross-reactivity with antibodies prepared against bovine pyruvate dehydrogenase. The phosphorylation of this protein in whole cells by externally added ATP is suppressed by inclusion in the incubation medium of carboxyatractyloside (CAT) and EDTA. These substances have no effect on ATP-dependent permeabilization, indicating that the phosphorylation of pyruvate dehydrogenase is not involved in this process.     

Accumulation of Maltose during Photosynthesis in Protoplasts Isolated from Spinach Leaves Treated with Mannose

When mannose was included in the enzyme incubation medium during the preparation of protoplasts from leaves of spinach, maltose was an early product of protoplast photosynthesis and, after 12 minutes, accounted for up to 15% of the ¹⁴C incorporated from ¹⁴CO₂. Maltose was not detected in protoplasts prepared in the normal enzyme medium. Rapid separation of cytoplasm and chloroplasts following exposure to ¹⁴CO₂ showed that maltose was present in both fractions. Direct measurements of [¹⁴C]maltose uptake indicated transport across the chloroplast envelope at rates similar to the transport of glucose. The source of maltose and site of its initial formation are discussed.