Purification of an Endogenous polynucleotide phosphorylase from Brevibacterium JM98A.

Polynucleotide phosphorylase was purified form Brevibacterium JM98A (ATCC 29895). Homopolynucleotides were arsenolysed in the order polyadenylate greater than polycytidylic acid greater than polyuridylic acid greater than polyguanylate. The products were ribonucleoside 5'-monophosphates.     

Coordination chemical studies on metalloenzymes. II. Kinetic behavior of various types of chelating agents towards bovine carbonic anhydrase.

In order to investigate the kinetics and mechanism of the removal of zinc ions from bovine carbonic anhydrase [EC 4.2.1.1] (BCA), several chelating agents with various stability constants were used to remove zinc from BCA. The second-order rate constants (kaap) of zinc removal from BCA were found to be in the following order; 2,6-pyridinedicarboxylic acid greater than 2-pyridinecarboxylic acid greater than 2,4-pyridinedicarboxylic acid greater than 2,3-pyridinedicarboxylic acid greater than or approximately 1,10-phenanthroline greater than or approximately 5-methyl-1,10-phenanthroline greater than 2,2'-bipyridine. With similar chelating agents the greater the stability constant, the faster was the rate of removal of zinc ions from BCA. With EDTA, trans-1,2-cyclohexanediaminetetraacetic acid, and nitrilotriacetic acid, the rate of zinc ion removal from the native enzyme was governed by the rate of spontaneous dissociation of zinc enzyme. The rate constants for the removal of zinc ions from BCA were governed by the affinity of the chelating agents for the metal ion and the conformation of the chelating agents. Based on these findings, reaction pathways for various chelating agents are proposed.     

Role of triglycerides in endothelial cell arachidonic acid metabolism

Arachidonic acid was incorporated into triglycerides by cultured bovine endothelial cells in a time- and concentration-dependent manner. At 75 microM or higher, more arachidonic acid was incorporated into triglycerides than into phospholipids. The triglyceride content of the cells increased as much as 5.5-fold, cytoplasmic inclusions appeared, and arachidonic acid comprised 22% of the triglyceride fatty acids. Triglyceride turnover occurred during subsequent maintenance culture; there was a 60% decrease in the radioactive arachidonic acid contained in triglycerides and a 40% decrease in triglyceride content in 6 hr. Most of the radioactivity was released into the medium as free fatty acid. The turnover of arachidonic acid, but not oleic acid in cellular triglycerides, decreased when supplemental fatty acid was added to the maintenance medium. Incorporation and turnover of radioactive arachidonic acid in triglycerides also was observed in human skin fibroblasts, 3T3-L1 cells, and MDCK cells. Other fatty acids were incorporated into triglycerides by the endothelial cells; the amounts after a 16-hr incubation with 50 microM fatty acid were 20:3 greater than 20:4 greater than 18:1 greater than 18:2 greater than 22:6 greater than 16:0 greater than 20:5. These findings indicate that triglyceride formation and turnover can play a role in the fatty acid metabolism of endothelial cells and that arachidonic acid can be stored in endothelial cell triglycerides.     

Lysis of Streptococcus mutans by hen egg white lysozyme and inorganic sodium salts.

Streptococcus mutans BHT was grown in a synthetic medium containing radioactive thymidine to monitor deoxyribonucleic acid release. Kinetic experiments demonstrated that although lysozyme alone could not liberate deoxyribonucleic acid, cellular deoxyribonucleic acid was liberated from lysozyme-treated cells by addition of low concentrations of inorganic sodium salts. When the salts were tested for their ability to dislodge cell-bound tritiated lysozyme, the extent of the initial release of enzyme by individual anions correlated with the anion potency for deoxyribonucleic acid liberation (SCN- greater than ClO4- greater than I- greater than Br- greater than NO3- greater than Cl- greater than F-), although the total amount of lysozyme dislodged did not correspond directly with cell lysis. Differences in the effectiveness of anions (SCN-, HCO3-, Cl- and F-) in potentiating cell lysis could be enhanced or minimized by varying the lysozyme, anion, and bacterial cell concentrations. As the anion concentration was increased for each enzyme concentration and cell concentration, the lysis increased, in some cases markedly, until maximum levels of released deoxyribonucleic acid were attained. The maximum levels of lysis of SCN- and HCO3- were similar and were greater than those for Cl- and F-. In addition, the maximum levels were observed to increase for each of the anions as the concentration of lysozyme increased.     

Lipid shape as a determinant of lipid composition in Clostridium butyricum. The effects of incorporation of various fatty acids on the ratios of the major ether lipids

The lipid composition of Clostridium butyricum is strongly influenced by the aliphatic chain compositions of the membrane lipids. Growth on cis-monounsaturated fatty acids in the absence of biotin was shown to affect the relative proportions of phosphatidylethanolamine, plasmenylethanolamine, and the glycerol acetal of plasmenylethanolamine most strongly, with smaller effects on the acidic lipids, phosphatidylglycerol and cardiolipin. The ratio of the glycerol acetal of plasmenylethanolamine to total phosphatidylethanolamine in cells grown on a series of fatty acids is shown to decrease in the following order; cis-vaccenic acid greater than or equal to oleic acid = C19-cyclopropane fatty acid greater than linoleic acid greater than petroselinic acid greater than elaidic acid greater than 14-methylhexadecanoic acid (anteiso-C17) greater than 12-methyltridecanoic acid (iso-C14). All fatty acids were extensively incorporated into the lipid acyl, alkenyl, and alkyl chains. There was considerable chain-elongation of the iso-C14 to iso-C16. The results are consistent with the hypothesis that the membrane lipid composition is strongly influenced by lipid shape and that the observed changes in lipid composition serve to stabilize the bilayer arrangement of the cell membrane.     

Characterization of GABAA receptor-mediated 36chloride uptake in rat brain synaptoneurosomes

gamma-Aminobutyric acid (GABA) receptor-mediated 36chloride (36Cl-) uptake was measured in synaptoneurosomes from rat brain. GABA and GABA agonists stimulated 36Cl- uptake in a concentration-dependent manner with the following order of potency: Muscimol greater than GABA greater than piperidine-4-sulfonic acid (P4S)greater than 4,5,6,7-tetrahydroisoxazolo-[5,4-c]pyridin-3-ol (THIP) = 3-aminopropanesulfonic acid (3APS) much greater than taurine. Both P4S and 3APS behaved as partial agonists, while the GABAB agonist, baclofen, was ineffective. The response to muscimol was inhibited by bicuculline and picrotoxin in a mixed competitive/non-competitive manner. Other inhibitors of GABA receptor-opened channels or non-neuronal anion channels such as penicillin, picrate, furosemide and disulfonic acid stilbenes also inhibited the response to muscimol. A regional variation in muscimol-stimulated 36Cl- uptake was observed; the largest responses were observed in the cerebral cortex, cerebellum and hippocampus, moderate responses were obtained in the striatum and hypothalamus and the smallest response was observed in the pons-medulla. GABA receptor-mediated 36Cl- uptake was also dependent on the anion present in the media. The muscimol response varied in media containing the following anions: Br- greater than Cl- greater than or equal to NO3- greater than I- greater than or equal to SCN- much greater than C3H5OO- greater than or equal to ClO4- greater than F-, consistent with the relative anion permeability through GABA receptor-gated anion channels and the enhancement of convulsant binding to the GABA receptor-gated Cl- channel.     

Fatty acid specificity of acyl-CoA synthetase in rat glomeruli

The fatty acid specificity of acyl-CoA synthetase in rat glomeruli for physiologically and pathologically important long-chain fatty acids was studied. The apparent Michaelis constants (Km) for substrate fatty acids increased in the order, linolenic less than linoleic less than eicosapentaenoic less than arachidonic less than oleic less than palmitic acid. The maximum velocities with these fatty acids decreased in the order, oleic greater than linoleic greater than palmitic (approximately equal to) linolenic greater than arachidonic greater than eicosapentaenoic acid. The syntheses of radioactive arachidonyl-CoA and palmitoyl-CoA from radioactive arachidonic and palmitic acid, respectively, were both inhibited by all fatty acids mentioned above including the substrate fatty acids, their inhibitory effects being inversely correlated with their apparent Km values. These results suggest that the enzyme in glomeruli has a unique specificity for fatty acids and that there is no arachidonic acid-specific acyl-CoA synthetase in glomeruli. The possible contribution of the glomerular enzyme with this specificity to the abnormal fatty acid levels in diabetic animals is discussed.     

Aromaticity at position 37 in human epidermal growth factor is not obligatory for activity.

The third disulfide loop (amino acids 33 to 42) of human epidermal growth factor (hEGF) encompasses the region of highest amino acid conservation among all of the EGF-like family of molecules. The importance of some of these highly conserved residues for the maintenance of biological activity, especially the aromatic amino acid tyrosine at position 37, has until now been considered essential on the basis of previous studies with the EGF-like molecule transforming growth factor alpha. Variants at the Tyr-37 position of hEGF were constructed by site-directed mutagenesis. The substituting amino acids were phenylalanine, histidine, serine, alanine, aspartic acid, arginine, and glycine. The variants were tested for their ability to competitively displace native [125I]hEGF from its receptor and to stimulate the protein-tyrosine kinase activity of the receptor; the order of efficacy of substituting amino acids was Phe greater than His greater than Ser greater than Ala greater than Asp greater than Arg greater than Gly in both assays. All were effective, with no or only moderate reduction in potency, in stimulating the incorporation of [3H]thymidine into acid-insoluble material of quiescent mouse A31 cells. Only Tyr-37----Ala, Tyr-37----Arg and Tyr-37----Gly were slightly less potent in the cell assay. Thus, neither tyrosine nor another aromatic amino acid at position 37 in hEGF is essential for full biological activity.     

Lipids of Leishmania promastigotes.

A chromatographic analysis of lipids of cultured promastigotes of Leishmania donovani, L. braziliensis, L. mexicana, L. tropica, L. enriettii, L. hertigi, L. adleri, and L. tarentolae showed that total lipids were 2--15% of dry wt, and neutral and polar lipids were 14--55% and 45--86% of total lipids. Major lipid classes were as follows: sterol ester, triacylglycerol, sterol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylinositol, phosphatidylcholine. Predominant fatty acids were 18:2 (n - 6) greater than 18:3 (n - 3) greater than 18:1 (n - 9) greater than 18:0 greater than 22:6 (n - 3) greater than 22:5 (n - 6) greater than 16:0 greater than 14:0 greater than 18:4 (n - 3) greater than 20:3 (n - 3). Some remarkable distributions of fatty acids among the phospholipid fractions were observed, as follows: diphosphatidylglycerol 18--33% 22:6 (n - 3); phosphatidylinositol 31--68% 18:1 (n - 9); phosphatidylcholine 13--41% 18:3 (n - 3). Alk-l-enyldiacyl glycerols, and alk-l-enylacyl and alkylacyl forms of phosphatidylethanolamine and of phosphatidylinositol were found, and their glyceryl ethers and fatty adehydes analyzed. Notable in the phosphatidylethanolamine of some leishmanias was a cyclopropane fatty acid (4--11%), identified as cis-9,10-methylene octadecanoic acid by chromatographic, and by mass and proton resonance spectrometric analyses. The comparative biochemistry of the cyclopropane fatty acid, characteristic of many prokaryotes, and of alpha-linolenic acid, characteristic of photosynthetic plants, are commented upon.     

Bile acid structure-activity relationship: evaluation of bile acid lipophilicity using 1-octanol/water partition coefficient and reverse phase HPLC

Two independent methods have been developed and compared to determine the lipophilicity of a representative series of naturally occurring bile acids (BA) in relation to their structure. The BA included cholic acid (CA), chenodeoxycholic acid (CDCA), ursodeoxycholic acid (UDCA), deoxycholic acid (DCA), hyodeoxycholic acid (HDCA), ursocholic acid (UCA), hyocholic acid (HCA), as well as their glycine and taurine amidates. Lipophilicity was determined using a 1-octanol/water shake-flask procedure and the experiments were performed at different pH and ionic strengths and at initial BA concentrations below their critical micellar concentrations (CMC) and the water solubility of the protonated form. The experimental data show that both the protonated (HA) and ionized (A-) forms of BA can distribute in 1-octanol, and consequently a partition coefficient for HA (logP' HA) and for A- (logP' A-) must be defined. An equation to predict a weighted apparent distribution coefficient (D) value as a function of pH and pKa has been developed and fits well with the experimental data. Differences between logP for protonated and ionized species for unconjugated BA were in the order of 1 log unit, which increased to 2 for glycine-amidate BA. The partition coefficient of the A- form increased with Na+ concentration and total ionic strength, suggesting an ion-pair mechanism for its partition into 1-octanol. Lipophilicity was also assessed using reverse phase chromatography (C-18-HPLC), and a capacity factor (K') for ionized species was determined. Despite a broad correlation with the logP data, some BA behaved differently. The logP values showed that the order of lipophilicity was DCA greater than CDCA greater than UDCA greater than HDCA greater than HCA greater than CA greater than UCA for both the protonated and ionized unconjugated and glycine-amidate BA, while the K' data showed an inversion for some BA, i.e., DCA greater than CDCA greater than CA greater than HCA greater than UDCA greater than HDCA greater than UCA. The logP data fitted well with other indirect measurements of BA monomeric lipophilicity such as albumin binding or accessible total hydrophobic surface area data calculated by energy minimization and molecular computer graphics. Differences between unconjugated and amidated BA are consistent with the presence of an amide bond and a lower pKa when pH dependence was studied. Capacity factors, on the other hand, were related to properties of BA micelles such as cholesterol-solubilizing capacity and membrane disruption, reflecting the BA detergency.(ABSTRACT TRUNCATED AT 400 WORDS)     

Maturation of membrane function: transport of amino acid by rat erythroid cells.

The membrane changes which occur during cellular maturation of erythroid cells have been investigated. The transport of alpha-aminoisobutyric acid, alanine, and N-methylated-alpha-aminoisobutyric acid have been studied in the erythroblastic leukemic cell, the reticulocyte, and the erythrocyte of the Long-Evans rat. The dependence of amino acid transport on extracellular sodium concentration was investigated. Erythrocytes were found to transport these amino acids only by Na-independent systems. The steady state distribution ratio was less than 1. Reticulocytes were found to transport alpha-aminoisobutyric acid and alanine by Na-dependent systems, but only small amounts of N-methylated-alpha-aminoisobutyric acid. Small amounts of these amino acids were transported by Na-independent systems. The steady state distribution ratio was greater than one for Na-dependent transport. The erythroblastic leukemia cell, a model immature erythroid cell, showed marked Na-dependence (greater than 90%) for alpha-aminoisobutyric acid and alanine transport, and greater than 80% for the Na-dependent transport of N-methyl-alpha-aminoisobutyric acid. The steady state distribution ratio for the Na-dependent transport was greater than 4. In the erythroblastic leukemic cell, at least three Na-dependent systems are present: one includes alanine and alpha-aminoisobutyric acid, but excludes N-methyl-alpha-aminoisobutyric acid; one is for alpha-aminoisobutyric acid, alanine and also N-methyl-alpha-aminoisobutyric acid; and one is for N-methyl-alpha-aminoisobutyric acid alone. In the reticulocyte, the number of Na-dependent systems are reduced to two: one for alpha-aminoisobutyric acid and alanine; one for N-methyl-alpha-aminoisobutyric acid. In the erythrocytes, no Na-dependent transport was found. Therefore, maturation of the blast cell to the mature erythrocyte is characterized by a systematic loss in the specificity and number of transport system for amino acids.     

Specific high affinity binding of lipoxygenase metabolites of arachidonic acid by liver fatty acid binding protein

Liver fatty acid binding protein (L-FABP) binds avidly the arachidonic acid metabolites, hydroperoxyeicosatetraenoic acids (HPETEs) and hydroxyeicosatetraenoic acids (HETEs). Binding of 15-[3H]HPETE was specific, saturable, reversible, and rapid. Protein specificity was indicated by the following order: L-FABP greater than bovine serum albumin greater than ovalbumin = beta-lactoglobulin greater than ribonuclease. Ligand specificity was evidenced by the following order of apparent competition: 15-HPETE greater than or equal to 5-HETE greater than or equal to 5-HPETE = oleic acid greater than 12-HETE greater than 12-HPETE greater than or equal to 15-HETE greater than prostaglandin E1 much greater than leukotriene C4 greater than prostaglandin E2 much greater than thromboxane B2 = leukotriene B4. Once bound, 15-HPETE was reversibly displaced. Ligand was recovered from the protein complex and confirmed to be 15-[3H]HPETE by TLC. L-FABP bound HPETE with a dissociation constant of 76 nM,5-HETE at 175 nM, and 15-HETE at 1.8 microM, and the reference fatty acids oleic acid at 1.2 microM and arachidonic acid at 1.7 microM. Thus, the affinity was approximately 16-fold greater for 15-HPETE, and 7-fold higher for 5-HETE, than for oleic acid. The need exists for studies of complexes of L-FABP with the HPETEs and HETEs in hepatocytes, especially since L-FABP has previously been associated with mitosis in normal hepatocytes, and shown to be the target protein of two liver carcinogens, and these arachidonic acid metabolites have been found to be able to modulate activities related to cell growth.     

Prostaglandin specific binding in hamster myometrial low speed supernatant.

A charcoal adsorption method was developed to measure specific prostaglandin binding in low speed supernates of hamster myometrial homogenates. This method was used to characterize and quantitate PGE-1-specific binding. The equilibrium binding constants and the concentration of specific PGE-1 binding sites were determined during the hamster estrous cycle. The apparent association constant for 12 different preparations was 1.16 plus or minus 0.08 times 10-9M-1. The concentration of PGE-1 specific binding sites was significantly higher on days 2 and 3 of the estrous cycle that it was on days 1 or 4. The competition for PGE-1 binding sites by PGE-2, PGF-2alpha, tpga-1 and various PGE-1 metabolites and derivatives was measured in hamster myometrial homogenates. Relative affinities of the natural prostaglandins for the PGE-1 binding sites, calculated by parallel line assay, were: PGE-2 greater than PGE-1 greater than PGA-1 greater than PGF-2alpha. For PGE-1 metabolites the relative affinities were: PGE-1 greater than 13,14-dihydro-PGE-1 greater than 13,14-dihydro-15-keto-PGE-1 greater than 15-keto-PGE-1. For the analogs and derivatives the compounds tested ranked as: 16,16-dimethyl-PGE-1 greater than PGE-1 methyl ester greater than 17-phenyl-18,19,20-trinor-PGE-1 greater than 15(S) 15-methyl-PGE-1 methyl ester. Arachidonic acid, bis-homo-gamma-linolenic acid and 7-oxa-13 prostynoic acid had relative affinities greater than 0.1 compared to PGE-1 equal 100. Indomethacin had a relative affinity of 0.4 compared to PGE-1.     

Identification and characterization of an anion-sensitive Mg2+-ATPase in pituitary secretory granule membranes

We have identified an anion-sensitive Mg2+-ATPase in adenohypophyseal secretory granule membranes. This enzyme is unaffected by sodium, ouabain, and calcium. By electron microscopic morphology, sedimentation properties, nucleotide substrate utilization, and marker enzyme studies, this activity is clearly shown to be intrinsic to the granule membranes. The kinetics for ATP saturation were complex, as curvilinear Lineweaver-Burk plots were obtained with 2 mM magnesium. However, an approach to linearity was obtained (Km for ATP, approximately 0.27 mM) with low concentrations of free magnesium. Many anions and anion-transport blockers significantly influenced enzyme activity. Stimulatory anions in decreasing order of potency were bisulfite greater than sulfite greater than isethionate greater than bicarbonate; Ka values were 2.5 mM for sulfite and 10.8 mM for bicarbonate. Acetate, borate, chloride, citrate, 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 2-(N-morpholino)ethanesulfonic acid, nitrite, oxalate, 1,3-piperazinediethanesulfonic acid, and sulfate were without major effect. Inhibitory anions in decreasing potency order were azide greater than thiocyanate greater than fluoride greater than nitrate. Anionic stimulation of the granule membrane Mg2+-ATPase linearized the Lineweaver-Burk plots by shifting the enzyme to its higher Km state. In addition, sulfite competitively reversed the produce inhibition exerted by ADP. Anion transport-blockers inhibited the enzyme; of those tested, the most potent was 4-acetamido-4-isothiocyano-stilbene-2,2'-disulfonic acid, with a Ki of 0.17 mM; pyridoxal phosphate, sulfisoxazole, and ethacrynic acid also inhibited enzyme activity. The protein-binding dye p-sulfobenzene-azo-o-sulfobenzene-azo-beta-naphthol-3,6-disulfonic acid, structurally similar to transport blockers, was a potent inhibitor, with a Ki of 2.8 mM. These data on pituitary secretory granule ATPase raise the possibility that the granule membranes may function in anion or proton transport, perhaps in relation to exocytosis and hormone secretion.     

Comparison of muscle proteinase activity among fish species

The effects of temperature during storage, portion of muscle and growth stage of fish on the activity level of carp muscle acid (cathepsin D, EC 3.4.23.5), neutral and alkaline proteinases were examined. Icing storage, freezer storage and portion of muscle did not affect each proteinase activity (P less than 0.05), but acid proteinase activity was affected by growth stage (P less than 0.05) and the level decreased from small to large fish. The activity levels of three kinds of proteinases were compared among species (P less than 0.05) and the following order was obtained. Acid proteinase, white croaker = rainbow trout = carp greater than sardine = common mackerel, sardine greater than lizardfish, common mackerel = lizardfish. Neutral proteinase, rainbow trout greater than carp = white croaker greater than lizardfish = sardine = common mackerel. Alkaline proteinase, rainbow trout = sardine greater than white croaker = carp = common mackerel greater than lizardfish.     

Selective incorporation of various C-22 polyunsaturated fatty acids in Ehrlich ascites tumor cells

Three 14C-labeled 22-carbon polyunsaturated fatty acids, 7,10,13,16-[14C]docosatetraenoic acid (22:4(n-6)), 7,10,13,16,19-[14C]docosapentaenoic acid (22:5(n-3)), and 4,7,10,13,16,19-[14C]docosahexaenoic acid (22:6(n-3)), were compared with [3H]arachidonic acid (20:4(n-6] and [14C]linoleic acid (18:2(n-6)) to characterize their incorporation into the lipids of Ehrlich ascites cells. The relatively rapid incorporation of the labeled 22-carbon acids into phosphatidic acid indicated that substantial amounts of these acids may be incorporated through the de novo pathway of phospholipid synthesis. In marked contrast to 20:4(n-6), the 22-carbon acids were incorporated much less into choline glycerophospholipids (CGP) and inositol glycerophospholipids (IGP). No selective preference was apparent for the (n-3) or (n-6) type of fatty acids. The amounts of the acids incorporated into diacylglycerophosphoethanolamine were in the order of: 22:6(n-3) greater than 20:4(n-6) much greater than 22:5(n-3) greater than or equal to 22:4(n-6) greater than 18:2(n-6), whereas for alkylacylglycerophosphoethanolamine they were in the order of: 22:4(n-6) greater than 22:6(n-3) greater than 22:5(n-3) much greater than 20:4(n-6) greater than 18:2(n-6). Of the mechanisms possibly responsible for the selective entry of 22-carbon acids into ethanolamine glycerophospholipids, the most reasonable explanation was that the cytidine-mediated ethanolamine phosphotransferase may have a unique double selectivity: for hexaenoic species of diacylglycerol and for 22-carbon polyunsaturated fatty acid-containing species of alkylacylglycerol. The relative distribution of fatty acids between newly incorporated and already maintained lipid classes suggested that IGP may function in Ehrlich cells as an intermediate pool for the retention of polyunsaturated fatty acids in glycerolipids.     

Shape of the fluidity gradient in the plasma membrane of living HeLa cells

The shape of the fluidity gradient of the outer hemi-leaflet of the plasma membrane of living HeLa cells was determined using a series of n-(9-anthroyloxy) fatty acid probes where n = 2, 3, 6, 7, 9, 10, 11, 12, and 16. Fluorescence uptake and steady-state anisotropy values were obtained with a flow cytometer capable of continuous recording over time of vertical and horizontal emission intensities, and of the output of these intensities as calculated anisotropy values. The fluorescence uptake of all of the membrane probes was rapid up to about 15 min. The magnitudes of the uptake of fluorescence were, for the n-(9-anthroyloxy) series, in the order 2 greater than 3 greater than 6 greater than 7 greater than 9 greater than 10 greater than 11 = 12 = 16. Anisotropy values were constant from 5 to 30 min after addition of the various probes, and the magnitudes were in the order 7 greater than 6 greater than 9 = 10 greater than 2 = 3 greater than 11 greater than 12 greater than 16, indicative of the shape of the fluidity gradient. No differences were noted between the values obtained with 12-(9-anthroyloxy) stearic acid and 12- (9-anthroyloxy) oleate. The kinetics of anisotropy exhibited by those probes with the anthroyloxy group in positions deeper than 9, where initially higher values declined until equilibrium was reached, were probably indicative of an energy barrier at the approximate depth sensed by 7 AS.     

Water relations of solute accumulation in Pseudomonas fluorescens

When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (aw) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool. This increase was not observed when the aw of the medium was reduced to 0.980 with sorbitol. Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium. In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 aw. The maintenance coefficients for glucose uptake were similar at both aw values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 aw.     

Water relations of solute accumulation in Pseudomonas fluorescens

When Pseudomonas fluorescens was grown in a glucose salts medium adjusted with NaCl to a water activity (a_w) value of 0.980, the intracellular glutamic acid concentration increased 23-fold and comprised 90% of the total amino acid pool. This increase was not observed when the a_w of the medium was reduced to 0.980 with sorbitol. Sorbitol was taken up rapidly over a 30 min period and accumulated intracellularly to a level approximately two-fold greater than the concentration in the growth medium. In continuous culture, the specific rate of glutamic acid production and glucose uptake was greater at 0.980 (NaCl) than at 0.997 a_w. The maintenance coefficients for glucose uptake were similar at both a_w values but were 2.4-fold greater for glutamic acid production at 0.980 (NaCl) than at 0.997 a_w.