Yeast Nem1-Spo7 Protein Phosphatase Activity on Pah1 Phosphatidate Phosphatase Is Specific for the Pho85-Pho80 Protein Kinase Phosphorylation Sites

Pah1 is the phosphatidate phosphatase in the yeast Saccharomyces cerevisiae that produces diacylglycerol for triacylglycerol synthesis and concurrently controls the levels of phosphatidate used for phospholipid synthesis. Phosphorylation and dephosphorylation of Pah1 regulate its subcellular location and phosphatidate phosphatase activity. Compared with its phosphorylation by multiple protein kinases, Pah1 is dephosphorylated by a protein phosphatase complex consisting of Nem1 (catalytic subunit) and Spo7 (regulatory subunit). In this work, we characterized the Nem1-Spo7 phosphatase complex for its enzymological, kinetic, and regulatory properties with phosphorylated Pah1. The dephosphorylation of Pah1 by Nem1-Spo7 phosphatase resulted in the stimulation (6-fold) of phosphatidate phosphatase activity. For Pah1 phosphorylated by the Pho85-Pho80 kinase complex, maximum Nem1-Spo7 phosphatase activity required Mg²⁺ ions (8 mm) and Triton X-100 (0.25 mm) at pH 5.0. The energy of activation for the reaction was 8.4 kcal/mol, and the enzyme was thermally labile at temperatures above 40 °C. The enzyme activity was inhibited by sodium vanadate, sodium fluoride, N-ethylmaleimide, and phenylglyoxal but was not significantly affected by lipids or nucleotides. Nem1-Spo7 phosphatase activity was dependent on the concentrations of Pah1 phosphorylated by Pho85-Pho80, Cdc28-cyclin B, PKA, and PKC with k_cat and K_m values of 0.29 s⁻¹ and 81 nm, 0.11 s⁻¹ and 127 nm, 0.10 s⁻¹ and 46 nm, and 0.02 s⁻¹ and 38 nm, respectively. Its specificity constant (k_cat/K_m) for Pah1 phosphorylated by Pho85-Pho80 was 1.6-, 4-, and 6-fold higher, respectively, than that phosphorylated by PKA, Cdc28-cyclin B, and PKC.     

The enzymes forming isopentenyl pyrophosphate from 5-phosphomevalonate (mevalonate 5-phosphate) in the latex of Hevea brasiliensis

1. Phosphomevalonate kinase and 5-pyrophosphomevalonate decarboxylase have been purified from the freeze-dried latex serum of the commercial rubber tree Hevea brasiliensis. 2. The phosphomevalonate kinase was acid- and heat-labile and required the presence of a thiol to maintain activity. 3. The 5-pyrophosphomevalonate decarboxylase was relatively acid-stable and more heat-stable than the phosphokinase. 4. Maximum activity of the phosphokinase was achieved at pH 7.2 with 0.2mm-5-phosphomevalonate (K_m 0.042mm), 2.0mm-ATP (K_m 0.19mm) and 8mm-Mg²⁺ at 40°C. The apparent activation energy was 14.8kcal/mol. 5. Maximum activity of 5-pyrophosphomevalonate decarboxylase was achieved at pH5.5–6.5 with 0.1mm-5-pyrophosphomevalonate (K_m 0.004mm), 1.5mm-ATP (K_m 0.12mm) and 2mm-Mg²⁺. The apparent activation energy was 13.7kcal/mol. The enzyme was somewhat sensitive to inhibition by its products, isopentenyl pyrophosphate and ADP.     

C-reactive Protein Exists in an NaCl Concentration-dependent Pentamer-Decamer Equilibrium in Physiological Buffer

C-reactive protein (CRP) is an acute phase protein of the pentraxin family that binds ligands in a Ca²⁺-dependent manner, and activates complement. Knowledge of its oligomeric state in solution and at surfaces is essential for functional studies. Analytical ultracentrifugation showed that CRP in 2 mm Ca²⁺ exhibits a rapid pentamer-decamer equilibrium. The proportion of decamer decreased with an increase in NaCl concentration. The sedimentation coefficients s_20,w⁰ of pentameric and decameric CRP were 6.4 S and in excess of 7.6 S, respectively. In the absence of Ca²⁺, CRP partially dissociates into its protomers and the NaCl concentration dependence of the pentamer-decamer equilibrium is much reduced. By x-ray scattering, the radius of gyration R_G values ranged from 3.7 nm for the pentamer to above 4.0 nm for the decamer. An averaged K_D value of 21 μm in solution (140 mm NaCl, 2 mm Ca²⁺) was determined by x-ray scattering and modeling based on crystal structures for the pentamer and decamer. Surface plasmon resonance showed that CRP self-associates on a surface with immobilized CRP with a similar K_D value of 23 μm (140 mm NaCl, 2 mm Ca²⁺), whereas CRP aggregates in low salt. It is concluded that CRP is reproducibly observed in a pentamer-decamer equilibrium in physiologically relevant concentrations both in solution and on surfaces. Both 2 mm Ca²⁺ and 140 mm NaCl are essential for the integrity of CRP in functional studies and understanding the role of CRP in the acute phase response.     

The metabolism of cyclic nucleotides in the guinea-pig pancreas. Cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase

Both cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase were recovered mainly from the supernatant fractions of guinea-pig pancreas, but a higher proportion of the activity of the former was associated with the pellet fractions. The activities in the supernatant were not separated by gel filtration, but were clearly separated by subsequent chromatography on an anion-exchange resin. The activities of cyclic AMP phosphodiesterase and cyclic GMP phosphodiesterase had high-affinity (K_m 6.5±1.1μm and 31.9±3.9μm respectively) and low-affinity (K_m 0.56±0.05mm and 0.32±0.03mm respectively) components. The activity of neither enzyme was affected by the pancreatic secretogens, cholecystokinin-pancreozymin, secretin and carbachol. Removal of ions by gel filtration resulted in a marked reduction in cyclic nucleotide phosphodiesterase activity, which could be restored by addition of Mg²⁺. Mn²⁺ (3mm) was as effective as Mg²⁺ (3mm) in the case of cyclic AMP phosphodiesterase, but was less than half as effective in the case of cyclic GMP phosphodiesterase. The metal-ion chelators, EDTA and EGTA, also decreased activity. Ca²⁺ (1mm) did not affect the activity of cyclic nucleotide phosphodiesterase when the concentration of Mg²⁺ was 3mm. At concentrations of Mg²⁺ between 0.1 and 1mm, 1mm-Ca²⁺ was activatory, and at concentrations of Mg²⁺ below 0.1mm, 1mm-Ca²⁺ was inhibitory. These results are discussed in terms of the possible significance of cyclic nucleotide phosphodiesterase in the physiological control of cyclic nucleotide concentrations during stimulus–secretion coupling.     

Stable changes to calcium fluxes in mitochondria isolated from rat livers perfused with α-adrenergic agonists and with glucagon

1. Human uterine cervical stroma was found to contain a Ca²⁺-independent neutral proteinase against casein and N-benzoyl-dl-arginine p-nitroanilide (Bz-dl-Arg-Nan). This enzyme was tightly bound to an insoluble material (20000g pellet) and was solubilized by high concentrations of NaCl or KCl. High concentrations of them in the reaction system, however, inhibited reversibly the activity of this enzyme. 2. The neutral proteinase was partially purified by extraction with NaCl, gel filtration on Sephadex G-200 and affinity chromatography on casein–Sepharose. 3. The optimal pH of this partially purified enzyme was 7.4–8.0 against casein and Bz-dl-Arg-Nan. The molecular weight of the enzyme was found to be about 1.4×10⁵ by gel filtration on Sephadex G-200. 4. The enzyme was significantly inhibited by di-isopropyl phosphorofluoridate (0.1mm). High concentration of phenylmethanesulphonyl fluoride (5mm), 7-amino-1-chloro-3-l-tosylamidoheptan-2-one (0.5mm), antipain (10μm) or leupeptin (10μm) was also found to be inhibitory, but chymostatin (40μg/ml), soya-bean trypsin inhibitor (2.5mg/ml), human plasma (10%, v/v), p-chloromercuribenzoate (1mm), EDTA (10mm) and 1-chloro-4-phenyl-3-l-tosylamidobutan-2-one (1mm) had no effect on the enzyme. 5. The neutral proteinase hydrolysed casein, Bz-dl-Arg-Nan and heat-denatured collagen, but was inactive towards native collagen and several synthetic substrates, such as 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-d-Arg, 3-carboxypropionyl-Ala-Ala-Ala p-nitroanilide and 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-d-Arg, and also proteoglycan. The enzyme did not act as a plasminogen activator. 6. These properties suggested that a neutral proteinase in the human uterine cervix was different from enzymes previously reported.     

Rapid stimulation by vasopressin, oxytocin and angiotensin II of glycogen degradation in hepatocyte suspensions

1. The hormonal control of glycogen breakdown was studied in hepatocytes isolated from livers of fed rats. 2. Glucose release was stimulated by [8-arginine]vasopressin (10pm–10nm), oxytocin (1nm–1μm), and angiotensin II (1nm–0.1μm). These responses are all at least as sensitive to hormone as is glucose output in the perfused rat liver. 3. The effect of these three hormones on glucose release was critically dependent on extracellular Ca²⁺, unlike that of glucagon. Half-maximal restoration of the vasopressin response occurred if 0.3mm-Ca²⁺ was added back to the incubation medium. 4. Glycogen breakdown was more than sufficient to account for the glucose released into the medium, in the absence or presence of hormones. Lactate release by hepatocytes was not affected by vasopressin, but was inhibited by glucagon. 5. If Ca²⁺ was omitted from the extracellular medium, vasopressin stimulated glycogenolysis, but not glucose release. 6. The phosphorylase a content of hepatocytes was increased by vasopressin, oxytocin and angiotensin II; minimum effective concentrations were 0.1pm, 0.1nm and 10pm respectively. This response was also dependent on Ca²⁺. 7. These results demonstrate that hepatocytes can respond to low concentrations of vasopressin and angiotensin II, i.e. these effects are likely to be relevant in the intact animal. The role of extracellular Ca²⁺ in the effects of these hormones on hepatic glycogenolysis and glucose release is discussed.     

Light and calcium interactions in chlorella inhibited by sodium chloride

Analysis of NaCl toxicity in Chlorella sorokiniana showed decreased growth rates, increased dry weight per cell, increased intracellular Na⁺ and Cl⁻, more total chlorophyll per cell, a decreased chlorophyll a to chlorophyll b ratio, increased rates of O₂ evolution, and decreased rates of CO₂ fixation when the extracellular concentration of NaCl was increased from zero to 0.3 m. Cultures did not grow at concentrations greater than 0.3 m NaCl unless 10 mm calcium salts were present. Inclusion of that concentration of Ca²⁺ extended the tolerance to 0.5 m NaCl before growth stopped. Increasing the light intensity from 1.2 to 9.4 mw/cm² increased growth rates for cultures in 0.10 to 0.45 m NaCl. At 14 mw/cm² added Ca²⁺ reduced growth rates of cultures in 0.3 m NaCl compared to controls without added Ca²⁺. Maximal growth rates for cultures in NaCl media were achieved by addition of 10 mm CaSO₄ and maintenance of the light intensity at 9.4 mw/cm². The maximal growth rate of the organism was 9.6 doublings/day achieved at 2.7 mw/cm² for control cultures. In 0.3 m NaCl the growth rate was 4.3 doublings/day at 2.7 mw/cm² and 8.2 doublings/day at 9.4 mw/cm² with 10 mm CaSO₄ added.     

Chronic Exposure to Excess Nutrients Left-shifts the Concentration Dependence of Glucose-stimulated Insulin Secretion in Pancreatic β-Cells

Hyperinsulinemia (HI) is elevated plasma insulin at basal glucose. Impaired glucose tolerance is associated with HI, although the exact cause and effect relationship remains poorly defined. We tested the hypothesis that HI can result from an intrinsic response of the β-cell to chronic exposure to excess nutrients, involving a shift in the concentration dependence of glucose-stimulated insulin secretion. INS-1 (832/13) cells were cultured in either a physiological (4 mm) or high (11 mm) glucose concentration with or without concomitant exposure to oleate. Isolated rat islets were also cultured with or without oleate. A clear hypersensitivity to submaximal glucose concentrations was evident in INS-1 cells cultured in excess nutrients such that the 25% of maximal (S_0.25) glucose-stimulated insulin secretion was significantly reduced in cells cultured in 11 mm glucose (S_0.25 = 3.5 mm) and 4 mm glucose with oleate (S_0.25 = 4.5 mm) compared with 4 mm glucose alone (S_0.25 = 5.7 mm). The magnitude of the left shift was linearly correlated with intracellular lipid stores in INS-1 cells (r² = 0.97). We observed no significant differences in the dose responses for glucose stimulation of respiration, NAD(P)H autofluorescence, or Ca²⁺ responses between left- and right-shifted β-cells. However, a left shift in the sensitivity of exocytosis to Ca²⁺ was documented in permeabilized INS-1 cells cultured in 11 versus 4 mm glucose (S_0.25 = 1.1 and 1.7 μm, respectively). Our results suggest that the sensitivity of exocytosis to triggering is modulated by a lipid component, the levels of which are influenced by the culture nutrient environment.     

Immobilized thylakoids in a cross-linked albumin matrix: effects of cations studied by electron microscopy, fluorescence emission, photoacoustic spectroscopy, and kinetic measurements

Immobilization of lettuce (Lactuca sativa) thylakoids has been performed by using glutaraldehyde and bovine serum albumin. Confirming previous reports, a stabilization of the O₂ evolution activity of the photosystem II (PSII) under storage and functional conditions has been observed. The present work is devoted to the role played by mono-and divalent cations, during the immobilization process itself, on the O₂ production. Four types of measurements have been employed: kinetic measurements, low temperature (77 K) fluorescence emission, photoacoustic (PA) spectroscopy, and electron microscopy observations. We show that the effect of glutaraldehyde is complex because it acts as an inhibitor, a stabilizing agent, and a cross-linking reactive. In the present studies, the thylakoids are immobilized within a polymeric insoluble albumin matrix. The highest activity yield and the best storage conditions are obtained when 0.15 mm Na⁺ (or K⁺), 1 mm Mg²⁺, and 0.1 mm Mn²⁺ are present in the resuspending media before the immobilization. Due to modifications of the ionic content during such a process, structural differences are observed on the stacking degree of thylakoids. No modification of the fluorescence and PA spectra after the immobilization are found. Furthermore, a correlation between activities and spectral changes have been shown: when the activities increase, the F₇₃₅ to F₆₉₅ ratio increases and the PA₆₇₆ to PA₄₄₀ ratio decreases.     

Deoxycytidylate deaminase. Properties of the enzyme from cultured kidney cells of baby hamster

dCMP deaminase was partially purified from BHK-21/C13 cells grown in culture. The molecular weight of the enzyme was estimated by gel filtration and gradient centrifugation to be 130000 and 115000 respectively. The enzyme had a pH optimum of 8.4. Its activity versus substrate concentration curve was sigmoid, the substrate concentration at half-maximal velocity being 4.4mm. dCTP activated the deaminase maximally at 40μm, gave a hyperbolic curve for activity versus dCMP concentration and a K_m value for dCMP of 0.91mm. dCTP activation required the presence of Mg²⁺ or Mn²⁺ ions. dTTP inhibited the deaminase maximally at 15μm; the inhibition required the presence of Mg²⁺ or Mn²⁺ ions. The enzyme was very heat-labile but could be markedly stabilized by dCTP at 0.125mm and ethylene glycol at 20% (v/v).     

The sedimentation behaviour of ribonuclease-active and -inactive ribosomes from bacteria

1. The `30s' and `50s' ribosomes from ribonuclease-active (Escherichia coli B) and -inactive (Pseudomonas fluorescens and Escherichia coli MRE600) bacteria have been studied in the ultracentrifuge. Charge anomalies were largely overcome by using sodium chloride–magnesium chloride solution, I 0·16, made 0–50mm with respect to Mg²⁺. 2. Differentiation of enzymic and physical breakdown at Mg²⁺ concentrations less than 5mm was made by comparing the properties of E. coli B and P. fluorescens ribosomes. 3. Ribonuclease-active ribosomes alone showed a transformation of `50s' into 40–43s components. This was combined with the release of a small amount of `5s' material which may be covalently bound soluble RNA. Other transformations of the `50s' into 34–37s components were observed in both ribonuclease-active and -inactive ribosomes at 1·0–2·5mm-Mg²⁺, and also with E. coli MRE600 when EDTA (0·2mm) was added to a solution in 0·16m-sodium chloride. 4. Degradation of ribonuclease-active E. coli B ribosomes at Mg²⁺ concentration 0·25mm or less was coincident with the formation of 16s and 21s ribonucleoprotein in P. fluorescens, and this suggested that complete dissociation of RNA from protein was not an essential prelude to breakdown of the RNA by the enzyme. 5. As high Cs⁺/Mg²⁺ ratios cause ribosomal degradation great care is necessary in the interpretation of equilibrium-density-gradient experiments in which high concentrations of caesium chloride or similar salts are used. 6. The importance of the RNA moiety in understanding the response of ribosomes to their ionic environment is discussed.     

Effect of ammonium and other ions on the glucose-dependent active transport of l-histidine in slices of rat-brain cortex

1. The influence of cations on the active transport into cells of rat-brain-cortex slices of l-histidine, an amino acid that is not metabolized by this tissue, has been studied. 2. Like other amino acids, l-histidine accumulated in the cells in the presence of glucose in concentrations up to over double that in the incubation medium. 3. The active transport of l-histidine was highest in a medium containing Ca²⁺ (3mm). The addition of K⁺ (27mm) led to a marked decrease in the intracellular concentration of l-histidine, though the oxygen uptake of the slices was higher. 4. The active l-histidine transport was inhibited by NH₄⁺. The inhibitory effect increased with the NH₄⁺ concentration, being about 25% at 8mm, 65% at 20mm, and 90% at 27 and 50mm. The oxygen uptake of the brain slices was depressed by only 25% by the highest NH₄⁺ concentration used, and less by lower concentrations.     

Separation and partial characterization of multiple ribonucleic Acid polymerases from soybean hypocotyl

Two major peaks of RNA polymerase activity have been routinely separated by diethylaminoethyl cellulose chromatography following solubilization from soybean (Glycine max L. var. Wayne) chromatin. The relative amounts of these two peaks depend upon the manner in which the chromatin is purified. Pelleting the chromatin through dense sucrose solutions results in not only a loss of total solubilized RNA polymerase activity but also a selective loss of the α-amanitin-sensitive form of the enzyme. Peak I elutes from a diethylaminoethyl cellulose column at a KCl concentration of approximately 0.27 m, is insensitive to α-amanitin and rifamycin, and has Mg²⁺ + Mn²⁺ optima of 5 mm and 1.25 mm, respectively. The enzyme is inhibited by KCl concentrations of about 0.03 m or greater. Peak II elutes from the column at a KCl concentration of approximately 0.35 m, is sensitive to α-amanitin, insensitive to rifamycin, and has Mg²⁺ + Mn²⁺ optima of 2 mm and 1.0 mm, respectively. Activity is inhibited by KCl concentrations of about 0.06 m or greater. Both enzymes prefer denatured calf thymus DNA, but peak II exhibits a stronger preference.     

Calcium Regulates Molecular Interactions of Otoferlin with Soluble NSF Attachment Protein Receptor (SNARE) Proteins Required for Hair Cell Exocytosis

Mutations in otoferlin, a C2 domain-containing ferlin family protein, cause non-syndromic hearing loss in humans (DFNB9 deafness). Furthermore, transmitter secretion of cochlear inner hair cells is compromised in mice lacking otoferlin. In the present study, we show that the C2F domain of otoferlin directly binds calcium (K_D = 267 μm) with diminished binding in a pachanga (D1767G) C2F mouse mutation. Calcium was found to differentially regulate binding of otoferlin C2 domains to target SNARE (t-SNARE) proteins and phospholipids. C2D–F domains interact with the syntaxin-1 t-SNARE motif with maximum binding within the range of 20–50 μm Ca²⁺. At 20 μm Ca²⁺, the dissociation rate was substantially lower, indicating increased binding (K_D = ∼10⁻⁹) compared with 0 μm Ca²⁺ (K_D = ∼10⁻⁸), suggesting a calcium-mediated stabilization of the C2 domain·t-SNARE complex. C2A and C2B interactions with t-SNAREs were insensitive to calcium. The C2F domain directly binds the t-SNARE SNAP-25 maximally at 100 μm and with reduction at 0 μm Ca²⁺, a pattern repeated for C2F domain interactions with phosphatidylinositol 4,5-bisphosphate. In contrast, C2F did not bind the vesicle SNARE protein synaptobrevin-1 (VAMP-1). Moreover, an antibody targeting otoferlin immunoprecipitated syntaxin-1 and SNAP-25 but not synaptobrevin-1. As opposed to an increase in binding with increased calcium, interactions between otoferlin C2F domain and intramolecular C2 domains occurred in the absence of calcium, consistent with intra-C2 domain interactions forming a “closed” tertiary structure at low calcium that “opens” as calcium increases. These results suggest a direct role for otoferlin in exocytosis and modulation of calcium-dependent membrane fusion.     

Partial Purification and Properties of d-Glucosamine 6-Phosphate N-Acetyltransferase from Phaseolus aureus

d-Glucosamine-6-P N-acetyltransferase (EC 2.3.1.4) from mung bean seeds (Phaseolus aureus) was purified 313-fold by protamine sulfate and isoelectric precipitation, ammonium sulfate and acetone fractionation, and CM Sephadex column chromatography. The partially purified enzyme was highly specific for d-glucosamine-6-P. Neither d-glucosamine nor d-galactosamine could replace this substrate. The partially purified enzyme preparation was inhibited up to 50% by 2 × 10⁻²m EDTA, indicating the requirement of a divalent cation. Among divalent metal ions tested, Mg²⁺ was required for maximum activity of the enzyme. Mn²⁺ and Zn²⁺ were inhibitory, while Co²⁺ had no effect on the enzyme activity. The pH optimum of the enzyme in sodium acetate and sodium citrate buffers was found to be 5.2. The effect of Mg²⁺ on the enzyme in sodium acetate and sodium citrate buffers was particularly noticeable in the range of optimum pH. Km values of 15.1 × 10⁻⁴m and 7.1 × 10⁻⁴m were obtained for d-glucosamine-6-P and acetyl CoA, respectively. The enzyme was completely inhibited by 1 × 10⁻⁴mp-hydroxymercuribenzoate, and this inhibition was partially reversed by l-cysteine; indicating the presence of sulfhydryl groups at or near the active site of the enzyme.     

The effect of all-trans-retinoic acid on the synthesis of epidermal cell-surface-associated carbohydrates

1. all-trans-Retinoic acid at concentrations greater than 10⁻⁷m stimulated the incorporation of d-[³H]glucosamine into 8m-urea/5% (w/v) sodium dodecyl sulphate extracts of 1m-CaCl₂-separated epidermis from pig ear skin slices cultured for 18h. The incorporation of ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected. 2. Electrophoresis of the solubilized epidermis showed increased incorporation of d-[³H]glucosamine into a high-molecular-weight glycosaminoglycan-containing peak when skin slices were cultured in the presence of 10⁻⁵m-all-trans-retinoic acid. The labelling of other epidermal components with d-[³H]glucosamine, ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected by 10⁻⁵m-all-trans-retinoic acid. 3. Trypsinization dispersed the epidermal cells and released 75–85% of the total d-[³H]glucosamine-labelled material in the glycosaminoglycan peak. Thus most of this material was extracellular in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 4. Increased labelling of extracellular epidermal glycosaminoglycans was also observed when human skin slices were treated with all-trans-retinoic acid, indicating a similar mechanism in both tissues. Increased labelling was also found when the epidermis was cultured in the absence of the dermis, suggesting a direct effect of all-trans-retinoic acid on the epidermis. 5. Increased incorporation of d-[³H]-glucosamine into extracellular epidermal glycosaminoglycans in 10⁻⁵m-all-trans-retinoic acid-treated skin slices was apparent after 4–8h in culture and continued up to 48h. all-trans-Retinoic acid (10⁻⁵m) did not affect the rate of degradation of this material in cultures `chased' with 5mm-unlabelled glucosamine after 4 or 18h. 6. Cellulose acetate electrophoresis at pH7.2 revealed that hyaluronic acid was the major labelled glycosaminoglycan (80–90%) in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 7. The labelling of epidermal plasma membranes isolated from d-[³H]glucosamine-labelled skin slices by sucrose density gradient centrifugation was similar in control and 10⁻⁵m-all-trans-retinoic acid-treated tissue. 8. The results indicate that increased synthesis of mainly extracellular glycosaminoglycans (largely hyaluronic acid) may be the first response of the epidermis to excess all-trans-retinoic acid.     

Glutamate, glutamine, aspartate, asparagine, glucose and ketone-body metabolism in chick intestinal brush-border cells

1. Suspensions of isolated chick jejunal columnar absorptive (brush-border) cells respired on endogenous substrates at a rate 40% higher than that shown by rat brush-border cells. 2. Added d-glucose (5 or 10mm), l-glutamine (2.5mm) and l-glutamate (2.5mm) were the only individual substrates which stimulated respiration by chick cells; l-aspartate (2.5 or 6.7mm), glutamate (6.7mm), glutamine (6.7mm), l-alanine (1 or 10mm), pyruvate (1 or 2mm), l-lactate (5 or 10mm), butyrate (10mm) and oleate (1mm) did not stimulate chick cell respiration; l-asparagine (6.7mm) inhibited slightly; glucose (5mm) stimulated more than did 10mm-glucose. 3. Acetoacetate (10mm) and d-3-hydroxybutyrate (10mm) were rapidly consumed but, in contrast to rat brush-border cells, did not stimulate respiration. 4. Glucose (10mm) was consumed more slowly than 5mm-glucose; the dominant product of glucose metabolism during vigorous respiration was lactate; the proportion of glucose converted to lactate was greater with 10mm- than with 5mm-glucose. 5. Glutamate and aspartate consumption rates decreased, and alanine and glutamine consumption rates increased when their initial concentrations were raised from 2.5 to 6.7 or 10mm. 6. The metabolic fate of glucose was little affected by concomitant metabolism of any one of aspartate, glutamate or glutamine except for an increased production of alanine; the glucose-stimulated respiration rate was unaffected by concomitant metabolism of these individual amino acids. 7. Chick cells produced very little alanine from aspartate and, in contrast to rat cells, likewise produced very little alanine from glutamate or glutamine; in chick cells alanine appeared to be predominantly a product of transmination of pyruvate derived from glucose metabolism. 8. In chick cells, glutamate and glutamine were formed from aspartate (2.5 or 6.7mm); aspartate and glutamine were formed from glutamate (2.5mm) but only aspartate from 6.7mm-glutamate; glutamate was the dominant product formed from glutamine (6.7mm) but aspartate only was formed from 2.5mm-glutamine. 9. Chick brush-border cells can thus both catabolize and synthesize glutamine; glutamine synthesis is always diminished by concomitant metabolism of glucose, presumably by allosteric inhibition of glutamine synthetase by alanine. 10. Proline was formed from glutamine (2.5mm) but not from glutamine (2.5mm)+glucose (5mm) and not from 2.5mm-glutamate; ornithine was formed from glutamine (2.5mm)+glucose (5.0mm) but not from glutamine alone; serine was formed from glutamine (2.5mm)+glucose (5mm) and from these two substrates plus aspartate (2.5mm). 11. Total intracellular adenine nucleotides (22μmol/g dry wt.) remained unchanged during incubation of chick cells with glucose. 12. Intracellular glutathione (0.7–0.8mm) was depleted by 40% during incubation of respiring chick cells without added substrates for 75min at 37°C; partial restoration of the lost glutathione was achieved by incubating cells with l-glutamate+l-cysteine+glycine.     

5-Oxoprolinase (l-Pyroglutamate Hydrolase) in Higher Plants: Partial Purification and Characterization of the Wheat Germ Enzyme

5-Oxoprolinase has been found to be widely distributed in higher plants. This enzyme catalyzes the ATP-dependent hydrolysis of 5-oxo-l-proline (l-pyrollidone carboxylate, l-pyroglutamate) to glutamate. The enzyme has been purified almost 60 fold from wheat germ (Triticum aestivum L). This enzyme requires a divalent cation, either Mn²⁺ or Mg²⁺, and a combination of both appears to be the most effective. There is also an absolute requirement for a monovalent cation best fulfilled by either NH₄⁺ or K⁺. The K_m for ATP is 0.4 mm and for 5-oxo-l-proline is 14 μm. A small amount of activity is observed when other purine nucleotides such as ITP and GTP replace ATP. The substitution of the pyrimidine nucleotides CTP and UTP for ATP yield almost completely inactive preparations. The enzyme appears to have an active sulfhydryl group since there is an increase in activity in the presence of dithioerythritol. Preincubation with reagents such as N-ethylmaleimide or iodoacetamide lead to complete inactivation. The presence of this enzyme leads to the speculation of the possible presence of a γ-glutamyl cycle in higher plants.     

Purification and Properties of Fructokinase from Developing Tubers of Potato (Solanum tuberosum L.)

Fructokinase has been purified from developing potato (Solanum tuberosum L.) tubers by a combination of hydrophobic interaction, affinity chromatography, and gel filtration. The protein has a native molecular mass of approximately 70 kD but is apparently a dimer. Ion-exchange chromatography and two-dimensional western blots resolved three major fructokinases, designated FK-I, FK-II, and FK-III in order of their elution from a Mono-Q column. Fructokinase activity proved labile when proteins were purified in the absence of fructose. Kinetically, FKs I, II, and III all have broad pH optima with peaks at about pH 8.5. The enzymes have a high specificity for fructose (K_m values ranging from 0.041 to 0.128 mm), and can utilize a range of nucleoside triphosphates. Unlike FKs I and II, FK-III is not inhibited by fructose concentrations in excess of 1 mm. MgADP inhibited activity of the three FKs (between 68 and 75% inhibition at 1.0 mm), whereas fructose 6-P caused inhibition at concentrations of 10 mm. There were no regulatory effects observed with a range of other metabolites. K⁺ (10 mm) activated FK-I by 4-fold and FKs II and III by only about 50%.