An in-line digestive cartridge increases enteral fat and vitamin absorption in a porcine model of short bowel syndrome.

BACKGROUND & AIMS: Short bowel syndrome (SBS) occurs after intestinal loss resulting in parenteral nutrition dependence and micronutrient deficiencies, which may lead to life-limiting complications. ALC-078 is a cartridge containing immobilized lipase that connects in-line with enteral feeding sets and digests fats in enteral nutrition (EN). In this study, we evaluate the efficacy of ALC-078 to improve fat and nutrient absorption in a porcine SBS model. METHODS: Fifteen male Yorkshire piglets were assessed. Animals were randomized to no intestinal resection (n = 5), 75% resection (n = 5), or 75% resection + ALC-078 (n = 5). After recovery, animals were treated for 14 days. Piglets received 60% of nutrition from continuous EN and 40% from chow. The degree of fat malabsorption was determined by the coefficient of fat absorption (CFA) following a 72-h stool collection. Body weight, fat-soluble vitamins, and nutritional markers were assessed. RESULTS: Adverse events were similar across the three groups (P = 1.00). ALC-078-treated animals had similar weight gain compared to resected piglets. Resected animals had a lower CFA compared to unresected controls (79.3% vs. 95.2%, P = 0.01) while there was no significant difference in the ALC-078 animals (87.1% vs. 95.2%, P = 0.19). Between Study Days 1 and 15, ALC-078 animals had increased concentrations of vitamin D (12.2 vs. 8.7 ng/mL, P = 0.0006), and vitamin E (4.3 vs. 2.5 mg/L, P = 0.03). These markers did not significantly change in untreated resected animals. CONCLUSION: ALC-078 increases the absorption of fat-soluble vitamins and may improve fat malabsorption. Future studies should determine whether ALC-078 can reduce PN dependence and if these findings translate to human patients with SBS.

Correlating amino acid conservation with function in tyrosyl-tRNA synthetase.

Sequence comparisons have been combined with mutational and kinetic analyses to elucidate how the catalytic mechanism of Bacillus stearothermophilus tyrosyl-tRNA synthetase evolved. Catalysis of tRNA(Tyr) aminoacylation by tyrosyl-tRNA synthetase involves two steps: activation of the tyrosine substrate by ATP to form an enzyme-bound tyrosyl-adenylate intermediate, and transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). Previous investigations indicate that the class I conserved KMSKS motif is involved in only the first step of the reaction (i.e. tyrosine activation). Here, we demonstrate that the class I conserved HIGH motif also is involved only in the tyrosine activation step. In contrast, one amino acid that is conserved in a subset of the class I aminoacyl-tRNA synthetases, Thr40, and two amino acids that are present only in tyrosyl-tRNA synthetases, Lys82 and Arg86, stabilize the transition states for both steps of the tRNA aminoacylation reaction. These results imply that stabilization of the transition state for the first step of the reaction by the class I aminoacyl-tRNA synthetases preceded stabilization of the transition state for the second step of the reaction. This is consistent with the hypothesis that the ability of aminoacyl-tRNA synthetases to catalyze the activation of amino acids with ATP preceded their ability to catalyze attachment of the amino acid to the 3' end of tRNA. We propose that the primordial aminoacyl-tRNA synthetases replaced a ribozyme whose function was to promote the reaction of amino acids and other small molecules with ATP.

Stabilization of the transition state for the transfer of tyrosine to tRNA(Tyr) by tyrosyl-tRNA synthetase.

Aminoacylation of tRNA(Tyr) involves two steps: (1) tyrosine activation to form the tyrosyl-adenylate intermediate; and (2) transfer of tyrosine from the tyrosyl-adenylate intermediate to tRNA(Tyr). In Bacillus stearothermophilus tyrosyl-tRNA synthetase, Asp78, Tyr169, and Gln173 have been shown to form hydrogen bonds with the alpha-ammonium group of the tyrosine substrate during the first step of the aminoacylation reaction. Asp194 and Gln195 stabilize the transition state complex for the first step of the reaction by hydrogen bonding with the 2'-hydroxyl group of AMP and the carboxylate oxygen atom of tyrosine, respectively. Here, the roles that Asp78, Tyr169, Gln173, Asp194, and Gln195 play in catalysis of the second step of the reaction are investigated. Pre-steady-state kinetic analyses of alanine variants at each of these positions shows that while the replacement of Gln173 by alanine does not affect the initial binding of the tRNA(Tyr) substrate, it destabilizes the transition state complex for the second step of the reaction by 2.3 kcal/mol. None of the other alanine substitutions affects either the initial binding of the tRNA(Tyr) substrate or the stability of the transition state for the second step of the aminoacylation reaction. Taken together, the results presented here and the accompanying paper are consistent with a concerted reaction mechanism for the transfer of tyrosine to tRNA(Tyr), and suggest that catalysis of the second step of tRNA(Tyr) aminoacylation involves stabilization of a transition state in which the scissile acylphosphate bond of the tyrosyl-adenylate species is strained. Cleavage of the scissile bond on the breakdown of the transition state alleviates this strain.

The 'KMSKS' motif in tyrosyl-tRNA synthetase participates in the initial binding of tRNA(Tyr).

Variants at each position of the 'KMSKS' signature motif in tyrosyl-tRNA synthetase have been analyzed to test the hypothesis that this motif is involved in catalysis of the second step of the aminoacylation reaction (i.e., the transfer of tyrosine from the enzyme-bound tyrosyl-adenylate intermediate to the tRNA(Tyr) substrate). Pre-steady-state kinetic studies show that while the rate constants for tyrosine transfer (k(4)) are similar to the wild-type value for all of the mobile loop variants, the K230A and K233A variants have increased dissociation constants (K(d)(tRNA)( )()= 2.4 and 1.7 microM, respectively) relative to the wild-type enzyme (K(d)(tRNA)( )()= 0.39 microM). In contrast, the K(d)(tRNA) values for the F231L, G232A, and T234A variants are similar to that of the wild-type enzyme. The K(d)(tRNA) value for a loop deletion variant, Delta(227-234), is similar to that for the K230A/K233A double mutant variant (3.4 and 3.0 microM, respectively). Double mutant free energy cycle analysis indicates there is a synergistic interaction between the side chains of K230 and K233 during the initial binding of tRNA(Tyr) (DeltaDeltaG(int) = -0.74 kcal/mol). These results suggest that while the 'KMSKS' motif is important for the initial binding of tRNA(Tyr) to tyrosyl-tRNA synthetase, it does not play a catalytic role in the second step of the reaction. These studies provide the first kinetic evidence that the 'KMSKS' motif plays a role in the initial binding of tRNA(Tyr) to tyrosyl-tRNA synthetase.

Pharmacologic characterization of botulinum toxin for basic science and medicine.

The use of Botulinum neurotoxin (BoNT) is increasing in both clinical and basic science. Clinically, intramuscular injection of nanogram quantities of BoNT is fast becoming the treatment of choice for a spectrum of disorders including movement disorders such as torticollis, blepharospasm, Meige Disease, and hemifacial spasm (Borodic et al., 1991, 1994a; Jankovic and Brin, 1991; Clarke, 1992). Neuroscientists are using BoNTs as tools to develop a better understanding of the mechanisms underlying the neurotransmitter release process. Consequently, our ability to accurately and reliably quantify the biologic activity of botulinum toxin has become more important than ever. The accurate measurement of the pharmacologic activity of BoNTs has become somewhat problematic with the most significant problems occurring with the clinical use of the toxins. The biologic activity of BoNTs has been measured using a variety of techniques including assessment of whole animal responses to in vitro effects on neurotransmitter release. The purpose of this review is to examine the approaches employed to characterize, quantify and investigate the actions of the BoNTs and to provide a guide to aid investigators in determining which of these methods is most appropriate for their particular application or use.

Human tyrosyl-tRNA synthetase shares amino acid sequence homology with a putative cytokine.

To test the hypothesis that tRNATyr recognition differs between bacterial and human tyrosyl-tRNA synthetases, we sequenced several clones identified as human tyrosyl-tRNA synthetase cDNAs by the Human Genome Project. We found that human tyrosyl-tRNA synthetase is composed of three domains: 1) an amino-terminal Rossmann fold domain that is responsible for formation of the activated E.Tyr-AMP intermediate and is conserved among bacteria, archeae, and eukaryotes; 2) a tRNA anticodon recognition domain that has not been conserved between bacteria and eukaryotes; and 3) a carboxyl-terminal domain that is unique to the human tyrosyl-tRNA synthetase and whose primary structure is 49% identical to the putative human cytokine endothelial monocyte-activating protein II, 50% identical to the carboxyl-terminal domain of methionyl-tRNA synthetase from Caenorhabditis elegans, and 43% identical to the carboxyl-terminal domain of Arc1p from Saccharomyces cerevisiae. The first two domains of the human tyrosyl-tRNA synthetase are 52, 36, and 16% identical to tyrosyl-tRNA synthetases from S. cerevisiae, Methanococcus jannaschii, and Bacillus stearothermophilus, respectively. Nine of fifteen amino acids known to be involved in the formation of the tyrosyl-adenylate complex in B. stearothermophilus are conserved across all of the organisms, whereas amino acids involved in the recognition of tRNATyr are not conserved. Kinetic analyses of recombinant human and B. stearothermophilus tyrosyl-tRNA synthetases expressed in Escherichia coli indicate that human tyrosyl-tRNA synthetase aminoacylates human but not B. stearothermophilus tRNATyr, and vice versa, supporting the original hypothesis. It is proposed that like endothelial monocyte-activating protein II and the carboxyl-terminal domain of Arc1p, the carboxyl-terminal domain of human tyrosyl-tRNA synthetase evolved from gene duplication of the carboxyl-terminal domain of methionyl-tRNA synthetase and may direct tRNA to the active site of the enzyme.

Analysis of the role of the KMSKS loop in the catalytic mechanism of the tyrosyl-tRNA synthetase using multimutant cycles.

A mobile loop in tyrosyl-tRNA synthetase, which corresponds to the KMSKS signature sequence of class I aminoacyl-tRNA synthetases, destabilizes the E.Tyr.ATP complex but stabilizes the following E.[Tyr-ATP]not equal to transition state for the formation of E.Tyr-AMP. Three amino acid residues in the mobile loop, K230, K233, and T234, are known to be primarily responsible for these effects. We now analyze the network of interactions between these three amino acids using multiple mutant free energy cycles. The complete characterization of the coupling energies within the mobile loop allows each of the steps leading to the formation of the transition state complex to be dissected into its energetic components. In particular, it is found that, in the absence of a functional mobile loop, there is synergistic coupling between the tyrosine and ATP substrates (i.e., each enhances the binding affinity of the other) which stabilizes the E.Tyr.ATP intermediate preceding the transition state complex. Thus, the mobile loop disrupts the synergism between the ATP and tyrosine substrates, using the ATP binding energy to stabilize the transition state for the reaction. Whereas the net effect of the mobile loop in the E.Tyr.ATP complex results from several conflicting side chain interactions that tend to offset each other, conflicting interactions in the E.[Tyr-ATP]not equal to transition state complex have been minimized and stabilizing pairwise interactions between the K230, K233, and T234 side chains are optimized. The tight coupling between the side chains of K230, K233, and T234 suggests that the mobile loop adopts a highly constrained conformation during formation of the transition state complex. These results quantitatively demonstrate the importance of side chain interactions in enzyme catalysis and illustrate the use of binding energy to stabilize the transition state of a reaction and the presence of unfavorable interactions to destabilize the ground state.

The median paralysis unit: a more pharmacologically relevant unit of biologic activity for botulinum toxin.

Although the LD50 has been used to quantify the biologically active toxin in clinical preparations of botulinum A toxin (Botox and Dysport), a discrepancy exists between the clinical potency of equivalent international units of different formulations of botulinum A toxin for multiple clinical indications. Our laboratory previously reported that a regional chemodenervation assay in the mouse could be utilized to detect the difference in the potencies of the clinical preparations of toxin [Pearce et al. (1994) Toxic. appl. Pharmac. 128, 69-77]. The purpose of this study was to quantify the regional paralysis produced by botulinum toxin and define a new pharmacologic/biologic unit of activity that more accurately reflects the mechanism of action of botulinum toxin in the clinical setting. Quantal analysis of regional paralysis revealed that the ED50, defined as the median paralysis unit (MPU) for Botox and Dysport, was 0.41 +/- 0.01 and 1.00 +/- 0.02 LD50 units, respectively. Differences in the potencies found in retrospective clinical studies comparing Botox and Dysport were accurately reflected, for the first time, by the dose of toxin expressed in terms of the MPU (median paralysis unit). The data suggested that the MPU may be a more appropriate measure of the biologic activity in therapeutic formulations of botulinum toxin.

Measurement of botulinum toxin activity: evaluation of the lethality assay.

The use of the mouse lethality assay for the estimation of the biologic activity of botulinum toxin was evaluated. The relationship between the number of animals, number of doses, and duration of the assay used to estimate the LD50 and the precision of the assay was investigated. The results of these studies demonstrated that the LD50 for botulinum toxin can be estimated with a high degree of precision (+/- 5%). The precision of the assay is not increased by using more than a 5-dose 50-animal assay or extending the duration of the assay beyond 72 hr. Estimates of the LD50 obtained at 48 hr were only slightly less precise but underestimated the LD50 by 15%. Analysis of the commercially available preparations of botulinum toxin with the mouse LD50 assay revealed significant discrepancies between the units of toxin in these preparations. In addition, a 2.67-fold difference in the relative potency of the two preparations of botulinum A toxin was observed using a regional chemodenervation assay that measures paralysis. The mouse LD50 assay could not detect this large difference in the potency of the two approved clinical preparations of botulinum toxin. The results of these studies demonstrate that although the mouse LD50 assay can be used to estimate the number of units of botulinum toxin with a high degree of precision this assay alone is not an adequate method for assessing the preclinical biological potency of botulinum toxin.

Involvement of threonine 234 in catalysis of tyrosyl adenylate formation by tyrosyl-tRNA synthetase.

There is a mobile loop in the tyrosyl-tRNA synthetase that contains the KMSKS signature sequence of class I aminoacyl-tRNA synthetases. As it has not been possible to determine the role of the mobile loop in catalysis from X-ray crystallographic studies, we are investigating its importance by a series of site-directed mutagenic and kinetic studies. Here we examine the role of threonine 234 (T234) in the catalysis of tyrosyl adenylate formation by tyrosyl-tRNA synthetase from Bacillus stearothermophilus. This residue is the carboxy-terminal residue in the signature sequence and is either a serine or threonine in eight of the ten class I aminoacyl-tRNA synthetases isolated from Escherichia coli. Kinetic analyses of tyrosyl adenylate formation in the mutant enzymes indicate that k3, the forward rate constant for the formation of tyrosyl adenylate, is reduced 500-fold on mutation of T234 to alanine. In contrast, mutation of T234 to serine results in only a 4-fold decrease in k3, suggesting that the loss of the hydroxyl group in the T234A mutant is responsible for its decreased reaction rate. Deletion of the hydroxyl group destabilizes the transition state for the formation of tyrosyl adenylate by 2.7 kcal/mol. The transition state is also destabilized by 1.4 kcal/mol on the mutation of K230 to alanine. The effects of mutation of both T234 and K230 to alanine are less than additive; there is a coupling energy of -1.3 kcal/mol in the transition state. The effects of mutating K230 and T234 to alanine are also nonadditive in the E.Tyr-AMP complex (coupling energy = -1.9 kcal/mol).(ABSTRACT TRUNCATED AT 250 WORDS)

Mutation of lysine 233 to alanine introduces positive cooperativity into tyrosyl-tRNA synthetase.

Tyrosyl-tRNA synthetase from Bacillus stearothermophilus is a dimeric enzyme which displays half-of-sites reactivity with respect to the binding of both tyrosine and ATP. The binding of both substrates follows Michaelis-Menten kinetics. Mutation of lysine 233 to alanine (K233A) decreases the affinity of the active subunit for ATP at both saturating and subsaturating tyrosine concentrations (from the Hill plot, kcat = 0.56 s-1, nH = 1.54, Kd = 372 mM at 50 microM tyrosine). In addition, this mutant displays sigmoidal kinetics (characteristic of positive cooperativity) with respect to the binding of ATP. These two effects can be reversed by the addition of NaCl (0.5 m final concentration) or by a second alanine mutation at either position K230 or T234. The effect of either NaCl or second site mutation is to increase the binding affinity of the K233A mutant for ATP (KATP values are 22 mM for the K233A mutant in the presence of 0.5 M NaCl, 0.16 mM for the K230A/K233A mutant, and 0.14 mM for the K233A/T234A mutant). With the restoration of the tight binding of ATP, Michaelis-Menten kinetics are restored since the kinetic analysis of tyrosyl adenylate formation involves only binding of ATP to the active subunit. It is likely that the physical mechanism for the positive cooperativity present in the K233A mutant actually exists in the wild-type enzyme but is not observed kinetically due to the initial binding of ATP to the active subunit. These results indicate that, in some cases, a decrease in substrate affinity is sufficient to introduce cooperativity into a noncooperative enzyme.

Mutational and kinetic analysis of a mobile loop in tyrosyl-tRNA synthetase.

The role of a mobile loop in tyrosyl-tRNA synthetase has been investigated by mutating each amino acid in the loop and kinetically analyzing the effect that each mutation has on the formation of the enzyme-bound tyrosyl adenylate intermediate. Kinetic analyses of mutations at three of the nine positions in the loop, K230, K233, and T234, have been reported elsewhere (Fersht et al., 1988; First & Fersht, 1993a,b). In this paper, the kinetic analyses of mutants in the remaining six positions, as well as a mutant in which the entire loop is deleted, are reported. With the exception of E235, which stabilizes the E.-[Tyr-ATP]++ and E.Tyr.ATP complexes by 1.0 and 1.2 kcal/mol, respectively, none of the remaining amino acids appears to be directly involved in the catalytic mechanism of the enzyme. Instead, mutation of these residues results in small alterations in the stability of E.Tyr.ATP, E.[Tyr-ATP]++ and E.Tyr.AMP.PPi complexes. The precise amino acid residues which stabilize each state vary, suggesting that the loop adopts different conformations in each of the complexes with the most highly constrained conformation being in the E.[Tyr-ATP]++ complex. Deletion of the loop reveals that the net effect of the loop in catalysis is two-fold: (1) to destabilize the E.Tyr.ATP complex preceding formation of the E.[Tyr.ATP]++ complex and (2) to stabilize the E.[Tyr-ATP]++ complex, indicating that the involvement of the loop in catalysis occurs at the expense of ATP-binding energy.(ABSTRACT TRUNCATED AT 250 WORDS)

Fluorescence energy transfer between cysteine 199 and cysteine 343: evidence for MgATP-dependent conformational change in the catalytic subunit of cAMP-dependent protein kinase.

The catalytic subunit of cAMP-dependent protein kinase has two cysteine residues, Cys 199 and Cys 343, which are protected against alkylation by MgATP [Nelson, N. C., & Taylor, S. S. (1981) J. Biol. Chem. 256, 3743]. While Cys 199 is in close proximity to the active site of the catalytic subunit and is probably directly protected against alkylation by MgATP, the mechanism by which MgATP prevents alkylation of Cys 343 is unclear. To determine whether MgATP directly protects Cys 343 from alkylation by being in close proximity to both Cys 199 and the MgATP binding site, fluorescence resonance energy transfer techniques were used to measure the distance between Cys 199 and Cys 343. Two different donor-acceptor pairs containing 4-[N-[(iodoacetoxy)ethyl]-N-methylamino]-7-nitrobenz-2-oxa-1,3-diazole at Cys 199 as the acceptor and either 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene-2, 8- dione or N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine at Cys 343 as the donor were prepared following the method described in the preceding paper [First, E. A., & Taylor, S. S. (1989) Biochemistry (preceding paper in this issue)]. From the efficiencies of fluorescence resonance energy transfer for each donor-acceptor pair, the distance between Cys 199 and Cys 343 was estimated to be between 31 and 52 A. Since Cys 199 is close to the MgATP binding site and since MgATP cannot extend beyond a distance of 16 A, it is unlikely that Cys 343 at a distance of at least 31 A from Cys 199 is in direct contact with the bound nucleotide.(ABSTRACT TRUNCATED AT 250 WORDS)

Selective modification of the catalytic subunit of cAMP-dependent protein kinase with sulfhydryl-specific fluorescent probes.

The catalytic subunit of cAMP-dependent protein kinase contains only two cysteine residues, and the side chains of both Cys 199 and Cys 343 are accessible. Modification of the catalytic subunit by a variety of sulfhydryl-specific reagents leads to the loss of enzymatic activity. The differential reactivity of the two sulfhydryl groups at pH 6.5 has been utilized to selectively modify each cysteine with the following fluorescent probes: 3,6,7-trimethyl-4-(bromomethyl)-1,5-diazabicyclo[3.3.0]octa-3,6-diene- 2,8-dione, N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine, and 4-[N-[(iodoacetoxy)ethyl]-N-methyl-amino]-7-nitrobenz-2-oxa-1,3-diazole. The most reactive cysteine is Cys 199, and exclusive modification of this residue was achieved with each reagent at pH 6.5. Modification of Cys 343 required reversible blocking of Cys 199 with 5,5'-dithiobis(2-nitrobenzoic acid) followed by reaction of Cys 343 with the fluorescent probe at pH 8.3. Treatment of this modified catalytic subunit with reducing reagent restored catalytic activity by unblocking Cys 199. In contrast, catalytic subunit that was selectively labeled at Cys 199 by the fluorescent probes was catalytically inactive. Even though Cys 199 is presumably close to the interaction site between the regulatory subunit and the catalytic subunit, all of the modified C-subunits retained the capacity to aggregate with the type II regulatory subunit in the absence of cAMP, and the resulting holoenzymes were dissociated in the presence of cAMP.(ABSTRACT TRUNCATED AT 250 WORDS)

CAMP-dependent protein kinase: prototype for a family of enzymes.

Protein kinases represent a diverse family of enzymes that play critical roles in regulation. The simplest and best-understood biochemically is the catalytic (C) subunit of cAMP-dependent protein kinase, which can serve as a framework for the entire family. The amino-terminal portion of the C subunit constitutes a nucleotide binding site based on affinity labeling, labeling of lysines, and a conserved triad of glycines. The region beyond this nucleotide fold also contains essential residues. Modification of Asp 184 with a hydrophobic carbodiimide leads to inactivation, and this residue may function as a general base in catalysis. Despite the diversity of the kinase family, all share a homologous catalytic core, and the residues essential for nucleotide binding or catalysis in the C subunit are invariant in every protein kinase. Affinity labeling and intersubunit cross-linking have localized a portion of the peptide binding site, and this region is variable in the kinase family. The crystal structure of the C subunit also is being solved. The C subunit is maintained in its inactive state by forming a holoenzyme complex with an inhibitory regulatory (R) subunit. This R subunit has a well-defined domain structure that includes two tandem cAMP binding domains at the carboxy-terminus, each of which is homologous to the catabolite gene activator protein in Escherichia coli. Affinity labeling with 8N3 cAMP has identified residues that are in close proximity to the cAMP binding sites and is consistent with models of the cAMP binding sites based on the coordinates of the CAP crystal structure. An expression vector was constructed for the RI subunit and several mutations have been introduced. These mutations address 1) the major site of photoaffinity labeling, 2) a conserved arginine in the cAMP binding site, and 3) the consequences of deleting the entire second cAMP binding domain.