Salt induced B----A transition of poly(dG).poly(dC) and the stabilization of A form by its methylation.

Raman spectra of poly(dG).poly(dC) have been observed in aqueous solutions at various ionic strengths, [NaCl] = 0.03 to 4 M, and at different temperatures, 10 to 60 degrees C. At 30 degrees C, and at [NaCl] = 0.03 M, it was found to have a B-form (with O4'endo-anti guanosine and C2'endo-anti cytidine), whereas, at [NaCl] = 4 M, an A form (with C3'endo-anti guanosine and C3'endo-anti cytidine). At 30 degrees C and [NaCl] = 1 M, namely at an intermediate state, a fraction of this molecules was considered to have a "heteronomous A" form (with O4'endo-anti guanosine and C3' endo-anti cytidine). At 60 degrees C and [NaCl] = 1 M, it assumes the B form, and at 10 degrees C and [NaCl] = 1 M, the A form. Cytosine-5-methylation was found to cause a marked stabilization of the A form. Even at [NaCl] = 0.1 M (at 30 degrees C), a substantial portion of poly(dG).poly(dm5C) was found to have a heteronomous form, in which the dG atrand is in the B form and the dC an A form; it never assumes a complete B form.     

Tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase: guanidine. HCl-induced unfolding and a low temperature requirement for refolding.

Guanidine x HCl (GdnHCl)-induced unfolding of tetrameric N(5)-(L-1-carboxyethyl)-L-ornithine synthase (CEOS; 141,300 M(r)) from Lactococcus lactis at pH 7.2 and 25 degrees C occurred in several phases. The enzyme was inactivated at approximately 1 M GdnHCl. A time-, temperature-, and concentration-dependent formation of soluble protein aggregates occurred at 0.5-1.5 M GdnHCl due to an increased exposure of apolar surfaces. A transition from tetramer to unfolded monomer was observed between 2 and 3.5 M GdnHCl (without observable dimer or trimer intermediates), as evidenced by tyrosyl and tryptophanyl fluorescence changes, sulfhydryl group exposure, loss of secondary structure, size-exclusion chromatography, and sedimentation equilibrium data. GdnHCl-induced dissociation and unfolding of tetrameric CEOS was concerted, and yields of reactivated CEOS by dilution from 5 M GdnHCl were improved when unfolding took place on ice rather than at 25 degrees C. Refolding and reconstitution of the enzyme were optimal at 相似文献   

N-(2-Carboxyethyl)chitosans: regioselective synthesis,characterisation and protolytic equilibria

N-(2-Carboxyethyl)chitosans were obtained by reaction of low molecular weight chitosan with a low degree of acetylation and 3-halopropionic acids under mild alkaline media (pH 8-9, NaHCO3) at 60 degrees C. The chemical structure of the derivatives obtained was determined by 1H and 13C NMR spectroscopies. It was found that alkylation of chitosan by 3-halopropionic acids proceeds exclusively at the amino groups. The products obtained are described in terms of their degrees of carboxyethylation and ratio of mono-, di-substitution and free amine content. The protonation constants of amino and carboxylate groups of a series of N-(2-carboxyethyl)chitosans were determined by pH-titration at ionic strength 0.1 M KNO3 and 25 degrees C.     

Binding kinetics of ecotropic (Moloney) murine leukemia retrovirus with NIH 3T3 cells.

A quantitative analysis of the binding kinetics of intact Moloney murine leukemia retrovirus (MoMuLV) particles with NIH 3T3 cells was performed with an immunofluorescence flow cytometry assay. The virus-cell binding equilibrium dissociation constant (KD), expressed in terms of virus particle concentration, was measured to be 8.5 (+/- 6.4) x 10(-12) M at 4 degrees C and was three- to sixfold lower at temperatures above 15 degrees C. The KD of virus binding is about 1,000-fold lower than the KD of purified MoMuLV envelope. The association rate constant was determined to be 2.5 (+/- 0.9) x 10(9) M-1 min-1 at 4 degrees C and was 5- to 10-fold higher at temperatures above 15 degrees C. The apparent dissociation rate constant at 4 degrees C was 1.1 (+/- 0.4) x 10(-3) min-1 and was doubled for every 10 degrees C increase in temperature over the range tested (15 to 37 degrees C).     

Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers

Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study hydrated N-lignocerylgalactosylsphingosine (NLGS) bilayers. DSC of fully hydrated NLGS shows an endothermic transition at 69-70 degrees C, immediately followed by an exothermic transition at 72-73 degrees C; further heating shows a high-temperature (Tc = 82 degrees C), high-enthalpy (delta H = 15.3 kcal/mol NLGS) transition. Heating to 75 degrees C, cooling to 20 degrees C and subsequent reheating shows no transitions at 69-73 degrees C; only the high-temperature (82 degrees C), high-enthalpy (15.3 kcal/mol) transition. Two exothermic transitions are observed on cooling; for the upper transition its temperature (about 65 degrees C) and enthalpy (about 6 kcal/mol NLGS) are essentially independent of cooling rate, whereas the lower transition exhibits marked changes in both temperature (30----60 degrees C) and enthalpy (2.2----9.5 kcal/mol NLGS) as the cooling rate decreases from 40 to 0.625 Cdeg/min. On reheating, the enthalpy of the 69-70 degrees C transition is dependent on the previous cooling rate. The DSC data provide clear evidence of conversions between metastable and stable forms. X-ray diffraction data recorded at 26, 75 and 93 degrees C show clearly that NLGS bilayer phases are present at all temperatures. The X-ray diffraction pattern at 75 degrees C shows a bilayer periodicity d = 65.4 A, and a number of sharp reflections in the wide-angle region indicative of a crystalline chain packing mode. This stable bilayer form converts to a liquid-crystal bilayer phase; at 93 degrees C, the bilayer periodicity d = 59.1 A, and a diffuse reflection at 1/4.6 A-1 is observed. The diffraction pattern at 22 degrees C represents a combination of the stable and metastable low-temperature bilayer forms. NLGS exhibits a complex pattern of thermotropic changes related to conversions between metastable (gel), stable (crystalline) and liquid-crystalline bilayer phases. The structure and thermotropic properties of NLGS are compared with those of hydrated N-palmitoylgalactosylsphingosine reported previously (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966).     

Effects of annealing on the polymorphic structure of starches from sweet potatoes (Ayamurasaki and Sunnyred cultivars) grown at various soil temperatures

Starches extracted from the sweet potato cultivars Sunnyred and Ayamurasaki grown at 15 or 33 degrees C (soil temperature) were annealed in excess water (3 mg starch/mL water) for different times (1, 4, 8 or 10h) at the temperatures 2-3 degrees K below the onset melting temperature. The structures of annealed starches, as well as their gelatinisation (melting) properties, were studied using high-sensitivity differential scanning calorimetry (HSDSC). In excess water, the single endothermic peak shifted to higher temperatures, while the melting (gelatinisation) enthalpy changed only very slightly, if any. The elevation of gelatinisation temperature was associated with increasing order/thickness of the crystalline lamellae. The only DSC endotherm identified in 0.6 M KCl for Sunnyred starch grown at 33 degrees C was attributed to A-type polymorphic structure. The multiple endothermic forms observed by DSC performed in 0.6M KCl for annealed starches from both cultivars grown at 15 degrees C provided evidence of a complex C-type (A- plus B-type) polymorphic structure of crystalline lamellae. The A:B-ratio of two polymorphic forms increased upon annealing due to partial transformation of B- to A-polymorph, which was time dependent. Long heating periods facilitated the maximal transformation of B- to A-polymorph associated with limited A:B ratio.     

The angles between the C(1)-, C(5)-, and C(9)-methyl bonds of the retinylidene chromophore and the membrane normal increase in the M intermediate of bacteriorhodopsin: direct determination with solid-state (2)H NMR.

The orientations of three methyl bonds of the retinylidene chromophore of bacteriorhodopsin were investigated in the M photointermediate using deuterium solid-state NMR ((2)H NMR). In this key intermediate, the chromophore has a 13-cis, 15-anti conformation and a deprotonated Schiff base. Purple membranes containing wild-type or mutant D96A bacteriorhodopsin were regenerated with retinals specifically deuterated in the methyl groups of either carbon C(1) or C(5) of the beta-ionone ring or carbon C(9) of the polyene chain. Oriented hydrated films were formed by drying concentrated suspensions on glass plates at 86% relative humidity. The lifetime of the M state was increased in the wild-type samples by applying a guanidine hydrochloride solution at pH 9.5 and in the D96A sample by raising the pH. (2)H NMR experiments were performed on the dark-adapted ground state (a 2:1 mixture of 13-cis, 15-syn and all-trans, 15-anti chromophores), the cryotrapped light-adapted state (all-trans, 15-anti), and the cryotrapped M intermediate (13-cis, 15-anti) at -50 degrees C. Bacteriorhodopsin was first completely converted to M under steady illumination of the hydrated films at +5 degrees C and then rapidly cooled to -50 degrees C in the dark. From a tilt series of the oriented sample in the magnetic field and an analysis of the (2)H NMR line shapes, the angles between the individual C-CD(3) bonds and the membrane normal could be determined even in the presence of a substantial degree of orientational disorder. While only minor differences were detected between dark- and light-adapted states, all three angles increase in the M state. This is consistent with an upward movement of the C(5)-C(13) part of the polyene chain toward the cytoplasmic surface or with increased torsional strain. The C(9)-CD(3) bond shows the largest orientational change of 7 degrees in M. This reorientation of the chromophore in the binding pocket provides direct structural support for previous suggestions (based on spectroscopic evidence) for a steric interaction in M between the C(9)-methyl group and Trp 182 in helix F.     

Thermal unfolding of dodecameric glutamine synthetase: inhibition of aggregation by urea.

Thermal unfolding of dodecameric manganese glutamine synthetase (622,000 M(r)) at pH 7 and approximately 0.02 ionic strength occurs in two observable steps: a small reversible transition (Tm approximately 42 degrees C; delta H approximately equal to 0.9 J/g) followed by a large irreversible transition (Tm approximately 81 degrees C; delta H approximately equal to 23.4 J/g) in which secondary structure is lost and soluble aggregates form. Secondary structure, hydrophobicity, and oligomeric structure of the equilibrium intermediate are the same as for the native protein, whereas some aromatic residues are more exposed. Urea (3 M) destabilizes the dodecamer (with a tertiary structure similar to that without urea at 55 degrees C) and inhibits aggregation accompanying unfolding at < or = 0.2 mg protein/mL. With increasing temperature (30-70 degrees C) or incubation times at 25 degrees C (5-35 h) in 3 M urea, only dodecamer and unfolded monomer are detected. In addition, the loss in enzyme secondary structure is pseudo-first-order (t1/2 = 1,030 s at 20.0 degrees C in 4.5 M urea). Differential scanning calorimetry of the enzyme in 3 M urea shows one endotherm (Tmax approximately 64 degrees C; delta H = 17 +/- 2 J/g). The enthalpy change for dissociation and unfolding agrees with that determined by urea titrations by isothermal calorimetry (delta H = 57 +/- 15 J/g; Zolkiewski M, Nosworthy NJ, Ginsburg A, 1995, Protein Sci 4: 1544-1552), after correcting for the binding of urea to protein sites exposed during unfolding (-42 J/g). Refolding and assembly to active enzyme occurs upon dilution of urea after thermal unfolding.     

Generic vapor heat treatments to control Maconellicoccus hirsutus (Homoptera: Pseudococcidae)

Vapor heat treatments were developed against life stages of the mealybug Maconellicoccus hirsutus (Green) (Homoptera: Pseudococcidae). Treatments tested were 47 degrees C for 5-50 min in 5-min increments and 49 degrees C for 3, 5, 8, 10, and 12 min. All tests were conducted with mixed age M. hirsutus on Chinese pea, Pisum sativum L. Treatment at 47 degrees C required 45 min to kill all M. hirsutus, whereas treatment at 49 degrees C required 10 min. The adult female and nymphal stages were the most heat tolerant at 47 degrees C, but the egg stage was the most heat tolerant at 49 degrees C. Use of the vapor heat treatments on other commodities will require achieving or exceeding the proper temperature and duration at all locations on the host where M. hirsutus may reside.     

Base protonation facilitates B-Z interconversions of poly(dG-dC) X poly(dG-dC)

Comparative studies on the salt titration and the related kinetics for poly(dG-dC) X poly(dG-dC) in pH 7.0 and 3.8 solutions clearly suggest that base protonation facilitates the kinetics of B-Z interconversion although the midpoint for such a transition in acidic solution (2.0-2.1 M NaCl) is only slightly lower than that of neutral pH. The rates for the salt-induced B to Z and the reverse actinomycin D induced Z to B transitions in pH 3.8 solutions are at least 1 order of magnitude faster than the corresponding pH 7.0 counterparts. The lowering of the B-Z transition barrier is most likely the consequence of duplex destabilization due to protonation as indicated by a striking decrease (approximately 40 degrees C) in melting temperature upon H+ binding in low salt. The thermal denaturation curve for poly(dG-dC) X poly(dG-dC) in a pH 3.8, 2.6 M NaCl solution indicates an extremely cooperative melting at 60.5 degrees C for protonated Z DNA, which is immediately followed by aggregate formation and subsequent hydrolysis to nucleotides at higher temperatures. The corresponding protonated B-form poly(dG-dC) X poly(dG-dC) in 1 M NaCl solution exhibits a melting temperature about 15 degrees C higher, suggesting further duplex destabilization upon Z formation.     

Crystal Structure Determination of New Antimitotic Agent Bis(p-fluorobenzyl)trisulfide

The purpose of this research was to investigate the physical characteristics and crystalline structure of bis(p-fluorobenzyl)trisulfide, a new anti-tumor agent. Methods used included X-ray single crystal diffraction, X-ray powder diffraction (XRPD), Fourier-transform infrared (FT-IR) spectroscopy, differential scanning calorimetric (DSC) and thermogravimetric (TG) analyses. The findings obtained with X-ray single crystal diffraction showed that a monoclinic unit cell was a = 12.266(1) A, b = 4.7757(4) A, c = 25.510(1) A, beta = 104.25(1) degrees ; cell volume = 1,448.4(2) A(3), Z = 4, and space group C2/c. The XRPD studies of the four crystalline samples, obtained by recrystallization from four different solvents, indicated that they had the same diffraction patterns. The diffraction pattern stimulated from the crystal structure data is in excellent agreement with the experimental results. In addition, the identical FT-IR spectra of the four crystalline samples revealed absorption bands corresponding to S-S and C-S stretching as well as the characteristic aromatic substitution. Five percent weight loss at 163.3 degrees C was observed when TG was used to study the decomposition process in the temperature range of 20-200 degrees C. DSC also allowed for the determination of onset temperatures at 60.4(1)-60.7(3) degrees C and peak temperatures at 62.1(3)-62.4(3) degrees C for the four crystalline samples studied. The results verified that the single crystal structure shared the same crystal form with the four crystalline samples investigated.     

Mechanisms for the spontaneous formation of covalently linked polymers of the terminal membranolytic complement protein (C9)

Purified human C9 spontaneously polymerizes upon prolonged incubation at 37 degrees C, and a fraction of these C9 polymers becomes resistant to dissociation by sodium dodecyl sulfate (SDS) and reducing agents. We examined possible mechanisms for this spontaneous covalent linking of C9. The following results are consistent with the conclusion that the formation of the covalently linked C9 polymer involves disulfide linking. 1) In addition to the SDS/dithiothreitol (DTT)-resistant C9 polymer (Mr = 950,000), disulfide-linked C9 dimers and trimers were formed upon incubation of C9 at 37 degrees C for 64 h. 2) The C9 polymer formed upon incubation at 37 degrees C for 64 h was resistant to dissociation by 6 M guanidine hydrochloride, 20 mM DTT but was dissociated by 6 M guanidine thiocyanate alone, yielding disulfide-linked C9 oligomers. 3) The formation of the SDS/DTT-resistant C9 polymer was completely inhibited by 1 mM iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), while DTNB enhanced the formation of disulfide-linked C9 oligomers. 4) A significant amount of free sulfhydryl group was detected in the polymerized C9 samples with various SH-specific reagents, though native C9 reacted with none of these reagents. In addition, inhibition by 1 mM iodoacetamide of C9 disulfide linking inhibited the self-association of C9 as analyzed by gel filtration on TSK-G4000 SW, whereas enhancement by 1mM DTNB of C9 disulfide linking enhanced C9 self-association. Thus, these results indicate that C9 disulfide linking that occurs upon C9 polymerization is an intrinsic property of C9 which is of importance in the formation of the stable C9 polymer structure.     

2-O-(beta-D-glucuronyl)-7-O-(2-amino-2-deoxy-alpha-D-glucopyranosyl)-L-glycero-D-manno-heptose: a constituent of the Bordetella pertussis endotoxin.

The presence of bound D-glucuronic acid in the endotoxin of Bordetella pertussis was demonstrated. The branched chain trisaccharide named in the title was isolated after hydrolysis of the endotoxin with 3 M HCl for 2 h at 100 degrees C. Its structure was established by chemical and enzymic degradation.     

Crystallization of trimeric recombinant human tumor necrosis factor (cachectin)

Crystals of tumor necrosis factor (TNF) have been obtained in two forms. Rhombohedral crystals grow in 1.8 to 2.0 M ammonium sulfite, pH 7.8 at 21 degrees C, and tetragonal crystals grow in 2.6 M magnesium sulfate, pH 5.5 at 25 degrees C. Analysis of TNF by isoelectric focusing under native and denaturing conditions indicates that TNF molecules exist as trimers in solution. The rhombohedral cachectin crystals belong to space group R3 and have unit cell constants a = b = c = 47.65 A and alpha = beta = gamma = 88.1 degrees. Density determinations and the space group indicate that the unit cell contains one 51,000-dalton trimer. These crystals are stable in the x-ray beam and diffract to at least 1.85 A but are apparently twinned by merohedry. The tetragonal crystals are space group P4(3)2(1)2 or its enantiomorph P4(1)2(1)2 and have unit cell constants a = b = 95.08, c = 117.49. The asymmetric unit contains one trimer; the crystals are stable in the x-ray beam and diffract to beyond 3 A.     

Stage-dependent hatching responses of rohu (Labeo rohita) embryos to different concentrations of cryoprotectants and temperatures

Hatching performances of three embryonic stages of postfertilization rohu (Labeo rohita) (9-, 12-, and 15-h) were examined after treatment with various concentrations (0.5-4.5M) of two cryoprotectants (methanol and propylene glycol) supplemented with 0.1M trehalose. Different lengths of storage (1-48 h) and temperature (-4 degrees C to ambient) were studied. Of the three stages of embryonic development, the 12-h stage proved to be the most suitable stage for low temperature storage, showing the highest percentage of hatch out (72+/-2%) with 2.0M methanol and 0.1M trehalose. Methanol was more useful for storage at higher temperatures and propylene glycol at subzero temperatures. The maximum possible duration of effective storage of 12-h embryos was 31h in 2.0M methanol at 0 degrees C. No hatch out was found beyond 31h of storage with all concentrations of methanol at 0 degrees C. The results of interactions was that the optimal concentration of methanol was 3.0M at 4 degrees C, 2.0M at 0 degrees C, and 1.5M at 4 degrees C. Among three embryonic stages 12-h stage showed better results in trehalose treatment than sucrose. Among all concentrations of trehalose tested 0.1M gave the maximal survival rate of the rohu embryos.     

Vitrification of buffalo (Bubalus bubalis) oocytes

The objective of the present study was to develop a method for the cryopreservation of buffalo oocytes by vitrification. Cumulus-oocyte complexes (COCs) were obtained from slaughterhouse ovaries. Prior to vitrification of COCs in the vitrification solution (VS) consisting of 4.5 M ethylene glycol, 3.4 M dimethyl sulfoxide, 5.56 mM glucose, 0.33 mM sodium pyruvate and 0.4% w/v bovine serum albumin in Dulbecco's phosphate buffered saline (DPBS), the COCs were exposed to the equilibration solution (50% VS v/v in DPBS) for 1 or 3 min at room temperature (25 to 30 degrees C). The COCs were then placed in 15-microL of VS and immediately loaded into 0.25-mL French straws, each containing 150 microL of 0.5 M sucrose in DPBS. The straws were placed in liquid nitrogen (LN2) vapor for 2 min, plunged and stored in LN2 for at least 7 d. The straws were thawed in warm water at 28 degrees C for 20 sec. For dilution, the COCs were equilibrated in 0.5 M sucrose in DPBS for 5 min and then washed 4 to 5 times in the washing medium (TCM-199+10% estrus buffalo serum). The proportion of oocytes recovered in a morphologically normal form was significantly higher (98 and 88%, respectively; P<0.05), and the proportion of oocytes recovered in a damaged form was significantly lower (2 and 12%, respectively; P<0.05) for the 3-min equilibration than for 1 min. For examining the in vitro developmental potential of vitrified-warmed oocytes, the oocytes were placed in 50-microL droplets (10 to 15 oocytes per droplet) of maturation medium (TCM-199+15% FBS+5 microg/mL FSH-P), covered with paraffin oil in a 35-mm Petri dish and cultured for 26 h in a CO2 incubator (5% CO2 in air) at 38.5 degrees C. Although the nuclear maturation rate did not differ between the 1- and 3-min equilibration periods (21.5+/-10.7 and 31.5+/-1.5%, respectively), the between-trial variation was very high for the 1-min period. This method of vitrification is simple and rapid, and can be useful for cryopreservation of buffalo oocytes.     

Intracomplex alkylation of octanucleotide pd(TGTTTGGC) by a 5'-(4-N-methyl-N-(2-chloroethyl)-amino)benzylphosphamide derivative of heptanucleotide pd(CCAAACA). Preparation of a covalent adduct and study of its spatial structure in an aqueous solution by two-dimensional (1)H-NMR spectroscopy]

Covalent adduct--the product of intracomplex alkylation at N-3-position of dC-8-nucleoside residue of target octanucleotide pd[TGTTTGGC] was completely synthesized by means of 4-[N-methyl-N-(2-chloroethyl)amino]benzyl-5'-phosphamido derivative of heptanucleotide pd[CCAAACA]. Its melting temperature was shown to be 70 degrees C. Tm did not depend on covalent adduct concentration and was by 40 degrees C higher than that for unmodified duplex pd[TGTTTGGC].pd[CCAAACA] at concentration of 0.5 x 10(-4) M. The spatial structure of the covalent adduct in aqueous solution was investigated by two-dimensional 3H-NMR spectroscopy. The assignment of oligonucleotide protons as well as protons of a modifying group was carried out using COSY, COSY-DQF and NOESY experiments. Conformational analysis of proton-proton coupling constants for H1', H2'a, H2'b and H3' protons showed the sugar residues to be in 2'-endo conformation. Analysis of NOE connectivities observed between the protons of the alkylating group and oligonucleotide protons yielded conclusion, regarding the 4-[N-methyl-N-(2-chloroethyl)amino]benzylamido 5'-residue being localized in the region of the lacked nucleoside residue of the heptanucleotide chain about 5 A apart from the dC-1 residue and from cytosine base of the alkylated dC-8 residue.     

Non-Enzymatic Glycosylation (or Glycation) and Inhibition of the Pig Heart Cytosolic Aspartate Aminotransferase by Glyceraldehyde 3-Phosphate

Glyceraldehyde 3-phosphate (Glyc3P), a glycolytic intermediate, non-enzymatically glycosylated (or glycated) and inhibited the pig heart cytoplasmic aspartate aminotransferase (cAAT). Glyc3P (5.0 mM) decreased cAAT activity by 47% after 1 min at 23 degrees C. cAAT activity remained unchanged after a 24 h incubation with either glucose 6-phosphate (5.0 mM) or ribose 5-phosphate (5.0 mM). Increasing the incubation pH from 6.4 to 7.8 or the incubation temperature from 23 degrees C to 50 degrees C enhanced Glyc3P's inhibitory effect on cAAT activity. Glyc3P (250-500 μM) decreased the thermal stability of cAAT as evidenced by lowering the T(m) or temperature that caused a 50% irreversible loss of cAAT activity (69 degrees C, control; 58.5 degrees C, 500 μM Glyc3P). Glyc3P decreased cAAT amino group content and increased glycation products, which were measured by adduct formation, fluorescence and protein crosslinking.     

A left-handed 3(1) helical conformation in the Alzheimer Abeta(12-28) peptide

We show for the first time that the secondary structure of the Alzheimer beta-peptide is in a temperature-dependent equilibrium between an extended left-handed 3(1) helix and a flexible random coil conformation. Circular dichroism spectra, recorded at 0.03 mM peptide concentration, show that the equilibrium is shifted towards increasing left-handed 3(1) helix structure towards lower temperatures. High resolution nuclear magnetic resonance (NMR) spectroscopy has been used to study the Alzheimer peptide fragment Abeta(12-28) in aqueous solution at 0 degrees C and higher temperatures. NMR translation diffusion measurements show that the observed peptide is in monomeric form. The chemical shift dispersion of the amide protons increases towards lower temperatures, in agreement with the increased population of a well-ordered secondary structure. The solvent exchange rates of the amide protons at 0 degrees C and pH 4.5 vary within at least two orders of magnitude. The lowest exchange rates (0.03-0.04 min(-1)) imply that the corresponding amide protons may be involved in hydrogen bonding with neighboring side chains.     

The possible existence of a heat-stable alkaline proteinase (HAP) in rat skeletal muscle

1. A unique caseinolytic activity was found in the crude extract from chicken and rat skeletal muscle. Hardly any activity was detected at physiological assay temperatures at pH 8.0 but did well at around 60 degrees C. 2. The activity partially purified from rat skeletal muscle showed optimum pH at around 8.0 at 60 degrees C. It hardly hydrolyzed casein below 50 degrees C, but in the presence of 5 M urea it showed relatively high activity at 30 degrees C. The activity was completely stable at 50 degrees C for 1 hr. 3. The activity seems to be contained in a high mol. wt (450,000) protein from the elution volume and is due to cysteine proteinase from the effect of inhibitors. 4. The above properties agreed with those of the heat-stable alkaline proteinase (HAP) of fish purified homogeneously by electrophoresis. This seems to suggest that HAP may also exist in rat skeletal muscle.