Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by nuclear magnetic resonance analysis of a single glucose derivative

A triple-tracer method was developed to provide absolute fluxes contributing to endogenous glucose production and hepatic tricarboxylic acid (TCA) cycle fluxes in 24-h-fasted rats by (2)H and (13)C nuclear magnetic resonance (NMR) analysis of a single glucose derivative. A primed, intravenous [3,4-(13)C(2)]glucose infusion was used to measure endogenous glucose production; intraperitoneal (2)H(2)O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal [U-(13)C(3)] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle flux. Plasma glucose was converted to monoacetone glucose (MAG), and a single (2)H and (13)C NMR spectrum of MAG provided the following metabolic data (all in units of micromol/kg/min; n = 6): endogenous glucose production (40.4+/-2.9), gluconeogenesis from glycerol (11.5+/-3.5), gluconeogenesis from the TCA cycle (67.3+/-5.6), glycogenolysis (1.0+/-0.8), pyruvate cycling (154.4+/-43.4), PEPCK flux (221.7+/-47.6), and TCA cycle flux (49.1+/-16.8). In a separate group of rats, glucose production was not different in the absence of (2)H(2)O and [U-(13)C]propionate, demonstrating that these tracers do not alter the measurement of glucose turnover.     

Simplified analysis of acetaminophen glucuronide for quantifying gluconeogenesis and glycogenolysis using deuterated water

Measurement of acetaminophen glucuronide (AG) ²H enrichment from deuterated water (²H₂O) by ²H nuclear magnetic resonance (NMR) analysis of its monoacetone glucose (MAG) derivative provides estimation of gluconeogenic and glycogenolytic contributions to endogenous glucose production (EGP). However, AG derivatization to MAG is laborious and unsuitable for high-throughput studies. An alternative derivative, 5-O-acetyl monoacetone glucuronolactone (MAGLA), was tested. Eleven healthy subjects ingested ²H₂O to 0.5% body water enrichment and 500 mg of acetaminophen. Plasma glucose and urinary glucuronide positional ²H enrichments were measured by ²H NMR spectroscopy of MAG and MAGLA, respectively. A Bland–Altman analysis indicated agreement at the 95% confidence level between glucose and glucuronide estimates.     

Noninvasive evaluation of liver metabolism by 2H and 13C NMR isotopomer analysis of human urine

Mammalian liver disposes of acetaminophen and other ingested xenobiotics by forming soluble glucuronides that are subsequently removed via renal filtration. When given in combination with the stable isotopes 2H and 13C, the glucuronide of acetaminophen isolated from urine provides a convenient "chemical biopsy" for evaluating intermediary metabolism in the liver. Here, we describe isolation and purification of urinary acetaminophen glucuronide and its conversion to monoacetone glucose (MAG). Subsequent 2H and 13C NMR analysis of MAG from normal volunteers after ingestion of 2H2O and [U-13C3]propionate allowed a noninvasive profiling of hepatic gluconeogenic pathways. The method should find use in metabolic studies of infants and other populations where blood sampling is either limited or problematic.     

Intraruminal infusion of n-butyric acid induces an increase of ruminal papillae size independent of IGF-1 system in castrated bulls

The objective of this study was to explore morphological alterations of rumen papillae induced by n-butyric acid in relation to the insulin-like growth factor (IGF) system in adult castrated bulls. Three animals fitted with rumen cannula were fed twice daily at a low and high nutritional level (LL and HL), i.e., at 1.1 x maintenance (M) and 1.6 x M, respectively. Diets contained artificial dried grass and concentrate (74:26 and 52:48). Bulls received no (B0) or daily intraruminal infusions of 500 g n-butyric acid (B500) over 14 d. The infusion started 1 h after the morning feeding (9:00) and lasted for 3.5 h. Thus, four treatments (BOLL, B500LL, BOHL, and B500HL) were compared. Blood and rumen mucosa samples from the atrium ruminis were taken at the last day of each period. Length, width and surface of rumen papillae were greater (p < 0.001) in BOHL than in BOLL. Treatment with n-butyric acid resulted in an increase of the papillae surface of 20-40% (p = 0.047) for both nutritional levels as compared to periods without n-butyric acid treatments. The higher nutritional level and intraruminal n-butyric acid infusion induced epithelial cell death. The percentage of proliferative cells was doubled by n-butyric acid treatment. The mRNA of IGF-1 and IGF type 1 receptor (IGF-1R), as well as IGF-1R binding capacity were unaffected by butyric acid treatments. The abundance of IGF-1 mRNA tended to be lower (p = 0.1) and IGF-1R abundance was lower (p = 0.03) in response to the HL. The plasma IGF-1 concentration was lower with butyric acid treatment (p < 0.01), but was unaffected by the nutritional level. In conclusion, under described experimental preconditions of daily short-time intraruminal n-butyric acid infusion alterations of rumen papillae morphology is not mediated by ruminal IGF type 1 receptor and by local IGF-1 expression in papillae in castrated bulls.     

Biofiltration of n-butyric acid for the control of odour

Odour control from pig production facilities is a significant concern due to increased public awareness and the development of more stringent legislation to control production. Although many technologies exist, biofiltration is still the most attractive due to its low maintenance and operating costs. One of the key odour components, n-butyric acid, was selected for a laboratory scale biofilter study. It was examined as a sole carbon substrate in order to investigate the effectiveness of biofiltration in reducing n-butyric acid concentration under different operating conditions using a moist enriched woodchip medium. Three superficial gas velocities; 38.2, 76.4, and 114.6 m x h(-1) were tested for n-butyric acid concentrations ranging from 0.13 to 3.1 g [n-butyric acid] m(-3) [air]. For superficial gas velocities 38.2, 76.4, and 114.6 m x h(-1), maximum elimination capacities (100% removal) of 148, 113 and 34.4 g x m(3) x h(-1), respectively, were achieved. Upon investigation of effective bed height, true elimination capacities (100% removal) of 230, 233 and 103 g x m(-3) x h(-1), respectively, were achieved at these superficial gas velocities. Averaged pressure drops for superficial gas velocities 38.2, 76.4, and 114.6 m x h(-1) were 30, 78 and 120 Pa, respectively. It was concluded that biofiltration is a viable technology for the removal of n-butyric acid from waste exhaust air, but near 100% removal efficiency is required due to the low odour detection threshold for this gaseous compound.     

MAIGO2 is involved in gibberellic acid, sugar, and heat shock responses during germination and seedling development in Arabidopsis

Our previous study has shown that MAIGO2 (MAG2) is a subunit of the Golgi/endoplasmic reticulum (ER) multi-subunit tethering complex, and is required for tolerance to general osmotic stresses and abscisic acid and response to ER stress during seed germination and early growth. MAG2 is crucial for multi-environmental stress responses. To verify this hypothesis, the response of mag2 mutants to gibberellic acid (GA), sugar, and heat shock was described in this study. The mag2 mutants showed defects during seed germination and early seedling development under treatments with the GA biosynthesis inhibitor paclobutrazol, sucrose, and glucose. MAG2 is also essential for basal thermotolerance. However, the MAG2 homolog (MAG2L) is not necessary for these responses. MAG2 is an important regulator in the response to multi-environmental stimuli, supposedly through its roles in Golgi/ER retrograde trafficking and ER stress response.     

Catalytic properties of MGAT3, a putative triacylgycerol synthase

Acyl-coenzyme A:monoacylglycerol acyltransferase 3 (MGAT3) is a member of the MGAT family of enzymes that catalyze the synthesis of diacylglycerol (DAG) from monoacylglycerol (MAG), a committed step in dietary fat absorption. Although named after the initial identification of its MGAT activity, MGAT3 shares higher sequence homology with acyl-coenzyme A:diacylglycerol acyltransferase 2 (DGAT2) than with other MGAT enzymes, suggesting that MGAT3 may also possess significant DGAT activity. This study compared the catalytic properties of MGAT3 with those of MGAT1 and MGAT2 enzymes using both MAG and DAG as substrates. Our results showed that in addition to the expected MGAT activity, the recombinant MGAT3 enzyme expressed in Sf-9 insect cells displayed a strong DGAT activity relative to that of MGAT1 and MGAT2 enzymes in the order MGAT3 > MGAT1 > MGAT2. In contrast, none of the three MGAT enzymes recognized biotinylated acyl-CoA or MAG as a substrate. Although MGAT3 possesses full DGAT activity, it differs from DGAT1 in catalytic properties and subcellular localization. The MGAT3 activity was sensitive to inhibition by the presence of 1% CHAPS, whereas DGAT1 activity was stimulated by the detergent. Consistent with high sequence homology with DGAT2, the MGAT3 enzyme demonstrated a similar subcellular distribution pattern to that of DGAT2, but not DGAT1, when expressed in COS-7 cells. Our data suggest that MGAT3 functions as a novel triacylglycerol (TAG) synthase that catalyzes efficiently the two consecutive acylation steps in TAG synthesis.     

Sn-2-monoacylglycerol, not glycerol, is preferentially utilised for triacylglycerol and phosphatidylcholine biosynthesis in Atlantic salmon (Salmo salar L.) intestine

Pathways of lipid resynthesis in the intestine of fish are relatively unknown. Various reports have suggested the existence of both sn-1,3-specific (pancreatic) and non-specific (bile salt-activated) lipase activity operating on dietary triacylglycerol (TAG) in the intestinal lumen of fish during digestion. Thus, sn-2-monoacylglycerol (2-MAG) and glycerol, respective hydrolytic products of each lipase, are absorbed and utilised for glycerolipid synthesis in enterocytes via two alternative routes: monoacylglycerol (MAG) and glycerol-3-phosphate (G3P) pathways. Despite different precursors, both pathways converge at the production of sn-1,2-diacylglycerol (1,2-DAG) where TAG or phosphatidylcholine (PC) synthesis can occur. To elucidate the relative activities of MAG and G3P pathways in Atlantic salmon enterocytes, intestinal segments were mounted in Ussing chambers where equimolar mixtures of sn-2-oleoyl-[1,2,3-(3)H]glycerol (2-MAG) and [(14)C(U)]glycerol, plus unlabelled 16:0 and 18:2n-6 as exogenous fatty acid sources, were delivered in bile salt-containing Ringer solution to the mucosa. The MAG pathway predominated, over the G3P pathway, synthesizing ca. 95% of total TAG and ca. 80% of total PC after a 3 h incubation period at 10 degrees C. Further, the 1,2-DAG branch point into TAG or PC was polarised towards TAG synthesis (6:1) via the MAG pathway but more evenly distributed between TAG and PC (1:1) via the G3P pathway. Effect of long-chain saturated, monounsaturated and polyunsaturated fatty acids on the synthesized TAG/PC ratio was assessed by individually exchanging 16:0, 18:1n-9 or 18:2n-6, for 16:0+18:2n-6, in mucosal solutions. TAG synthesis was influenced considerably more than PC synthesis, via either pathway, by exogenous fatty acids utilised. 18:1n-9 significantly stimulated TAG synthesis via the MAG pathway yielding a TAG/PC ratio of 12:1. Alternatively, 18:2n-6 stimulated TAG synthesis the most via the G3P pathway (TAG/PC=4:1). 16:0 significantly attenuated TAG synthesis via either pathway. Micellar fatty acid species also significantly affected intestinal active transport mechanisms as shown by decreasing transepithelial potential (TEP) and short-circuit current (SSC) with increasing fatty acid unsaturation. The epithelial integrity was, however, not compromised after 3 h of exposure to any of the fatty acids. The implications of these findings on dietary fatty acid composition and enterocytic lipid droplet accumulation are discussed.     

l-Arginine increases AMPK phosphorylation and the oxidation of energy substrates in hepatocytes,skeletal muscle cells,and adipocytes

Previous work has shown that dietary l-arginine (Arg) supplementation reduced white fat mass in obese rats. The present study was conducted with cell models to define direct effects of Arg on energy-substrate oxidation in hepatocytes, skeletal muscle cells, and adipocytes. BNL CL.2 mouse hepatocytes, C2C12 mouse myotubes, and 3T3-L1 mouse adipocytes were treated with different extracellular concentrations of Arg (0, 15, 50, 100 and 400 µM) or 400 µM Arg?+?0.5 mM N^G-nitro-l-arginine methyl ester (L-NAME; an NOS inhibitor) for 48 h. Increasing Arg concentrations in culture medium dose-dependently enhanced (P?<?0.05) the oxidation of glucose and oleic acid to CO₂ in all three cell types, lactate release from C2C12 cells, and the incorporation of oleic acid into esterified lipids in BNL CL.2 and 3T3-L1 cells. Arg at 400 µM also stimulated (P?<?0.05) the phosphorylation of AMP-activated protein kinase (AMPK) in all three cell types and increased (P?<?0.05) NO production in C2C12 and BNL CL.2 cells. The inhibition of NOS by L-NAME moderately reduced (P?<?0.05) glucose and oleic acid oxidation, lactate release, and the phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in BNL CL.2 cells, but had no effect (P?>?0.05) on these variables in C2C12 or 3T3-L1 cells. Collectively, these results indicate that Arg increased AMPK activity and energy-substrate oxidation in BNL CL.2, C2C12, and 3T3-L1 cells through both NO-dependent and NO-independent mechanisms.

     

Structure-based discovery of human L-xylulose reductase inhibitors from database screening and molecular docking

Human L-xylulose reductase (XR) is an enzyme of the glucuronic acid/uronate cycle of glucose metabolism and is a possible target for treatment of the long-term complications of diabetes. In this study we utilised the molecular modelling program DOCK to analyse the 249,071 compounds of the National Cancer Institute Database and retrieved those compounds with high predicted affinity for XR. Several carboxylic acid-based compounds were tested and shown to inhibit XR. These included nicotinic acid (IC50=100 microM), benzoic acid (IC50=29 microM) and their derivatives. These results extend and improve upon the activities of known, commercially available inhibitors of XR such as the aliphatic fatty acid n-butyric acid (IC50=64 microM). To optimise the interaction between the inhibitor and the holoenzyme, the program GRID was used to design de novo compounds based on the inhibitor benzoic acid. The inclusion of a hydroxy-phenyl group and a phosphate to the benzoic acid molecule increased the net binding energy by 1.3- and 2.4-fold, respectively. The resultant compounds may produce inhibitors with improved specificity for XR.     

A comparison of the behavioural responses of the haematophagous bug, Triatoma infestans, to synthetic homologues of two naturally occurring chemicals (n- and iso-butyric acid)

ABSTRACT. Responses of adult Triatoma infestans Klug (Reduviidae) to iso-butyric acid (which is secreted by the Brindleys glands of this species) and its isomer n-butyric acid (which is not) were tested in an olfactometer. Bugs avoided both these chemicals with a response that involved klinotactic and orthokinetic components. The avoidance response to iso-butyric acid showed a marked diminution at high concentrations. Avoidance of n-butyric acid, however, was inversely proportional to the log of its concentration. This difference in response is discussed in relation to the communicative function of these two chemicals.     

Intraruminal infusion of n-butyric acid induces an increase of ruminal papillae size independent of IGF-1 system in castrated bulls

Abstract

The objective of this study was to explore morphological alterations of rumen papillae induced by n-butyric acid in relation to the insulin-like growth factor (IGF) system in adult castrated bulls. Three animals fitted with rumen cannula were fed twice daily at a low and high nutritional level (LL and HL), i.e., at 1.1 × maintenance (M) and 1.6 × M, respectively. Diets contained artificial dried grass and concentrate (74:26 and 52:48). Bulls received no (B0) or daily intraruminal infusions of 500 g n-butyric acid (B500) over 14 d. The infusion started 1 h after the morning feeding (9:00) and lasted for 3.5 h. Thus, four treatments (B0LL, B500LL, B0HL, and B500HL) were compared. Blood and rumen mucosa samples from the atrium ruminis were taken at the last day of each period. Length, width and surface of rumen papillae were greater (p < 0.001) in B0HL than in B0LL. Treatment with n-butyric acid resulted in an increase of the papillae surface of 20 – 40% (p = 0.047) for both nutritional levels as compared to periods without n-butyric acid treatments. The higher nutritional level and intraruminal n-butyric acid infusion induced epithelial cell death. The percentage of proliferative cells was doubled by n-butyric acid treatment. The mRNA of IGF-1 and IGF type 1 receptor (IGF-1R), as well as IGF-1R binding capacity were unaffected by butyric acid treatments. The abundance of IGF-1 mRNA tended to be lower (p = 0.1) and IGF-1R abundance was lower (p = 0.03) in response to the HL. The plasma IGF-1 concentration was lower with butyric acid treatment (p < 0.01), but was unaffected by the nutritional level. In conclusion, under described experimental preconditions of daily short-time intraruminal n-butyric acid infusion alterations of rumen papillae morphology is not mediated by ruminal IGF type 1 receptor and by local IGF-1 expression in papillae in castrated bulls.     

Identification, purification, and characterization of monoacylglycerol acyltransferase from developing peanut cotyledons

Biosynthesis of diacylglycerols in plants occurs mainly through the acylation of lysophosphatidic acid in the microsomal membranes. Here we describe the first identification of diacylglycerol biosynthetic activity in the soluble fraction of developing oilseeds. This activity was NaF-insensitive and acyl-CoA-dependent. Diacylglycerol formation was catalyzed by monoacylglycerol (MAG) acyltransferase (EC ) that transferred an acyl moiety from acyl-CoA to MAG. The enzyme was purified by successive chromatographic separations on octyl-Sepharose, blue-Sepharose, Superdex-75, and palmitoyl-CoA-agarose to apparent homogeneity from developing peanut (Arachis hypogaea) cotyledons. The enzyme was purified to 6,608-fold with the final specific activity of 15.86 nmol min(-1) mg(-1). The purified enzyme was electrophoretically homogeneous, and its molecular mass was 43,000 daltons. The purified MAG acyltransferase was specific for MAG and did not utilize any other acyl acceptor such as glycerol, glycerol-3-phosphate, lysophosphatidic acid, and lysophosphatidylcholine. The K(m) values for 1-palmitoylglycerol and 1-oleoylglycerol were 16.39 and 5.65 micrometer, respectively. The K(m) values for 2-monoacylglycerols were 2- to 4-fold higher than that of the corresponding 1-monoacylglycerol. The apparent K(m) values for palmitoyl-, stearoyl-, and oleoyl-CoAs were 17.54, 25.66, and 9.35 micrometer, respectively. Fatty acids, phospholipids, and sphingosine at low concentrations stimulated the enzyme activity. The identification of MAG acyltransferase in oilseeds suggests the presence of a regulatory link between signal transduction and synthesis of complex lipids in plants.     

Preparation of curcumin-carrying nanoemulsions using mono- and diacylglycerides enzymatically structured with bioactive fatty acids

Abstract

Nanoemulsions (NE) are employed as carrier systems of lipophilic active compounds that have low bioavailability and need to be protected from the environment; such as curcumin. In this study, enzymatically prepared monoacylglycerides (MAG) and diacylglycerides (DAG) structured with three bioactive lipids: conjugated linoleic acid (CLA), medium chain fatty acids (MCFA), and ω-3 fatty acids (ω-3?FA). The highest amounts of MAG and DAG containing ω-3 (68.8%) were obtained at 75?°C, 3:1 substrate molar ratios (SMR), 15% of Novozyme 435 and 400?rpm for 90?min. MAG and DAG containing CLA (96.84%) were successfully produced at 40?°C, 1:1 w/w, 15% of lipase RMLM and 400?rpm, for 60?min. Finally, MCFA (95.16%) were prepared at 50?°C with a substrates ratio of 1:1 w/w, 5% of Novozyme 435, and 300?rpm for 30?min. Seven nanoemulsions were formulated using: MAG-ω-3, DAG-ω-3, MAG-CLA, and DAG-CLA individually, as well as reaction mixtures containing MAG and DAG, and MCFA. All exhibited desirable characteristics of average particle size (d?<?200?nm), polydispersity index (PDI; <0.2) and zeta potential (≈?30?mV) and in most cases, the NE maintained their physical properties for up to 5 weeks. It was also determined that when using the reaction mixture containing a high percentage of MAG and DAG of each lipid, the resulting NE exhibited better performance.

• Practical applications

• Nanoemulsions are a new and novel carrier for the transportation of bioactive compounds that can be affected during digestion. Then, the use of the enzyme-catalysed synthesis of structured lipids as emulsifiers is an alternative for increasing the bioavailability of compounds such as curcumin.

     

Myelin-associated glycoprotein interacts with ganglioside GT1b. A mechanism for neurite outgrowth inhibition

Myelin-associated glycoprotein (MAG) is expressed on myelinating glia and inhibits neurite outgrowth from post-natal neurons. MAG has a sialic acid binding site in its N-terminal domain and binds to specific sialylated glycans and gangliosides present on the surface of neurons, but the significance of these interactions in the effect of MAG on neurite outgrowth is unclear. Here we present evidence to suggest that recognition of sialylated glycans is essential for inhibition of neurite outgrowth by MAG. Arginine 118 on MAG is known to make a key contact with sialic acid. We show that mutation of this residue reduces the potency of MAG inhibitory activity but that residual activity is also a result of carbohydrate recognition. We then go on to investigate gangliosides GT1b and GD1a as candidate MAG receptors. We show that MAG specifically binds both gangliosides and that both are expressed on the surface of MAG-responsive neurons. Furthermore, antibody cross-linking of cell surface GT1b, but not GD1a, mimics the effect of MAG, in that neurite outgrowth is inhibited through activation of Rho kinase. These data strongly suggest that interaction with GT1b on the neuronal cell surface is a potential mechanism for inhibition of neurite outgrowth by MAG.     

Select nutrients in the ovine uterine lumen. IX. Differential effects of arginine, leucine, glutamine, and glucose on interferon tau, ornithine decarboxylase, and nitric oxide synthase in the ovine conceptus

Nutrients are primary requirements for development of conceptuses (embryo and extraembryonic membranes), including protein synthesis. We have shown that arginine (Arg), leucine (Leu), and glucose stimulate protein synthesis through phosphorylation of MTOR signaling molecules, thereby increasing proliferation of ovine trophectoderm cells. This study determined whether Arg, Leu, glutamine (Gln), and glucose influence gene expression and protein synthesis in explant cultures of ovine conceptuses recovered from ewes on Day 16 of pregnancy. Conceptuses were deprived of select nutrients and then cultured with either Arg, Leu, Gln, or glucose for 18 h, after which they were analyzed for abundance of MTOR, RPS6K, RPS6, EIF4EBP1 (also known as 4EBP1), IFNT, NOS2, NOS3, GCH1, and ODC1 mRNAs and proteins. Levels of MTOR, RPS6K, RPS6, and EIF4EBP1 mRNAs were not affected by treatment with any of the select nutrients. Similarly, expression of IFNT, NOS2, NOS3, and ODC1 mRNAs were not different. Interestingly, GCH1 mRNA levels increased in response to Arg treatment. Importantly, Arg, Leu, Gln, and glucose increased the abundance of phosphorylated MTOR, RPS6K, RPS6, and EIF4EBP1 proteins as well as NOS and ODC1 proteins, but only Arg increased the abundance of IFNT protein. These findings indicate that Arg, Leu, Gln, and glucose stimulate translation of mRNAs to increase synthesis of proteins through phosphorylation and activation of components of the MTOR signaling pathway. Increases in abundance of IFNT protein (the pregnancy recognition signal), NOS2, NOS3 and GCH1 for conversion of Arg to nitric oxide, and ODC1 for synthesis of polyamines are all important for growth and development of the ovine conceptus during pregnancy.     

Blood chemistry changes in food-deprived herring gulls

Blood chemistry changes caused by food-stress was measured in fledgling herring gulls (Larus argentatus). Increases of corticosterone, T3, free fatty acids, glucose, cholesterol and alpha-amino n-butyric acid were observed. Significant decreases were found in the concentrations of 14 of the 26 free amino acids studied.     

Effects of cod bradykinin and its analogs on vascular and intestinal smooth muscle of the Atlantic cod, Gadus morhua.

The effects of [Arg(0),Trp(5),Leu(8)]-BK (cod [Arg(0)]BK) on vascular preparations from branches of the cod celiac artery and on longitudinal smooth muscle preparations from the cod intestine were investigated. Cod [Arg(0)]BK (3 x 10(-8) M) caused a relaxation of the celiac artery precontracted with adrenaline. The relaxation was abolished by the cyclooxygenase inhibitor indomethacin, suggesting that the effect is mediated through the release of prostaglandins, but there was no evidence for the involvement of leukotrienes or nitric oxide in the response. In the intestinal preparations, cod [Arg(0)]BK produced concentration-dependent contractions (pD(2) = 8.28 +/- 0.16). Experiments with N-terminally and C-terminally truncated analogs and with alanine-substituted analogs of cod [Arg(0)]BK demonstrate that the central amino acid Gly(4) and the C-terminal amino acids Leu(8) and Arg(9) are the most important in determining the conformation of the peptide that interacts with the receptor. The results indicate that the ligand binding properties of the cod BK receptor are considerably different from the receptor present in trout tissues and may resemble those of the mammalian B(2) receptor more closely.     

Structural and functional characterization of mutants of recombinant single-chain urokinase-type plasminogen activator obtained by site-specific mutagenesis of Lys158, Ile159 and Ile160

Single-chain urokinase-type plasminogen activator (scu-PA) is converted to urokinase by hydrolysis of the Lys158-Ile159 peptide bond. Site-directed mutagenesis of Lys158 to Gly or Glu yields plasmin-resistant mutants with a 10-20-fold reduced catalytic efficiency for the activation of plasminogen [Nelles et al. (1987) J. Biol. Chem. 262, 5682-5689]. In the present study, we have further evaluated the enzymatic properties of derivatives of recombinant scu-PA (rscu-PA), produced by site-directed mutagenesis of Lys158, Ile159 or Ile160, in order to obtain additional information on the structure/function relations underlying the enzymatic properties of the single- and two-chain u-PA moieties. [Arg158]rscu-PA (rscu-PA with Lys158 substituted with Arg) appeared to be indistinguishable from wild-type rscu-PA with respect to plasminogen-activating potential (catalytic efficiency k2/Km = 0.21 mM-1 s-1 versus 0.64 mM-1 s-1), conversion to active two-chain urokinase by plasmin (k2/Km = 0.13 microM-1 s-1 versus 0.28 microM-1 s-1), as well as its specific activity (48,000 IU/mg as compared to 60,000 IU/mg) and its fibrinolytic potential in a plasma medium (50% lysis in 2 h with 2.8 micrograms/ml versus 2.1 micrograms/ml). [Pro159]rscu-PA (Ile159 substituted with Pro) and [Gly159]rscu-PA (Ile159 converted to Gly) are virtually inactive towards plasminogen (k2/Km less than 0.004 mM-1 s-1). They are however converted to inactive two-chain derivatives by plasmin following cleavage of the Arg156-Phe157 peptide bond in [Pro159]rscu-PA and of the Lys158-Gly159 peptide bond in [Gly159]rscu-PA. [Gly158,Lys160]rscu-PA (with Lys158 converted to Gly and Ile160 to Lys) has a low catalytic efficiency towards plasminogen both as a single-chain form (k2/Km = 0.012 mM-1 s-1) and as the two-chain derivative (k2/Km = 0.13 mM-1 s-1) generated by cleavage of both the Arg156-Phe157 and/or the Lys160-Gly161 peptide bonds by plasmin. These findings suggest that the enzymatic properties of rscu-PA are critically dependent on the amino acids in position 158 (requirement for Arg or Lys) and position 159 (requirement for Ile). Conversion of the basic amino acid in position 158 results in a 10-20-fold reduction of the catalytic efficiency of the single-chain molecule but yields a fully active two-chain derivative. The presence of Ile in position 159 is not only a primary determinant for the activity of the two-chain derivative, but also of the single-chain precursor. Cleavage of the Arg156-Phe157 or the Lys160-Gly161 peptide bonds by plasmin yields inactive two-chain derivatives.