Alterations of phospholipid metabolism by phorbol esters and fatty acids occur by different intracellular mechanisms in cultured glioma, neuroblastoma, and hybrid cells

Differences between the influences of phorbol esters (such as 4 beta-12-O-tetradecanoylphorbol 13-acetate) and of fatty acids (such as oleic acid) on the synthesis and turnover of phosphatidylcholine (PtdCho) and other phospholipids have been studied in glioma (C6), neuroblastoma (N1E-115), and hybrid (NG108-15) cells in culture using [methyl-3H]choline, [32P]Pi, [1,2-14C]ethanolamine, or 1-14C-labeled fatty acids as lipid precursors. 100-500 microM oleic acid stimulated PtdCho synthesis 3- to 5-fold in all three cell lines, but had little influence on chase of choline label following a 24-h pulse. Phorbol ester (50-200 nM) stimulated PtdCho synthesis 1.5- to 3-fold in C6 cells, was without effect in N1E-115 cells, and had intermediate effects on NG108-15 cells. Phorbol ester stimulated both uptake of extracellular choline and synthesis of PtdCho, whereas fatty acid stimulated only synthesis. Release of radioactivity from 24-h pulse-labeled PtdCho to the medium was enhanced by phorbol ester in C6 cells. Incorporation of [32P]Pi, primarily into PtdCho, was stimulated, whereas utilization of [1,2-14C]ethanolamine or 1-14C-fatty acid was little altered by phorbol ester. C6 cells "down-regulated" with phorbol ester lost the stimulatory response of subsequent treatment with phorbol esters on PtdCho synthesis, but the response to fatty acid was enhanced. Fatty acid had little influence on the relative binding of phorbol ester or "translocation" of phorbol ester binding sites. Accordingly, metabolism of phospholipids in these cultured cells of neural origin is markedly influenced by cell type, phospholipid class, condition of incubation medium, and nature of stimulator. Phorbol esters and fatty acids appear to enhance phospholipid synthesis and turnover by distinct intracellular mechanisms.     

On the mechanism of the acid-catalyzed hydrolysis of uridine to uracil. Evidence for 6-hydroxy-5,6-dihydrouridine intermediates

In acidic media, the 5,6-double bond of uridine is rapidly hydrated to give a small amount of 6-hydroxy-5,6-dihydrouridine (Urd-H2O), the mechanism of which is known from studies of the acid-catalyzed dehydration of Urd-H2O (Prior, J. J., Maley, J., and Santi, D. V. (1984) J. Biol. Chem. 258, 2422-2428). In addition to dehydration, Urd-H2O also undergoes direct hydrolysis of the N-glycosidic bond in acidic solution. The kinetics of the above reaction demonstrates that Urd-H2O, or an intermediate in the pathway leading from Urd to Urd-H2O, is kinetically competent to account for the hydrolysis of the N-glycosidic bond of Urd. The hydrolysis of (1'-2H)Urd proceeds with an alpha-secondary deuterium isotope effect of kH/kD of 1.11 at 25 degrees C. This isotope effect is sufficiently large to implicate carbonium ion character at the 1'-carbon during hydrolysis but, since it is not the maximal value expected, suggests that N-glycoside cleavage is rate-determining with a transition state intermediate between reactant and products. Importantly, the hydrolysis of [6-3H]Urd proceeds with a substantial inverse secondary isotope effect of kT/kH = 1.15 at 25 degrees C which indicates some degree of sp2 to sp3 rehybridization of C-6 of the pyrimidine moiety during hydrolysis. From the data available, it appears that an important pathway in the hydrolysis of the N-glycoside bond of Urd involves either spontaneous cleavage of Urd which is protonated at the 5-carbon or a protonated species of Urd-H2O. The studies described here, together with the known susceptibility of the 6-position of pyrimidine heterocycles toward nucleophiles, permits the proposal of chemically reasonable mechanisms for enzyme-catalyzed cleavage of N-glycosidic bonds of pyrimidines.     

The Seven Carbohydrate Bioengineering Meeting,Braunschweig, Germany,April, 22–25, 2007

Stereocontrol in bakers' yeast reduction can be achieved by introduction of a sulfur functional group into substrates. α-Methylthio-β-keto esters are reduced to give exclusively (3S)-3-hydroxy esters. α-Substituted β-keto thiol esters and dithioesters afford (2R,3S)-3-hydroxy esters with high diastereo-and enantioselectivity. Ketones possessing 1,3-dithiane, phenylsulfenyl, or phenylsulfonyl groups at the α-position are transformed also into the corresponding (S)-secondary alcohols. Optically pure (S)-(phenylsulfinyl)acetones can be obtained by kinetic resolution of racemic derivatives with the yeast. Diastereo- and enantioselective reduction of 1,2-diketones leading into (1S,2S)-1,2-diol derivatives can be also achieved by introduction of 1,3-dithiane, phenylsulfenyl or phenylsulfonyl groups into the α-position. Reductions of carbon-carbon double bond of sulfur-functionalized prenyl derivatives provide both chiral (R)- and (S)-C₅-building blocks for terpenoid synthesis. The utility of the reduction products as chiral building blocks is demonstrated in the synthesis of biologically active natural products such as pheromones, sugars, antibiotics etc. by functional group transformation and carbon-carbon bond formation reactions with the aid of sulfur functional groups.     

Catalysis by human leukocyte elastase: III. Steady-state kinetics for the hydrolysis of p-nitrophenyl esters

Steady-state kinetic parameters were determined at pH 7.4 and 25 degrees C for the human leukocyte elastase-catalyzed hydrolysis of several N-carbobenzoxy-L-amino acid p-nitrophenyl esters. The substrate specificity for these esters was quite broad, and included the Gly, Phe, and Tyr derivatives. Together with reports of a much narrower P-1 specificity for peptide-based substrates, these results suggest that interactions remote from the scissle bond between enzyme and substrate regulate primary specificity. Also, it was found that kc and kc/Km did not exhibit the same dependence on substrate structure. This is interpreted to suggest that there are significant differences in P-1 specificity between acylation and deacylation for leukocyte elastase-catalyzed reactions.     

Bradykinin and its Gly6 analogue are substrates of cyclophilin: a fluorine-19 magnetization transfer study

Fluorine-19 magnetization transfer experiments have been used to determine the rates of cis/trans isomerization about the X-Pro7 peptide bond in [p-fluoro-Phe8]bradykinin (cis/trans ratio approximately 0.1) and its Gly6 analogue (cis/trans ratio approximately 0.4). The measurements were carried out both prior to and after the addition of cyclophilin, which has recently been shown to have peptidyl-proline cis/trans isomerase activity and is the apparent target enzyme of the immunosuppressive agent cyclosporin A. Magnetization transfer measurements over the temperature range 40-75 degrees C in the absence of enzyme give activation energies of 22.8 and 23.0 kcal/mol for [p-fluoro-Phe8]bradykinin and its Gly6 analogue, respectively. The values for the uncatalyzed cis----trans rate constant, kc, are determined by extrapolation to be 4.8 x 10(-2) and 2.1 x 10(-2) s-1 for the two peptides at 25 degrees C. The enzyme-catalyzed enhancement of the cis/trans interconversion rate was proportional to added cyclophilin concentration and was strongly sequence specific, with bradykinin a much better substrate than [Gly6]bradykinin. At a peptide concentration of 2.2 mM, the catalytic activity expressed as kc per micromolar cyclophilin was determined to be 1.2 s-1/microM for [p-fluoro-Phe8]bradykinin and 0.13 s-1/microM for the Gly6 analogue. The increased cis----trans interconversion rates were strongly inhibited by cyclosporin A and the 6-(methylalanine) derivative, which bind to cyclophilin, but not by the 1-(tetrahydrofurfuryl) derivative of cyclosporin that binds weakly.     

Substrate specificity of regiospecific desaturation of aliphatic compounds by a mutant Rhodococcus strain

Substrate specificity of cis-desaturation of alipahtic compounds by resting cells of a mutant, Rhodococcus sp. strain KSM-MT66, was examined. Among substrates tested, the rhodococcal cells were able to convert n-alkanes (C13-C19), 1-chloroalkanes (C16 and C18), ethyl fatty acids (C14-C17) and alkyl (C1-C4) esters of palmitic acid to their corresponding unsaturated products of cis configuration. The products from n-alkanes and 1-chloroalkanes had a double bond mainly at the 9th carbon from their terminal methyl groups, and the products from acyl fatty acids had a double bond mainly at the 6th carbon from their carbonyl carbons.     

Effects of various non-esterified fatty acids on the transfer of cholesteryl esters from HDL to LDL induced by the cholesteryl ester transfer protein

The effects of saturated, monounsaturated and polyunsaturated non-esterified fatty acids on the rate of transfer of radiolabeled cholesteryl esters from high density lipoproteins (HDL) to low density lipoproteins (LDL), induced by the cholesteryl ester transfer protein (CETP), have been studied. Human high-density lipoproteins-subfraction 3 (HDL3) containing radiolabeled cholesteryl esters were incubated with LDL at 37 degrees C with or without CETP and in the absence or in the presence of non-esterified fatty acids. Less than 6% of the total radioactivity was recovered in the LDL fraction after incubation of HDL3, and LDL for 3 h at 37 degrees C in the absence of CETP, regardless of whether or not non-esterified fatty acids were added. The addition of CETP to the incubation mixture induced a time-dependent redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. Non-esterified fatty acids were found to alter the rate of transfer of cholesteryl esters induced by CETP. While short chain saturated non-esterified fatty acids (caprylic and capric acids) had no effect on the rate of transfer of cholesteryl esters, the medium and long chain ones (lauric, myristic, palmitic and stearic acids) significantly increased the CETP-mediated transfers from HDL3 to LDL. At low concentrations, unsaturated fatty acids also stimulated the CETP-mediated redistribution of radiolabeled cholesteryl esters from HDL3 to LDL. As the concentration of either oleic, linoleic or arachidonic acids increased to higher levels, a significant proportion of fatty acids remained unassociated with lipoprotein particles. Under these circumstances the transfer process was inhibited. These results show that non-esterified fatty acids can modulate the CETP-mediated transfer of cholesteryl esters from HDL to LDL and that this effect is dependent on both the length and the degree of unsaturation of their monomeric carbon chain.     

The thermal transitions and structural properties of 5alpha-cholestan-3beta-ol esters of aliphatic acids.

5 alpha-Cholestan-3 beta-ol esters of aliphatic acids undergo both enantiotropic and monotropic changes of state. Ten saturated and three unsaturated esters have been examined by differential scanning calorimetry and polarizing microscopy to determine transition temperatures, enthalpies, and entropies. The results are compared with an analogous series of cholesterol esters. All esters of even-numbered n-alkanoic acids from C2 to C20 melt from a crystalline state to an isotropic liquid. The crystalline state has been studied by X-ray powder diffraction. The C8 to C20 esters have progressively increasing crystalline melting transition temperatures from 76 to 99 degrees C and possess similar X-ray powder diffraction patterns, suggesting that these compounds form an isostructural series. Esters of C2, C4, and C6 acids exhibit polymorphism. Crystalline cholestanol oleate melts to an isotropic liquid, whereas cholestanol linoleate and linolenate fail to crystallize, even after several months at -20 degrees C. Esters of the even-numbered saturated acids from C4 to C14 form monotropic cholesteric liquid crystalline phases. Esters C10, C12, and C14 form smectic liquid crystalline phases. Cholestanol oleate, linoleate, and linolenate form both cholesteric and smectic mesophases. The lower smectic to cholesteric and cholesteric to isotropic transition temperatures of the cholestanol esters compared to the corresponding transition temperatures of the analogous cholesterol esters suggest that the delta 5 double bond in cholesterol increases the thermal stability of the mesophases of cholesterol esters.     

Cholesterol esterification in rabbit plasma

1. When [4-(14)C]cholesterol, attached to beta-globulin or dispersed with Tween 20, was incubated with fresh rabbit (New Zealand albino females) plasma, 30-47% esterification was observed. The optimum pH was 6.8. This esterification was accomplished by the transfer of fatty acids from the C-2 position of lecithin (phosphatidylcholine) to cholesterol. 2. There was no evidence that triglycerides or free fatty acids participated directly in this reaction. Lecithins with labelled palmitic acid, oleic acid and linoleic acid in the 2-position yielded 3.2, 4.8 and 6.8% of cholesteryl esters respectively. This pattern reflects that which is normally observed in the cholesteryl esters of rabbit plasma and supports the concept that plasma cholesteryl esters originate from the plasma. 3. Snake venom (containing phospholipase A), sulphoevernan [an alpha-(1-->3,1-->4)-sulphopolyglucan with 12% sulphur], thiol-blocking agents (p-chloromercuribenzoate and N-ethylmaleimide), or an atherogenic diet (stock diet supplemented with 1% cholesterol for 8 weeks) were all effective inhibitors of this cholesterol esterification.     

Inhibition of malic enzyme by S-oxalylglutathione, a probable in vivo effector

Various oxalyl thiol esters (RSCOCOO-), especially S-oxalylglutathione (GS-Ox), were found to be very effective inhibitors of chicken liver malic enzyme. When the conditions are similar to those encountered physiologically [high reduced nicotinamide adenine dinucleotide phosphate (NADPH) concentrations], inhibition is detectable with less than 1 microM concentrations of GS-Ox. The amount of inhibition is not reversed by excess glutathione, thus indicating that it is not due to oxalyl transfer to some enzymic thiol group with release of glutathione. Detailed kinetic studies show that the inhibition by GS-Ox can be treated as a simple reversible binding to the enzyme; the double reciprocal plot patterns indicate that the inhibition is linear noncompetitive (mixed type), vs. both L-malate in the oxidative decarboxylation reaction and pyruvate in the reverse reaction. At pH 7.4 and 25 degrees C in the presence of 100-200 microM NADPH, the Kis and Kii values for GS-Ox are 0.7 and 5 microM, respectively, and are the same for reactions run in either direction. The high specificity for GS-Ox is indicated by the observation that, under similar conditions, the Kis values for S-oxalyl coenzyme A and S-oxalyl-N-acetylcysteamine are 40 and 150 microM, respectively. Such high specificity indicates that the enzyme has evolved a specific binding site for the glutathione part of GS-Ox. The current results, when considered in conjunction with recent evidence that oxalyl thiol esters are present in animal tissues at concentrations up to 50 microM, imply that GS-Ox is an important in vivo regulator of malic enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Glycosphingolipids: 2H NMR study of the influence of carbohydrate headgroup structure on ceramide acyl chain behavior in glycolipid-phospholipid bilayers

Galactosyl- and glucosylceramide, globoside, and dihydrolactosylceramide, bearing [2,2-2H2]stearic acid, have been studied at a concentration of 10 mol% in bilayers of dimyristoylphosphatidylcholine by 2H NMR. The quadrupolar splitting delta vQ of the C2 deuterons were measured at several temperatures in the range of 30-60 degrees C. Spin-lattice relaxation times T1 of C2 deuterons were determined in the same temperature range for all lipids but globoside. T1 values at 30 and 50 degrees C were unexpectedly short (6-8 ms), indicating reduced mobility of the ceramide acyl chains compared to that of the host phospholipid. At all temperatures, both delta vQ and T1 were essentially identical for the monoglycosylated species, GalCer and GlcCer, indicating that the order and dynamics of the upper portion of the fatty acyl chain are insensitive to this small change in the headgroup structure. In the case of globoside, where the glycolipid headgroup is equivalent to that of GlcCer extended by three sugar residues, values for the quadrupolar splittings associated with the acyl chain C2-position were very close to those obtained for Gal- and GlcCer. In contrast, the delta vQ values obtained for the diglycosyl species, LacCer, were significantly different at all temperatures. This different behavior of LacCer relative to that of the other glycolipids most likely originates from an orientational change of the acyl chain at the C2-position due to the absence of a 4,5 double bond in dihydrosphingosine. T1 values for the GlcCer and GalCer systems increased with temperature, indicating that the motions responsible for relaxation were in the short correlation time regime.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of methylamine on the reaction of alpha 2-macroglobulin with enzymes.

The kinetics of reaction of alpha 2-macroglobulin (alpha 2M) with thrombin and with trypsin were studied in the presence and absence of methylamine. The rate of enzyme-induced thiol release was found to be the same whether or not amine was present. The result suggests that covalent bond formation and enzyme-catalyzed amine incorporation proceed via a common (enzyme-dependent) rate-determining step. The reaction of lysyl-modified enzymes (which show poor covalent binding with alpha 2M) was similarly unaffected by amine, indicating that enzyme-catalyzed steps were also rate determining for hydrolysis of the thiol ester. The products of the reactions were analyzed by native and denaturing gel electrophoresis. Methylamine did not affect the total binding of enzyme to alpha 2M but did cause a substantial decrease in covalent binding. Surprisingly, not all covalent complexes were affected by the presence of amine: complexes in which enzyme was covalently bound to one half-molecule increased compared to the reaction with no amine; complexes in which two half-molecules are cross-linked by two bonds to a single enzyme were substantially reduced, however. The results are consistent with a mechanism of reaction in which an enzyme-dependent step is rate determining. This step is accompanied by activation of two thiol esters. One of these reacts immediately with the bound enzyme (or may be hydrolyzed if the enzyme amine groups are blocked). The other activated center is capable of reaction with external nucleophiles such as methylamine.     

Lipids of ocular tissues: VII. Positional distribution of the fatty acids in the phospholipids of bovine retina rod outer segments

The positional distribution of the fatty acids in the major phospholipids of bovine retina rod outer segments was determined. Phosphatidyl ethanolamine and phosphatidyl serine have mostly saturated acids in the 1-position and docosahexaenoic acid in position 2. These phospholipids contain 94 and 79%, respectively, of polyun-saturated acids in the 2-position. Phosphatidyl choline contains mostly saturated acids in the 1-position, but has significant quantities of palmitic in the 2-position along with docosahexaenoic acid. The levels of docosahexaenoic acid in rod outer segment phospholipids are among the highest yet reported for membrane phospholipids, amounting to 23% in phosphatidyl choline, 39% in phosphatidyl ethanolamine, and 45% in phosphatidyl serine.     

Diesters of alkane diols and 2-hydroxy fatty acids: identification and discrimination of isomers with the aid of NMR spectroscopy.

High resolution nuclear magnetic resonance spectroscopy has been shown to be extremely useful for the identification and discrimination of naturally occurring diesters of 1,2- and 2,3-alkanediols as well as for fatty alkyl esters of acylated 2-hydroxy fatty acids. A comparison of 220 MHz spectra of 1,2 and erythro- 2,3-alkanediol diesters exhibits the following distinguishing features: (1) two non-equivalent methylene protons from the glycol group of 1,2-alkanediol diesters resonate at 3.87 ppm and 4.17 ppm respectively while these resonances are completely absent in the spectrum of 2,3-isomer; (2) methylene protons adjacent to esther carbonyl groups appear as two overlapping triplets at 2.22 ppm in 1,2-alkanediol diesters while the corresponding protons in the 2,3-isomer are displayed as two partially overlapping triplets centered at 2.15 ppm and 2.2 ppm respectively; and (3) methyl protons adjacent to glycol group in 2,3-isomer appear as downfield doublet at 1.13 ppm; this downfield doublet is not shown by 1,2-alkanediol diesters. Erythro- and threo-2,3-alkanediol diesters have also been distinguished from each other; two alpha-methylenes in erythro isomers appear as partially overlapping triplets while these protons in threo isomer display an apparent quartet centered at 2.22 ppm. Fatty alkyl esters of acylated 2-hydroxy fatty acids display a triplet at 4.79 for 2-position methylene proton, a distinguishing feature not shown by diacyl alkanediols. A distinction between diester lipids and other classes of neutral lipids has also been achieved by the study of nuclear magnetic resonance spectra, particularly in the region of 3-6 ppm.     

Enzymic properties of thermopsin

The specificity of thermopsin, a thermostable acid protease from Sulfolobus acidocaldarius, was studied using oxidized insulin B chain as substrate followed by peptide isolation and identification. The following bonds were hydrolyzed: Leu-Val, Leu-Tyr, Phe-Phe, Phe-Tyr, and Tyr-Thr. Thus, the specificity of thermopsin is similar to that of pepsin, that is, it prefers large hydrophobic residues at both sides of the scissile bond. We confirmed this by the use of a synthetic substrate, Lys-Pro-Ala-Glu-Phe-p-nitro-phenylalanyl-Ala-Leu, which was cleaved by thermopsin between Phe and p-nitro-phenylalanyl. Using this substrate, enzyme inhibition and kinetic properties of thermopsin have been studied. Thermopsin optimally hydrolyzes this substrate at 75 degrees C and pH 2 with Km and kcat values under these conditions of 5.3 x 10(-5) M and 14.3 s-1, respectively. Pepstatin competitively inhibits thermopsin with a Ki of 2 x 10(-7) M. Other known aspartic protease inhibitors, diazoacetylnorleucine ethyl ester and 1,2-epoxy-3-(p-nitrophenoxy)propane inhibited thermopsin only slowly and with nonspecific reactions. Although thermopsin contains a single cysteine, iodoacetic acid and p-chloromercuric benzoate had no effect on activity. Mercuric chloride inhibited the enzyme, and the inhibition was reversible by mercaptoethanol. However, the enzyme was not labeled by [14C]iodoacetic acid either before or after sodium dodecyl sulfate denaturation. Thus, the thiol group is likely blocked, and the inhibition effect of mercuric ion is unrelated to the thiol group. These observations suggest that thermopsin has a different active site than the aspartic protease family but may have a similar transition state structure. The temperature dependence of Km and kcat was studied for thermopsin hydrolysis of the synthetic substrate between 26-78 degrees C. Both parameters increased with temperature, and the rise of kcat value was particularly sharp above 65 degrees C. Hydrolysis activity measured at high substrate concentration has a maximum at 76 degrees C, which is near the physiological temperature for the optimal growth of this organism. Thus, thermopsin appears to function best at high temperature and high substrate concentration. It may be utilized by the organism to response to the presence of high substrate concentration in the medium. Thermopsin is also competitively inhibited by urea, acetamide, and phenylalaninamide with Ki values of 0.5, 0.4, and 0.01 M, respectively.     

Urea and ethylene glycol-facilitated transport systems in the human red cell membrane. Saturation, competition, and asymmetry

The equilibrium exchange of [14C]urea and ethylene glycol was measured using a new type of fast flow system. Approximately equal volumes of saline and air were mixed to form a segmented fluid stream into which 14C-loaded red cells are injected. The stream flows through three filter chambers which allow sampling of the 14C in the extracellular fluid at three time points. The chambers are designed so that they do not disrupt the segmented bubble pattern. The alternating air and saline segments prevent laminar dispersion in the flowing stream and ensure good mixing at the injection and sampling sites. The equilibrium exchange of both urea and ethylene glycol showed saturation kinetics. The maximum permeability (Po) measured in the limit of zero solute concentration is 1.6 X 10(-3) cm/s for urea and 4.8 X 10(-4) cm/s for ethylene glycol (T = 23 degrees C). The apparent dissociation constant (Km) was 218 mM for urea and 175 mM for ethylene glycol. The Po for thiourea is 2.3 X 10(-6) cm/s and the Km is 19 mM. Urea and thiourea inhibit the transport of each other and the inhibition constant (KI) is approximately equal to the Km for both compounds. 53 other analogues of urea were screened for their inhibition of urea or thiourea transport. Several analogues [e.g., 1-(3,4-dichloro-phenyl)-2-thiourea] had a KI in the range of 0.03 mM. The affinity of the inhibitor increased as it was made more hydrophobic. The urea analogues did not significantly inhibit the ethylene glycol or osmotic permeability. Glycerol inhibited ethylene glycol permeability with a KI of 1,200 mM.     

A short-chain acyl-CoA: 1-alkyl-2-acyl-sn-glycerol acyltrasnferase from a microsomal fraction of the rabbit Harderian gland.

The 3-position of the alkyldiacylglycerols from the pink portion of the rabbit harderian gland is occupied exclusively by isovaleric acid. We describe a microsomal 1-alkyl-2-acyl-sn-glycerol acyltransferase from this gland which specifically incorporates short-chain acyl-CoA's into the 3-position of alkylacyl-glycerols. The enzyme is most active in the presence of CoA esters with chain lengths similar to isovaleric acid and is inactive in the presence of acetyl CoA and long-chain acyl-CoA's. No evidence was found for an enzyme that would transfer long-chain acyl-CoA's to the same substrate. The specificity of this acyltransferase can account for the exclusion of long-chain acyl moieties from the 3-position of the alkyldiacylglycerols in the harderian gland of rabbits.     

Substrate Specificity of Regiospecific Desaturation of Aliphatic Compounds by a Mutant Rhodococcus Strain

Substrate specificity of cis-desaturation of alipahtic compounds by resting cells of a mutant, Rhodococcus sp. strain KSM-MT66, was examined. Among substrates tested, the rhodococcal cells were able to convert n-alkanes (C13-C19), 1-chloroalkanes (C16 and C18), ethyl fatty acids (C14-C17) and alkyl (C1-C4) esters of palmitic acid to their corresponding unsaturated products of cis configuration. The products from n-alkanes and 1-chloroalkanes had a double bond mainly at the 9th carbon from their terminal methyl groups, and the products from acyl fatty acids had a double bond mainly at the 6th carbon from their carbonyl carbons.     

Characterization of a protease from Thermoactinomyces vulgaris (thermitase). 3. Substrate specificity and properties of partially purified thermitase]

During the process of cultivation of Th. vulgaris several proteases are formed. In the present investigation the extensively purified major component was used. The substrate specificity was determined by means of 7 proteins, 7 amino acid esters, 5 fatty acid esters and 15 amino acid 4-nitroanilides. Among the protein substrates tested, urea denaturated hemoglobin was split best, followed by gelatin, casein, field bean protein, serum albumin and gluten. The weakest rate of hydrolysis was observed with elastin. In contrast to this acetyl-(L-ala)3-methylester, that is a substrate for elastase, was split best from all the esters tested. Only 8% of this activity could be found with the chymotrypsin substrates acetyl-L-tyr-ethylester and acetyl-L-phe-ethylester and 1% of the above activity with the trypsin substrates tosyl-L-arg-methylester and benzoyl-L-arg-methylester. The fatty acid esters and the p-nitroanilides were hydrolyzed much more slowly. The pH-optimum of thermitase was found in the weakly alkaline region of pH 7 to 9. There were only small differences between the individual high and low molecular substrates. The temperature optimum was between 60 and 75 degrees C for esters and p-nitroanilides as substrates and at 90 degrees C for casein. It should be mentioned that the enzyme was quickly inactivated at temperatures above 70 degrees C.