Reversal of H-reflex operant conditioning in the rat

After developing a rapid gel filtration method to prepare pure and stable apoenzyme forms of D-amino acid oxidase from the yeast Rhodotorula gracilis, we carried out comparative kinetic studies on the reconstitution to holoenzyme (with FAD) of the intact (40 kDa) and proteolyzed (38.3 kDa) apoenzyme forms of this oxidase. Changes in catalytic activity and flavin and protein fluorescence revealed that in both cases reconstitution was biphasic. The proteolyzed enzyme was catalytically competent, but unlike the intact form was unable to dimerize following formation of the apoprotein-FAD complex. We present evidence that reconstitution of holoenzyme from apoenzyme plus FAD does not involve dimerization, and that dimerization is not necessary for expression of DAAO activity. We propose that both apoenzyme forms share a common reconstitution mechanism, which includes a step of conformational interconversion of an enzymatically active intermediate to the final holoenzyme.     

Interaction of pyridoxal 5'-phosphate with tryptophan-139 at the subunit interface of dimeric D-amino acid transaminase

The crystal structure of dimeric bacterial D-amino acid transaminase shows that the indole rings of the two Trp-139 side chains face each other in the subunit interface about 10 angstroms from the coenzyme, pyridoxal 5'-phosphate. To determine whether it has a role in the catalytic efficiency of the enzyme or interacts with the coenzyme, Trp-139 has been substituted by several different types of amino acids, and the properties of these recombinant mutant enzymes have been compared to the wild-type enzyme. In the native wild-type holoenzyme, the fluorescence of one of the three Trp residues per monomer is almost completely quenched, probably due to its interaction with PLP since in the native wild-type apoenzyme devoid of PLP, tryptophan fluorescence is not quenched. Upon reconstitution of this apoenzyme with PLP, the tryptophan fluorescence is quenched to about the same extent as it is in the native wild-type enzyme. The site of fluorescence quenching is Trp-139 since the W139F mutant in which Trp-139 is replaced by Phe has about the same amount of fluorescence as the wild-type enzyme. The circular dichroism spectra of the holo and the apo forms of both the wild-type and the W139F enzymes in the far-ultraviolet show about the same degree of ellipticity, consistent with the absence of extensive global changes in protein structure. Furthermore, comparison of the circular dichroism spectrum of the W139F enzyme at 280 nm with the corresponding spectral region of the wild-type enzyme suggests a restricted microenvironment for Trp-139 in the latter enzyme. The functional importance of Trp-139 is also demonstrated by the finding that its replacement by Phe, His, Pro, or Ala gives mutant enzymes that are optimally active at temperatures below that of the wild-type enzyme and undergo the E-PLP --> E-PMP transition as a function of D-Ala concentration with reduced efficiency. The results suggest that a fully functional dimeric interface with the two juxtaposed indole rings of Trp-139 is important for optimal catalytic function and maximum thermostability of the enzyme and, furthermore, that there might be energy transfer between Trp-139 and coenzyme PLP.     

UV resonance Raman-selective amide vibrational enhancement: quantitative methodology for determining protein secondary structure

We have directly determined the amide band resonance Raman spectra of the "average" pure alpha-helix, beta-sheet, and unordered secondary structures by exciting within the amide pi-->pi* transitions at 206.5 nm. The Raman spectra are dominated by the amide bands of the peptide backbone. We have empirically determined the average pure alpha-helix, beta-sheet, and unordered resonance Raman spectra from the amide resonance Raman spectra of 13 proteins with well-known X-ray crystal structures. We demonstrate that we can simultaneously utilize the amide I, II, and III bands and the Calpha-H amide bending vibrations of these average secondary structure spectra to directly determine protein secondary structure. The UV Raman method appears to be complementary, and in some cases superior, to the existing methods, such as CD, VCD, and absorption spectroscopy. In addition, the spectra are immune to the light-scattering artifacts that plague CD, VCD, and IR absorption measurements. Thus, it will be possible to examine proteins in micelles and other scattering media.     

An apyrase from Mimosa pudica contains N5,N10-methenyl tetrahydrofolate and is stimulated by light

An apyrase (NTP/NDPase) implicated in the response of Mimosa pudica to stimuli, such as touch, has been cloned, sequenced and expressed in Escherichia coli. While purifying and characterizing this enzyme, it was observed that a chromophore is associated with it, having absorption in the ultraviolet-A/blue region of the spectrum. The absorbance maximum of the chromophore, purified from the enzyme complex by gel filtration and HPLC, is around 350 nm. The chromophore has been identified as N5,N10-methenyl tetrahydrofolate (MTHF) by comparing the excitation and emission spectra of synthetic MTHF and the isolated cofactor, and by reconstitution of the enzyme complex with synthetic MTHF. Upon excitation with light (350 nm), an increase of apyrase activity was observed in the purified or reconstituted holoenzyme but not in the apoenzyme. The wavelength dependence of the light stimulation matched well with the fluorescence excitation spectra of the cofactor, MTHF. Possible implications of the results for signal transduction in M. pudica have been discussed.     

Optical spectroscopy of nicotinoprotein alcohol dehydrogenase from Amycolatopsis methanolica: a comparison with horse liver alcohol dehydrogenase and UDP-galactose epimerase

The NADH absorbance spectrum of nicotinoprotein (NADH-containing) alcohol dehydrogenase from Amycolatopsis methanolica has a maximum at 326 nm. Reduced enzyme-bound pyridine dinucleotide could be reversibly oxidized by acetaldehyde. The fluorescence excitation spectrum for NADH bound to the enzyme has a maximum at 325 nm. Upon excitation at 290 nm, energy transfer from tryptophan to enzyme-bound NADH was negligible. The fluorescence emission spectrum (excitation at 325 nm) for NADH bound to the enzyme has a maximum at 422 nm. The fluorescence intensity is enhanced by a factor of 3 upon binding of isobutyramide (Kd = 59 microM). Isobutyramide acts as competitive inhibitor (Ki = 46 microM) with respect to the electron acceptor NDMA (N,N-dimethyl-p-nitrosoaniline), which binds to the enzyme containing the reduced cofactor. The nonreactive substrate analogue trifluoroethanol acts as a competitive inhibitor with respect to the substrate ethanol (Ki = 1.6 microM), which binds to the enzyme containing the oxidized cofactor. Far-UV circular dichroism spectra of the enzyme containing NADH and the enzyme containing NAD+ were identical, indicating that no major conformational changes occur upon oxidation or reduction of the cofactor. Near-UV circular dichroism spectra of NADH bound to the enzyme have a minimum at 323 nm (Deltaepsilon = -8.6 M-1 cm-1). The fluorescence anisotropy decay of enzyme-bound NADH showed no rotational freedom of the NADH cofactor. This implies a rigid environment as well as lack of motion of the fluorophore. The average fluorescence lifetime of NADH bound to the enzyme is 0.29 ns at 20 degreesC and could be resolved into at least three components (in the range 0.13-0.96 ns). Upon binding of isobutyramide to the enzyme-containing NADH, the average excited-state lifetime increased to 1.02 ns and could be resolved into two components (0.37 and 1.11 ns). The optical spectra of NADH bound to nicotinoprotein alcohol dehydrogenase have blue-shifted maxima compared to other NADH-dehydrogenase complexes, but comparable to that observed for NADH bound to horse liver alcohol dehydrogenase. The fluorescence lifetime of NADH bound to the nicotinoprotein is very short compared to enzyme-bound NADH complexes, also compared to NADH bound to horse liver alcohol dehydrogenase. The cofactor-protein interaction in the nicotinoprotein alcohol dehydrogenase active site is more rigid and apolar than that in horse liver alcohol dehydrogenase. The optical properties of NADH bound to nicotinoprotein alcohol dehydrogenase differ considerably from NADH (tightly) bound to UDP-galactose epimerase from Escherichia coli. This indicates that although both enzymes have NAD(H) as nonexchangeable cofactor, the NADH binding sites are quite different.     

Quinohaemoprotein ethanol dehydrogenase from Comamonas testosteroni. Purification, characterization, and reconstitution of the apoenzyme with pyrroloquinoline quinone analogues

Pyrroloquinoline-quinone(PQQ)-free quinohaemoprotein ethanol dehydrogenase (QH-EDH) apoenzyme was isolated from ethanol-grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N-terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could be separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo-QH-EDH and holo-QH-EDH showed g values typical for low-spin haem c proteins. In partially oxidized holo-QH-EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o-quinone/o-quinol moiety is not essential for binding but it is for activity.     

Crystal structure of eucaryotic E3, lipoamide dehydrogenase from yeast

The crystal structure of eucaryotic lipoamide dehydrogenase from yeast has been determined by an X-ray analysis at 2.7 (partially at 2.4) A resolution. The enzyme has two identical subunits related by a pseudo twofold symmetry. The tertiary structure is similar to those of other procaryotic enzymes. The active site, consisting of FAD, Cys44, and Cys49 from one subunit and His457' from the other subunit, is highly conserved. This enzyme is directly bound to the core protein E2 of the 2-oxoglutarate dehydrogenase complex, whereas it is bound to the pyruvate dehydrogenase complex through a protein X. The calculated electrostatic potential suggests two characteristic regions for binding with these two proteins.     

Fluorescence-energy transfer in human estradiol 17 beta-dehydrogenase-NADPH complex and studies on the coenzyme binding,

Fluorescence spectroscopy was used to examine the interaction between human estradiol 17 beta-dehydrogenase (estrogenic 17 beta-hydroxysteroid dehydrogenase, 17 beta-HSD) and the cofactor NADPH. After the binding of NADPH to the enzyme, there was an emission enhancement at 436 nm following an excitation at 295 nm, as compared to the cofactor alone. This phenomenon was attributed to a radiationless transfer of excitation energy from 17 beta-HSD to the enzyme-bound cofactor. The distance of 2.69 nm, between the bound NADPH and the sole tryptophan residue (Trp46) within one subunit, has been determined using fluorescence energy transfer. This result coincides very well with the same distance, recently calculated from the crystallographic coordinates obtained by Ghosh et al. [Ghosh, D., Pletnev, V. Z., Zhu, D.-W., Wawrzak, Z., Duax, W. L., Pangborn, W., Labrie, F. & Lin, S.-X. (1995) Structure 3, 503-513]. Compared to free NADPH, the fluorescence emission of enzyme-bound NADPH was increased in intensity and its maximum blue-shifted from 457 nm to 436 nm. Binding of NADPH to 17 beta-HSD was studied by fluorescence titration. The enzyme binds two molecules of NADPH with a Kd = 0.73 +/- 0.2 microM. The dissociation constant was further confirmed by the method of coenzyme protection against cold inactivation of the enzyme. The binding was little altered in the presence of estradiol-17 beta. The environment of tryptophan residues on the surface of the enzyme is discussed.     

FAD-mediated enzymatic conversion of NAD+ to NADH: application to chiral synthesis of L-lactate

Electroenzymatic reduction of NAD+ to NADH for subsequent use in enzymatic synthesis has been carried out at carbon electrodes bearing lipoamide dehydrogenase (LiDH) immobilized under a Nafion film. The self-mediated electron transfer was made possible by an excess of flavin adenine dinucleotide (FAD) entrapped together with LiDH. Results were compared to those obtained with a similar electrode containing both LiDH and a polymeric form of FAD (pFAD) prepared by anodic polymerization of FAD.     

Structural changes of horseradish peroxidase in presence of low concentrations of urea

The presence of very low concentrations of the widely used denaturant urea induces structural changes in the monomeric heme-containing enzyme, horseradish peroxidase (HRP). Structural alterations in the protein were reflected in quenching studies of tryptophan fluorescence using the widely used quencher acrylamide. Stern-Volmer quenching constants measured in presence of urea, even in concentrations below 100 mm, were higher than those measured in absence of the denaturant. The fluorescence emission maximum of 1, 8-ANS, used as a probe for monitoring conformational changes in the enzyme, was blue-shifted from 530 nm in aqueous buffer to 518 nm when incorporated in native HRP. This blue shift increased further by 3 nm in presence of HRP preincubated with 100 mm urea, whereupon it steadily decreased with increasing urea concentration to become zero at 8 m urea. The mean fluorescence lifetime of 1,8-ANS incorporated in HRP was much higher than that of ANS in aqueous buffer, and showed continuous variation with the concentration of urea in which the enzyme was incubated. Systematic changes in the microenvironment of the heme moiety in HRP were also reflected in the visible CD spectra of the enzyme incubated with low concentrations of urea. These results are consistent with those of our earlier studies performed with the denaturant guanidinium chloride and indicate structural relaxation of HRP, with retention of enzymatic activity and native-like secondary structure, in presence of millimolar concentrations of urea.     

Mutation of an active site residue of tryptophan synthase (beta-serine 377) alters cofactor chemistry

To better understand how an enzyme controls cofactor chemistry, we have changed a tryptophan synthase residue that interacts with the pyridine nitrogen of the pyridoxal phosphate cofactor from a neutral Ser (beta-Ser377) to a negatively charged Asp or Glu. The spectroscopic properties of the mutant enzymes are altered and become similar to those of tryptophanase and aspartate aminotransferase, enzymes in which an Asp residue interacts with the pyridine nitrogen of pyridoxal phosphate. The absorption spectrum of each mutant enzyme undergoes a pH-dependent change (pKa approximately 7.7) from a form with a protonated internal aldimine nitrogen (lambdamax = 416 nm) to a deprotonated form (lambdamax = 336 nm), whereas the absorption spectra of the wild type tryptophan synthase beta2 subunit and alpha2 beta2 complex are pH-independent. The reaction of the S377D alpha2 beta2 complex with L-serine, L-tryptophan, and other substrates results in the accumulation of pronounced absorption bands (lambdamax = 498-510 nm) ascribed to quinonoid intermediates. We propose that the engineered Asp or Glu residue changes the cofactor chemistry by stabilizing the protonated pyridine nitrogen of pyridoxal phosphate, reducing the pKa of the internal aldimine nitrogen and promoting formation of quinonoid intermediates.     

Time-resolved EPR studies with DNA photolyase: excited-state FADH0 abstracts an electron from Trp-306 to generate FADH-, the catalytically active form of the cofactor

Photolyase repairs UV-induced cyclobutane-pyrimidine dimers in DNA by photoinduced electron transfer. The enzyme isolated from Escherichia coli contains 5,10-methenyltetrahydrofolate, which functions as the light-harvesting chromophore, and fully reduced flavin adenine dinucleotide (FAD), which functions as the redox catalyst. During enzyme preparation, the flavin is oxidized to FADH0, which is catalytically inert. Illumination of the enzyme with 300- to 600-nm light converts the flavin to the fully reduced form in a reaction that involves photooxidation of an amino acid in the apoenzyme. The results of earlier optical studies had indicated that the redox-active amino acid in this photoactivation process was tryptophan. We have now used time-resolved electron paramagnetic resonance (EPR) spectroscopy to investigate the photoactivation reaction. Excitation of the flavin-radical-containing inactive enzyme produces a spin-polarized radical that we identify by 2H and 15N labeling as originating from a tryptophan residue, confirming the inferences from the optical work. These results and Trp-->Phe replacement by site-directed mutagenesis reveal that flavin radical photoreduction is achieved by electron abstraction from Trp-306 by the excited-state FADH0. Analysis of the hyperfine couplings and spin density distribution deduced from the isotopic-labeling results shows that the product of the light-driven redox chemistry is the Trp-306 cation radical. The results strongly suggest that the active form of photolyase contains FADH- and not FADH2.     

The Na(+)-translocating NADH:ubiquinone oxidoreductase from the marine bacterium Vibrio alginolyticus contains FAD but not FMN

The Na(+)-translocating NADH:ubiquinone oxidoreductase from Vibrio alginolyticus was extracted from the bacterial membranes and purified by ion exchange chromatographic procedures. The enzyme catalyzed NADH oxidation by suitable electron acceptors, e.g. menadione, and the Na+ and NADH-dependent reduction of ubiquinone-1. Four dominant bands and a number of minor bands were visible on SDS-PAGE that could be part of the enzyme complex. Flavin analyses indicated the presence of FAD but no FMN in the purified enzyme. FAD but no FMN were also present in V. alginolyticus membranes. FAD is therefore a prosthetic group of the Na(+)-translocating NADH:ubiquinone oxidoreductase and FMN is not present in the enzyme. The FAD was copurified with the NADH dehydrogenase. The purified enzyme exhibited an absorption spectrum with a maximum at 450 nm that is typical for a flavoprotein. Upon incubation with NADH this absorption disappeared indicating reduction of the enzyme-bound FAD.     

Human regulatory subunit RI beta of cAMP-dependent protein kinases: expression, holoenzyme formation and microinjection into living cells

The human regulatory subunit RI beta of cAMP-dependent protein kinases was expressed in Escherichia coli as a fusion protein with glutathione S-transferase. Purification was performed by affinity chromatography on glutathione-agarose beads after cleavage with thrombin. The human recombinant RI beta protein migrated at 55 kDa on SDS-PAGE and displayed immunoreactivity with an anti-human RI beta antiserum. Furthermore, the purified recombinant RI beta protein was shown to exist as a dimer that was able to form holoenzyme with the catalytic subunit C alpha. The rate of RI beta 2C alpha 2 holoenzyme formation was faster in the presence than in the absence of MgATP. The kinase activity measured before and after adding cAMP to the holoenzyme showed that the presence of cAMP resulted in holoenzyme dissociation and release of active C alpha-subunit, due to cAMP binding to RI beta. Compared to a RI alpha 2C alpha 2 holoenzyme, the RI beta 2C alpha 2 holoenzyme exhibited a more than twofold higher sensitivity to cAMP. The subcellular localization of RI beta was analyzed in quiescent REF-52 fibroblasts and Wistar rat thyroid (WRT) cells after microinjection of fluorescently labeled proteins into the cytoplasm. A cytoplasmic distribution was observed when free RI beta was injected, whereas free C alpha injected into the cytoplasm appeared in the nucleus. When holoenzymes with labeled RI beta and unlabeled C alpha, or unlabeled RI beta and labeled C alpha, were injected, unstimulated cells showed fluorescence in the cytoplasm of both cell types. REF-52 cells stimulated with 8-bromo-cAMP (8-Br-cAMP) and WRT cells treated with thyrotropin (TSH) showed fluorescence mainly in the cytoplasm when RI beta was the labeled subunit of the in vivo dissociated holoenzyme. In contrast, nuclear fluorescence was evident from the release and translocation of labeled C alpha from the holoenzyme complex after stimulation with 8-Br-cAMP or TSH.     

The actigraph data analysis software: I. A novel approach to scoring and interpreting sleep-wake activity

Intersubunit cross-linked creatine kinase (CK) has been prepared with the cross-linking reagent dithiobis(succinimidyl propionate) (DTSP). Unfolding of cross-linked CK during denaturation by guanidine hydrochloride (GuHCl), as monitored by intrinsic fluorescence, circular dichroism and fluorescence of the hydrophobic probe, 1-aniline-naphthalene-8-sulfonate (ANS), occurs in two stages with increasing GuHCl concentration. The process is similar to that of the unmodified enzyme, but in the second stage, conformational changes of the cross-linked enzyme need higher concentration of GuHCl, suggesting that there is a stable intermediate during its unfolding transition and the intermediate is stabilized by intersubunit cross-linkage.     

Biosynthetic incorporation of tryptophan analogues into staphylococcal nuclease: effect of 5-hydroxytryptophan and 7-azatryptophan on structure and stability

5-Hydroxytryptophan (5HW) and 7-azatryptophan (7AW) are analogue of tryptophan that potentially can be incorporated biosynthetically into proteins and used as spectroscopic probes for studying protein-DNA and protein-protein complexes. The utility of these probes will depend on the extent to which they can be incorporated and the demonstration that they cause minimal perturbation of a protein's structure and stability. To investigate these factors in a model protein, we have incorporated 5HW and 7AW biosynthetically into staphylococcal nuclease A, using a trp auxotroph Escherichia coli expression system containing the temperature-sensitive lambda cI repressor, Both tryptophan analogues are incorporated into the protein with good efficiency. From analysis of absorption spectra, we estimate approximately 95% incorporation of 5HW into position 140 of nuclease, and we estimate approximately 98% incorporation of 7AW, CD spectra of the nuclease variants are similar to that of the tryptophan-containing protein, indicating that the degree of secondary structure is not changed by the tryptophan analogues. Steady-state fluorescence data show emission maxima of 338 nm for 5HW-containing nuclease and 355 nm for 7AW-containing nuclease. Time-resolved fluorescence intensity and anisotropy measurements indicate that the incorporated 5HW residue, like tryptophan at position 140, has a dominant rotational correlation time that is approximately the value expected for global rotation of the protein. Guanidine-hydrochloride-induced unfolding studies show the unfolding transition to be two-state for 5HW-containing protein, with a free energy change for unfolding that is equal to that of the tryptophan-containing protein. In contrast, the guanidine-hydrochloride-induced unfolding of 7AW-containing nuclease appears to show a non-two-state transition, with the apparent stability of the protein being less than that of the tryptophan form.     

Membrane permeability transition as induced by dysfunction of the electron transport chain

A novel activity producing gamma-aminobutyric acid (GABA) from L-ornithine in the presence of NAD(P)+ was found in the crude extract of L-ornithine-induced Hafnia alvei, in addition to L-ornithine decarboxylase (ODC) activity. The reaction system for the former activity consisted of two enzymes, L-ornithine oxidase (decarboxylating, OOD) and gamma-aminobutyraldehyde (GABL) dehydrogenase (GDH). OOD catalyzed the conversion of L-ornithine into GABL, CO2, NH3, and H2O2 in the presence of O2, and GDH dehydrogenated GABL to GABA in the presence of NAD(P)+. OOD, purified to homogeneity, had a high ODC activity and the activity ratio of ODC to OOD was almost constant throughout the purification (ODC/ OOD=160:1). The molecular mass of the OOD was about 230 kDa, probably consisting of three identical subunits of a 77 kDa peptide, and OOD had an absorption maximum at 420 nm as well as at 278 nm, the specific absorption for an enzyme containing pyridoxal phosphate (PLP). The content of PLP was estimated at about 1 mol per subunit. OOD was specific to L-ornithine, and other L-amino acids and polyamines including putrescine were inert. The enzyme was activated by PLP, but not by pyridoxamine 5'-phosphate, FAD, FMN, or pyrroloquinoline quinone, and it was inactivated by hydrazine, semicarbazide, and hydroxylamine. The holoenzyme can be resolved to the apoenzyme by incubation with hydroxylamine, and reconstituted with PLP. These properties of OOD were almost the same as those of ODC separately purified to homogeneity from H. alvei. Zn2+ and Cu2+, butanedione, and sodium borohydride inhibited both OOD and ODC in a similar manner. The OOD reaction required O2 and only the ODC reaction proceeded under anaerobic conditions. The substitution of air for oxygen in the reaction vessel and the addition of catalase-H2O, enhanced only the OOD reaction, resulting in an increase of the ratio of OOD/ODC to 1:30 and 1:4.1, respectively. These results suggested that OOD and ODC are identical and that the former is a side reaction of the latter in the presence of O2.     

Secondary structure analysis of individual transmembrane segments of the nicotinic acetylcholine receptor by circular dichroism and Fourier transform infrared spectroscopy

Circular dichroism (CD) and attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy are used to establish the secondary structure of peptides containing one or more transmembrane segments (M1-M4) of the Torpedo californica nicotinic acetylcholine receptor (AChR). Peptides containing the M2-M3 and M1-M2-M3 transmembrane segments of the AChR beta-subunit and the M4 segment of the alpha- and gamma-subunits were isolated from proteolytic digests of receptor subunits, purified, and reconstituted into lipid vesicles. For each peptide, an amide I vibrational frequency centered between 1650 and 1656 cm-1 and negative CD absorption bands at 208 and 222 nm indicate that the peptide is largely alpha-helical. In addition, the CD spectrum of a tryptic peptide of the alpha-subunit containing the M1 segment is also consistent with a largely alpha-helical structure. However, secondary structure analysis of the alpha-M1 CD spectrum indicates the presence of other structures, suggesting that the M1 segment may represent either a distorted alpha-helix, likely the consequence of several proline residues, or may not be entirely alpha-helical. Overall, these findings are consistent with studies that indicate that the transmembrane region of the AChR comprises predominantly, if not exclusively, membrane-spanning alpha-helices.     

Properties and subunit structure of pig heart pyruvate dehydrogenase

Pyruvate dehydrogenase [EC 1.2.4.1] was separated from the pyruvate dehydrogenase complex and its molecular weight was estimated to be about 150,000 by sedimentation equilibrium methods. The enzyme was dissociated into two subunits (alpha and beta), with estimated molecular weights of 41,000 (alpha) and 36,000 (beta), respectively, by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. The subunits were separated by phosphocellulose column chromatography and their chemical properties were examined. The subunit structure of the pyruvate dehydrogenase was assigned as alpha2beta2. The content of right-handed alpha-helix in the enzyme molecule was estimated to be about 29 and 28% by optical rotatory dispersion and by circular dichroism, respectively. The enzyme contained no thiamine-PP, and its dehydrogenase activity was completely dependent on added thiamine-PP and partially dependent on added Mg2+ and Ca2+. The Km value of pyruvate dehydrogenase for thiamine diphosphate was estimated to be 6.5 X 10(-5) M in the presence of Mg2+ or Ca2+. The enzyme showed highly specific activity for thiamine-PP dependent oxidation of both pyruvate and alpha-ketobutyrate, but it also showed some activity with alpha-ketovalerate, alpha-ketoisocaproate, and alpha-ketoisovalerate. The pyruvate dehydrogenase activity was strongly inhibited by bivalent heavy metal ions and by sulfhydryl inhibitors; and the enzyme molecule contained 27 moles of 5,5'-dithiobis(2-nitrobenzoic acid)-reactive sulfhydryl groups and a total of 36 moles of sulfhydryl groups. The inhibitory effect of p-chloromercuribenzoate was prevented by preincubating the enzyme with thiamine-PP plus pyruvate. The structure of pyruvate dehydrogenase necessary for formation of the complex is also reported.     

Characterization of CD44-mediated hyaluronan binding by renal tubular epithelial cells

Sedimentation equilibrium studies of dilute solutions of tryptophan synthase reveal dissociation from the holoenzyme form, alpha 2 beta 2, into mixtures of alpha beta 2, small amounts of beta 2, and alpha as well as the original alpha 2 beta 2 holoenzyme. The holoenzyme form is stabilized by pyridoxal 5'-phosphate. A new sedimentation equilibrium analytical procedure shows the dissociation of the second alpha subunit to be negatively cooperative. The analytical procedure calculates theoretical error profiles with assumed values of the dissociation constant, k, and a cooperativity parameter until a match is made between one of the theoretical profiles and that computed from experimental data. The latter profile is calculated with an experimentally determined k and assumed values of the cooperativity parameter.