Identification and Characterization of New Members of Vacuolar H<Superscript>+</Superscript>-Pyrophosphatase Family from <Emphasis Type="Italic">Oryza sativa</Emphasis> Genome

The vacuolar H⁺-pyrophosphatase (V-PPase) is an electrogenic H⁺ pump localized in the plant vacuolar membrane. V-PPase from many species has been characterized previously and the corresponding genes/cDNAs have been cloned. Cloning of the V-PPase genes from many plant species has revealed conserved motifs that may correspond to catalytic sites. The completion of the entire DNA sequence of Oryza sativa (430 Mb) presented an opportunity to study the structure and function of V-PPase proteins, and also to identify new members of this family in Oryza sativa. Our analysis identified three novel V-PPase proteins in the Oryza sativa genome that contain functional domains typical of V-PPase. We have designated them as OVP3 to OVP5. The new predicted OVPs have chromosomal locations different from previously characterized V-PPases (OVP1 and OVP2) located on chromosome 6. They all contain three characteristic motifs of V-PPase and also a conserved motif [DE]YYTS, specific to type I V-PPases and involved in coupling PP_i hydrolysis to H⁺ translocation.     

Molecular cloning,characterization and expression of Mn-superoxide dismutase from the rubber tree (Hevea brasiliensis)

The gene and the RNA from Arabidopsis thaliana for the plastid-located glycerol-3-phosphate acyltransferase (GPAT; EC 2.3.1.15) and their encoded product have been studied. The gene (designated ATS1) was isolated by screening a DASH genomic library for cross-hybridization with a radiolabeled probe prepared from cDNA for GPAT from squash. cDNA clones representing the mRNA were isolated by screening a ZAPII cDNA library for hybridization with a radiolabeled probe prepared from a DNA fragment of ATS1. The nucleotide sequences of the gene and the cDNA were determined, and the 5 end of the RNA was mapped by primer extension. Sequences similar to the TATA box, polyadenylation sequences and intron-splicing sequences were found at the expected locations. The pre-mRNA was 3288 nucleotides long and contained 5 and 3-untranslated sequences of 57 and 442 nucleotides, respectively. The coding sequence of 1377 nucleotides was interrupted by 11 introns of 1412 nucleotides in total and the 3-untranslated sequence contained another intron of 94 nucleotides. The open-reading frame encoded a polypeptide of 459 amino acid residues, the amino acid sequence of which was highly homologous to those of precursors to plastid-located GPATs from squash and pea. The enzymatic activity of a gene product that was over-produced in Escherichia coli confirmed the indentity of the gene.Abbreviations ACP acyl carrier protein - GPAT glycerol-3-phosphate acyltransferase - IPTG isopropyl--thiogalactopyranoside.     

Sodium efflux from perfused giant algal cells

Internodal cells of the giant alga Chara corallina were perfused internally to replace the native cytoplasm, tonoplast and vacuole with artificial cytoplasm. Sodium efflux from perfused cells, measured by including ²²Na in the perfusion media, was increased by increasing the internal sodium concentration and by decreasing the external pH, and was inhibited by external application of the renal diuretic amiloride. The sodium efflux was markedly ATP-dependent, with a 50-fold decrease in efflux observed after perfusion with media lacking ATP. Efflux in the presence of ATP was reduced by 33% by inclusion of 10 M N,N-dicyclohexylcarbodiimide in the perfusion medium. The membrane potential of the perfused cells approximated that of intact cells from the same culture. It is suggested that sodium efflux in perfused Chara cells proceeds via a secondary antiporter with protons, regulated by ATP in a catalytic role and with the proton motive force acting as the energy source.Abbreviations DCCD N,N-dicyclohexylcarbodiimide - EGTA ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid - Mes 2-(N-morpholino)ethanesulphonic acid - Mops 3(N-morpholino)propanesulphonic acid - Taps tris(hydroxymethyl)methylaminopropanesulphonic acid     

Role of metal ferrocyanides in chemical evolution

Adsorption of several ribose and 2-deoxyribose 5-nucleotides on zinc- and copper ferrocyanides has been studied at a neutral pH of 7.01. The Langmuir adsorption isotherm was used to determine the values of K_L and X_m. Both types of nucleotides, ribose and 2-deoxyribose, showed similar adsorption behavior on zinc- and copper ferrocyanides. Zinc ferrocyanide showed larger adsorption as compared to copper ferrocyanide. Purine nucleotides adsorbed more than pyrimidine nucleotides on both the metal ferrocyanides probably because of an additional binding site in the imidazole ring in purines. Results of the present study suggest the importance of metal ferrocyanides and metal ions in stabilization of nucleotides during processes of prebiotic condensation reactions.     

Optimization of the synthesis of porcine somatotropin in Escherichia coli

We report on the influence of choice of promoter and RNA polymerase, 5-untranslated regions and ribosome binding sites, codon usage, leader peptide coding sequences and poly A tail in the 3-untranslated region on the synthesis of porcine somatotropin (PST) in Escherichia coli. A total of 12 different constructs were tested in this study for the production of porcine somatotropin (PST) in E. coli. Several factors have significant effects on PST synthesis. In the presence of a strong promoter and a strong ribosome binding site, the next most important factor seems to be the combination of sequences at the 5-end of the mRNA including both the 5-untranslated region and the start of the coding sequence. Codon usage in the 5-coding sequence per se is not important in determining the level of PST synthesis where high level expression is achieved from a strong ribosome binding site. However, where low level synthesis of recombinant PST (rPST) is achieved, codon usage in the 5-coding sequence is important in determining the level of PST synthesis. Leader sequences dramatically reduce the level of PST synthesis. The presence of a poly A tail in the 3-untranslated region has no significant effect on PST synthesis.     

Facile synthesis of 2′-substituted lactoses

Isopropylidenation of lactose with 2,2-dimethoxypropane in the presence ofp-toluenesulfonic acid gave two products, which were identified by¹H- and¹³C-NMR as 2,35,63,4-tri-O-isopropylidenelactose dimethyl acetal (1) and its 6-O-(2-methoxy)-isopropyl derivative (2). These products were used for the synthesis of 2-O-methyllactose (7), 2,6-di-O-methyllactose (9) and 2-O-benzyllactose (13).     

Synthesis of oligoguanylates on oligocytidylate templates

Summary Short oligocytidylates can act as templates for the self-condensation of guanosine 5-phosphorimidazolide. In the absence of a catalytic metal ion or in the presence of Pb²⁺ a noticeable template effect is already observed with the dimer and the yield of long oligomers reaches a plateau with a hexamer template. Short templates give oligomers longers than the template length. The products are predominantly 2-5 linked for the Pb²⁺-catalyzed reaction while mixed linkages are observed in the uncatalyzed reaction.In the presence of Zn²⁺, a template effect is first observed with the pentamer and is maximal by the heptamer. The products are predominantly 3-5 linked. Oligomers shorter than or as long as the template are obtained in substantial yield, and longer products in much lower yields.Abbreviations G Guanosine - Gp guanosine 2(3)-phosphate - pG guanosine 5-phosphate - Gp! guanosine cyclic 2,3-phosphate - ImpG guanosine 5-phosphorimidazolide - ImpG^* [8-¹⁴C]-guanosine 5-phosphorimidazolide - pGp 5-phosphoguanosine 2(3)-phosphate - G²pG guanylyl-[2-5]-guanosine - G³pG guanylyl-[3-5]-guanosine - ImpGpG 5-phosphorimidazolide of GpG - (pG)_n (n = 2,3) oligomers of pG - GppG P₁, P₂-diguanosine 5-diphosphate - GppGpG 5-[guanosine 5-pyrophosphate] of GpG - NH₂pG guanosine 5-phosphoramidate - (pG)₄₊ tetramer and higher oligoguanylates with 5 terminal phosphate - oligo(G) oligoguanylate - Cp cytidine 2(3)-phosphate - Cp! cytidine cyclic 2,3-phosphate - (Cp)_n–1 Cp! (n= 2,3,4) oligocytidylates terminated by 5-OH groups and 2,3-cyclic phosphates - oligo(C) oligocytidylate - poly(C) polycytidylic acid - poly(U) polyuridylic acid - poly(C,G) random copolymer of C and G - BAP bacterial alkaline phosphatase (E. coli) - EDTA ethylenediaminetetraacetic acid - R_f chromatographic mobility     

Analogues of NADP+ as inhibitors and coenzymes for NADP+ malic enzyme from maize leaves

Structural analogues of the NADP⁺ were studied as potential coenzymes and inhibitors for NADP⁺ dependent malic enzyme from Zea mays L. leaves. Results showed that 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate ( NADP⁺), 3-acetylpyridine-adenine dinucleotide phosphate (APADP⁺), nicotinamide-hypoxanthine dinucleotide phosphate (NHDP⁺) and -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate (23NADPc⁺) act as alternate coenzymes for the enzyme and that there is little variation in the values of the Michaelis constants and only a threefold variation in V_max for the five nucleotides. On the other hand, thionicotinamide-adenine dinucleotide phosphate (SNADP⁺), 3-aminopyridine-adenine dinucleotide phosphate (AADP⁺), adenosine 2-monophosphate (2AMP) and adenosine 2: 3-cyclic monophosphate (23AMPc) were competitive inhibitors with respect to NADP⁺, while -nicotinamide adenine dinucleotide 3-phosphate (3NADP⁺), NAD⁺, adenosine 3-monophosphate (3AMP), adenosine 2: 5-cyclic monophosphate (25AMPc), 5AMP, 5ADP, 5ATP and adenosine act as non-competitive inhibitors. These results, together with results of semiempirical self-consistent field-molecular orbitals calculations, suggest that the 2-phosphate group is crucial for the nucleotide binding to the enzyme, whereas the charge density on the C₄ atom of the pyridine ring is the major factor that governs the coenzyme activity.Abbreviations NADP⁺ 1, N⁶-etheno-nicotinamide adenine dinucleotide phosphate - NHDP⁺ nicotinamide-hypoxanthine dinucleotide phosphate - APADP⁺ 3-acetylpyridine-adenine dinucleotide phosphate - SNADP⁺ thionicotinamide-adenine dinucleotide phosphate - AADP⁺ 3-aminopyridine-adenine dinucleotide phosphate - 23NADPc⁺ -nicotinamide adenine dinucleotide 2: 3-cyclic monophosphate - 3NADP⁺ -nicotinamide adenine dinucleotide 3-phosphate - 2AMP adenosine 2-monophosphate - 3AMP adenosine 3-monophosphate - 23AMPc adenosine 2: 3 monophosphate cyclic - A adenosine - RuBP ribulose 1,5-bisphosphate - SCF-MO Self-Consistent Field-Molecular Orbitals (method)     

Abundance and half-life of the distinct oat phytochrome A3 and A4 mRNAs

Gene-preferential oligonucleotide probes were used to determined the relative abundance and half-lives of distinct oat phytochrome A (PHYA) mRNAs. Oat PHYA mRNAs are highly conserved in the 5-untranslated region and the coding region, but the 3-untranslated region has an overall lower sequence conservation and was the source of gene-preferential probes. PHYA3 mRNA was estimated to be ca. 61% of the oat PHYA mRNA pool present in poly(A)⁺ RNA from dark-grown seedlings. The half-lives for PHYA3 and PHYA4 mRNAs were both estimated to be ca. 30 min, and a similar short half-life was estimated for the average PHYA mRNA. Sequence comparisons of PHYA mRNAs from four grass species identified conserved sequences within the 5- and 3-untranslated regions that might be important for PHYA mRNA degradation.     

Enzymic synthesis,chemical characterisation and Sda activity of GalNAcß1-4[NeuAcα2-3]Galß1-4GlcNAc and GalNAcß1-4[NeuAcα2-3]Galß1-4Glc

The tetrasaccharides GalNAcß1-4[NeuAc2-3]Galß1-4Glc and GalNAcß1-4[NeuAc2-3]Galß1-4GlcNAc were synthesised by enzymic transfer of GalNAc from UDP-GalNAc to 3-sialyllactose (NeuAc2-3Galß1-4Glc) and 3-sialyl-N-acetyllactosamine (NeuAc2-3Galß1-4GlcNAc). The structures of the products were established by methylation and¹H-500 MHz NMR spectroscopy. In Sd^a serological tests the product formed with 3-sialyl-N-acetyllactosamine was highly active whereas that formed with 3-sialyllactose had only weak activity.     

Two-dimensional 1H NMR study of two cyclobutane type photodimers of thymidylyl-(3′→5′)-thymidine

2D NMR spectroscopy and J coupling constant analysis are applied to resolve the structure of two photoproducts of thymidylyl-(35)-thymidine. These products are cyclobutane type thymine dimers possessing the cis-syn (the predominant one) and trans-syn geometry. The cis-syn is formed in an ANTI-ANTI conformation about the N-glycosyl linkages and resembles the normal base-stacked configuration. The glycosidic conformation in solution of the 5 terminal fragment differs from the crystal in which the less common SYN conformation is observed. In this isomer only the sugar pucker of the 3 terminal fragment is changed substantially with respect to the dinucleotide. The trans-syn isomer is formed in a SYN-ANTI glycosidic conformation. In this isomer the sugar puckers of both deoxyribose rings are affected and a preference for a pure 2-endo conformation is observed.Abbreviations dTpdT 2-deoxythymidylyl-(35)-2-deoxythymidine - dTp[]dT cyclobutane type photodimers of dTpdT - dTp- and dTp[]- their 5' terminal fragments (fragment A) - -pdT and-[]pdT their 3 terminal fragments (fragment B) - RP-HPLC reversed-phase high-performance liquid chromatography - COSY two-dimensional correlated spectroscopy - 2D NOE two-dimensional nuclear Overhauser spectroscopy     

Regioselective preparation of 2′, 3′-di-O-acylribonucleosides carrying lipophilic acyl groups through a lipase-catalysed alcoholysis

Candida antarctica B lipase-catalysed alcoholysis of 2, 3, 5-tri-O-hexanoyluridine (1a), 2, 3, 5-tri-O-dodecanoyluridine (1b), 2, 3, 5-tri-O-hexanoylinosine (1c) and 2, 3, 5-tri-O-dodecanoylinosine (1d) proceeded regioselectively to produce the corresponding 2, 3-di-O-acylribonucleosides 2a–d, providing a simple and efficient access to these new lipophilic compounds. Contrasting to the alcoholysis, enzymatic hydrolysis of 1a–d using different enzymes and experimental conditions did not proceed regioselectively.     

Cyanamide mediated syntheses under plausible primitive earth conditions

Summary When an aqueous solution (pH 7.0) of deoxythymidine 5-phosphate, 4-amino-5-imidazolecarboxamide and cyanamide was dried and heated for 18 h at 60°C, P¹, P²-dideoxythymidine 5-pyrophosphate (I) was formed in a 58% yield. Oligonucleotides were not detected in the reaction product. Under conditions employed in the above reaction, (I) was shown to be stable. In prebiotic polymerization reactions employing deoxythymidine 5-triphosphate as the polymerizing species, (I) could therefore function as a primer and minimize the formation of cyclic nucleotides.Abbreviations dT deoxythymidine - dTMP deoxythymidine 5-phosphate - dTppT P¹, P²-dideoxythymidine 5-pyrophosphate - dTTP deoxythymidine 5-triphosphate - AICA 4-amino-5-imidazolecarboxamide     

The effects of carbodiimides on functions associated with the energy-conservation mechanism in beef heart sub-mitochondrial particles

N,N-di-n-propyl-,N,Ndi-n-butyl-,N,N-di-n-pentyl-,N,Ndi-n-hexyl-,N,Ndi-n-octoyl-,N,N-dibenzhydryl-, andN,N-dibenzhydrylcarbodiimides were synthesized. They were all effective inhibitors (2 nmoles carbodiimide per milligram protein) of the ATP-driven reduction of NAD by succinate and the ATP-driven transhydrogenase activities catalyzed by beef heart submitochondrial particles (SMP). They had no effect on the nonenergylinked transhydrogenase and stimulated the succinate-driven aerobic transhydrogenase activity of beef heart SMP. It was concluded that they exert their effects by reacting with theN,N-dicyclohexylcarbodiimide-binding protein. Water-soluble carbodiimides were not effective inhibitors.     

Molecular cloning of the cDNA encoding a stress-inducible protein phosphatase 1 (PP1) catalytic subunit from French bean (Phaseolus vulgaris L.)

A cDNA showing high sequence similarity (>70%) to plant protein phosphatase 1 catalytic subunit variants from other species has been isolated from a cDNA library derived from mRNAs expressed in elicitor-treated suspension-cultured cells. The clone appears to be a near full-length 1431 bp with a 172 bp 5-untranslated region and a 317 bp 3-untranslated region. The open reading frame, determined by sequence similarity, codes for a protein with predicted M _r of 35552. Alternatively an ATG situated to the 5 end of the putative start site would increase the protein size by 6 amino acids.The mRNA for Pvpp1 was shown to be rapidly induced by elicitor treatment of suspension-cultured cells of French bean. The cloned cDNA represents one of the few examples of a gene product that is probably involved in dephosphorylation events arising after the initial responses to biotic stress.Abbreviations PAL phenylalanine ammonia-lyase - PP1 protein phosphatase 1 - Pvpp1 Phaseolus vulgaris protein phosphatase 1     

Structure and function of the yeast vacuolar membrane proton ATPase

Our current work on a vacuolar membrane proton ATPase in the yeastSaccharomyces cerevisiae has revealed that it is a third type of H⁺-translocating ATPase in the organism. A three-subunit ATPase, which has been purified to near homogeneity from vacuolar membrane vesicles, shares with the native, membrane-bound enzyme common enzymological properties of substrate specificities and inhibitor sensitivities and are clearly distinct from two established types of proton ATPase, the mitochondrial F₀F₁-type ATP synthase and the plasma membrane E₁E₂-type H⁺-ATPase. The vacuolar membrane H⁺-ATPase is composed of three major subunits, subunita (M _r =67 kDa),b (57kDa), andc (20 kDa). Subunita is the catalytic site and subunitc functions as a channel for proton translocation in the enzyme complex. The function of subunitb has not yet been identified. The functional molecular masses of the H⁺-ATPase under two kinetic conditions have been determined to be 0.9–1.1×10⁵ daltons for single-cycle hydrolysis of ATP and 4.1–5.3×10⁵ daltons for multicycle hydrolysis of ATP, respectively.N,N-Dicyclohexylcarbodiimide does not inhibit the former reaction but strongly inhibits the latter reaction. The kinetics of single-cycle hydrolysis of ATP indicates the formation of an enzyme-ATP complex and subsequent hydrolysis of the bound ATP to ADP and Pi at a 7-chloro-4-nitrobenzo-2-oxa-1,3-diazolesensitive catalytic site. Cloning of structural genes for the three subunits of the H⁺-ATPase (VMA1, VMA2, andVMA3) and their nucleotide sequence determination have been accomplished, which provide greater advantages for molecular biological studies on the structure-function relationship and biogenesis of the enzyme complex. Bioenergetic aspects of the vacuole as a main, acidic compartment ensuring ionic homeostasis in the cytosol have been described.Abbreviations CCCP carbonyl cyanidem-chlorophenyl hydrazone - DCCD N,N-dicyclohexylcarbondiimide - DES diethylstilbestrol - DIDS 4,4-diisothiocyano-2,2-stilbene disulfonic acid - NBD-Cl 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole - Pi inorganic phosphate - SDS sodium dodecylsulfate - SF6847 3,5-di-tert-butyl-4-hydroxybenzylidenemalononitrile - SITS 4-acetamide-4-isothiocyanatostilbene-2,2-disulfonic acid - ZW3-14 N-tetradecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate     

Chemo-enzymatic synthesis of 2′-O-methoxyethyl ribonucleosides using a phosphodiesterase from Serratia marcescens

An enzyme able to cleave the 3,5-phosphate ring of 2-methoxyethyl cyclic nucleotides (3,5-cyclic nucleotide phosphodiesterase, EC 3.1.4.17) from Serratia marcescens DSM 30121 was used to deprotect the cyclic phosphate nucleotides after chemical alkylation. The process yielded 2-O-alkylated nucleosides used as building blocks of antisense oligonucleotides for subsequent potential applications in therapeutics (antisense oligonucleotide synthesis) and diagnostics. The phosphodiesterase from the Gram-negative enteric bacterium S. marcescens was selected on account of the broad substrate range and high activity of the enzyme. The protein was purified by heat-treatment of the crude cell-free extract, followed by column chromatography (gel filtration). It was characterised and showed optimal activity at a broad pH range (pH 6.8–9.4, with a peak at ca. pH 8.5) and at a temperature of 60–65°C. No metal ions were required for activity, although Ba²⁺ was an activator. Conversion of 2-O-methoxyethyl cAMP into the corresponding nucleoside derivative on a multi-gram scale was successfully performed in two steps, using the S. marcescens enzyme in conjunction with a commercially available alkaline phosphatase from Escherichia coli.     

Negative-ion fast atom bombardment and electrospray ionization tandem mass spectrometry for characterization of sulfated and sialyl Lewis-type glycosphingolipids

Structural characterization of sulfated and sialyl Lewis (Le)-type glycosphingolipids performed by fast atom bombardment (FAB) and electrospray ionization (ESI) mass spectrometry is described. Both FAB and ESI collision-induced dissociation tandem mass spectrometry (CID-MS/MS) of acidic glycosphingolipids allowed identification of the sulfated or sialyl sugar, and provided information on the saccharide chain sequence. The negative-ion tandem FABMS of sulfated Le-type glycosphingolipids having the non-reducing end trisaccharide ion as the precursor can be used to differentiate the Le^a- and Le^X-type oligosaccharides. The ESI CID-MS/MS of multiple-charged ions provided even more detailed structural information, and some of the useful daughter ions appeared with higherm/z values than the precusor because of a lower charge-state. These methodologies can be applied to the structural analyses of glycoconjugates with much larger molecular masses and higher polarity, such as the poly-sulfated and sialyl analogues.Abbreviations CID collision-induced dissociation - ESI electrospray ionization - FABMS fast atom bombardment mass spectrometry - Fuc fucose - Gal galactose - GlcNAc N-acetylglucosamine - Le Lewis - Le^a Lewis^a - Le^X Lewis^X - MS/MS mass spectrometry/mass spectrometry - NeuAc N-acetylneuraminic acid - 3-SO₄-Le^a 3-sulfated Le^a pentaosyl ceramide - 3-SO₄-Le^X 3-sulfated Le^X pentaosyl ceramide - 2,3-SO₄-Le^X 2,3-disulfated Le^X pentaosyl ceramide - 3-S-Le^a 3-sialyl Le^a pentaosyl ceramide - 3-S-Le^x 3-sialyl Le^X heptaosyl ceramide - 3-S-Le^X-Le^X 3-sialyl-Le^x-Le^x octaosyl ceramide.     

Nucleotide sequence analysis of a cDNA clone encoding the 34K movement protein gene of odontoglossum ringspot virus,ORSV-Cy,the Korean isolate

The partial nucleotide sequence of the 3-terminal region of the Korean isolate of odontoglossum ringspot tobamovirus (ORSV-Cy) from cool-growing Cymbidium was determined. The sequence contained a full length open reading frame (ORF) coding for the viral cell-to-cell movement protein (MP). The ORF was located upstream of the coat protein gene and 105 nucleotides longer than that of tobacco mosaic virus (TMV). The ORF predicts a polypeptide chain of 303 amino acids with a molecular weight of 33573. The ORF contained a similar region of conserved sequence motif of tobamoviruses and putative assembly origin of the viral RNA was located at about 1,100 nucleotides away from the 3 end. The predicted amino acid sequence for the MP gene of ORSV-Cy is more closely related to pepper mild mottle virus (PMMV), TMV-vulgare and TMV-Rakkyo than to tobacco mild green mosaic virus (TMGMV), TMV-L, cowpea strain of TMV (SHMV), and cucumber green mottle mosaic virus (CGMMV).     

Rotational diffusion tensor of nucleic acids from 13C NMR relaxation

Rotational diffusion properties have been derived for the DNA dodecamer d(CGCGAATTCGCG)₂ from ¹³C R₁ and R₁ measurements on the C₁, C₃, and C₄ carbons in samples uniformly enriched in ¹³C. The narrow range of C-H bond vector orientations relative to the DNA axis make the analysis particularly sensitive to small structural deviations. As a result, the R₁/R₁ ratios are found to fit poorly to the crystal structures of this dodecamer, but well to a recent solution NMR structure, determined in liquid crystalline media, even though globally the structures are quite similar. A fit of the R₁/R₁ ratios to the solution structure is optimal for an axially symmetric rotational diffusion model, with a diffusion anisotropy, D_||/D, of 2.1±0.4, and an overall rotational correlation time, (2D_||+4D)^–1, of 3.35 ns at 35 °C in D₂O, in excellent agreement with values obtained from hydrodynamic modeling.