Domains in lambda Cro repressor. A calorimetric study.

Thermodynamic properties of a mutant lambda Cro repressor with Cys replacing Val55 were studied calorimetrically. Formation of the S-S cross-link between neighboring Cys55 residues in this dimeric molecule leads to stabilization of a structure formed by the C-terminal parts of the two polypeptide chains, which behave as a single cooperative domain upon protein denaturation by heating. This composite domain is very stable at neutral pH and disrupts at 110 degrees C. The S-S-cross-linked tryptic fragment (residues 22-66), which includes this C-terminal domain, has similar stability. The N-terminal parts of the polypeptide chains do not form any stable structure when isolated, but in S-S-cross-linked dimer, they form a single cooperative block which melts in an all-or-none way 9 degrees C higher than the un-cross-linked protein. The observed cooperation of the distant N-terminal parts in dimer raises questions regarding lambda Cro repressor structure in solution.     

Effects of Na2SO4 on hydrophobic and electrostatic interactions between amphipathic alpha-helices.

The effects of 2 molal Na2SO4 at neutral pH on hydrophobic and electrostatic interactions between amphipathic alpha-helices were investigated by circular dichroism spectroscopy. The amphipathic peptides that were studied included LEK (acetyl-LEELKKKLEELKKKLEEL-NH2) and LEE (acetyl-LEELEEELEELEEELEEL-NH2). In phosphate buffer at neutral pH, only LEK adopted a predominantly alpha-helical conformation, attributable to glu-lys+ interactions where a major contribution is evidently a hydrogen bond (Biochemistry 32: 9668-9676). Despite the presence of lys+ in the e and g' positions of the abcdefg heptad repeat, LEK exhibited mean-residue ellipticities at 222 nm ([theta]222) which were dependent on peptide concentration, indicating the presence of a coiled coil. In the presence of 2 molal Na2SO4 at 25-75 degrees C, the helical content of LEK increased, with the greatest increase observed at 75 degrees C. The value of the ellipticity ratio R ([theta]222/[theta]208) of LEK in 2 molal Na2SO4 also increased, indicating a stronger interhelical association. At 50 degrees C and 75 degrees C, LEK remained predominantly alpha-helical. In phosphate buffer at neutral pH, LEE was mainly random coil. In the presence of 2 molal Na2SO4, however, the peptide formed alpha-helices that associated to form a coiled coil. At 50 degrees C and 75 degrees C, LEE became predominantly random coil but the remaining alpha-helices were still associating. These results are consistent with the strengthening of interhelical hydrophobic interactions and the absence of screening of helix-stabilizing and helix-destabilizing electrostatic interactions in amphipathic alpha-helices by Na2SO4.     

Heterostereocomplexes prepared from d-PLA and l-PLA and leuprolide. II. Release of leuprolide

Reversible stereoselective complexes were spontaneously obtained from mixing acetonitrile solutions of enatiomeric d-poly(lactic acid) (d-PLA), l-poly(lactic acid) (l-PLA), and leuprolide, a l-configured nonapeptide LHRH analogue. The complex spontaneously aggregated and precipitated in high yields (>90%) from acetonitrile solution, forming uniform, porous microparticles. The stereocomplex microparticles showed a continuous release of the interlocked peptide for a period of one to three months under physiological conditions. Various factors, including method of complex formation, molecular weight of PLA, leuprolide:polymer and d-PLA:l-PLA complex ratios, and additives, influenced the release pattern of leuprolide from the stereocomplexes. Continuous release of leuprolide for over 100 days was observed for certain stereocomplex compositions. In vivo evaluation of the leuprolide loaded stereocomplexes in rats by monitoring testosterone levels in the blood of rats after subcutaneous injection showed low testosterone levels for over 42 days.     

Stability of equine lysozyme. I. Thermal unfolding behaviour.

The thermal denaturation of Ca(2+)- and apo-forms of equine lysozyme was followed by using far and near UV circular dichroism and intrinsic fluorescence methods. The difference found between the temperature dependence of the ellipticity at 222 nm and 287 nm, which show two stages in the thermal transition, and those at 228 nm and 294 nm, which indicate only one stage over a wide range of temperatures reflects that different subdivisions of the protein molecule are characterized by a different stability, cooperativity and pathway of denaturation. The first transition, reflected in the increase of the ellipticity at 222 nm and 287 nm, coincides with the transition detected by fluorescence and occurs at 30-50 degrees C for the apo-form and at 50-60 degrees C for the Ca(2+)-form of lysozyme. It seems to correlate with the transfer of some tryptophan residues to a more hydrophobic environment and with a local rearrangement of the tertiary and secondary structures. The unfolding transition detected by the decrease of the ellipticity at all wavelengths occurs nearly in the same temperature region for the apo- and Ca(2+)-forms, i.e. 50-80 degrees C and 55-80 degrees C, respectively. The presence of a Ca(2+)-binding loop in equine lysozyme may be partly responsible for the drastic destabilization of its structure as a whole both in the presence but especially in the absence of Ca2+ in comparison with hen and human lysozymes.     

Synthesis and properties of defined DNA oligomers containing base mispairs involving 2-aminopurine.

DNA heptamers containing the mutagenic base analogue 2-aminopurine (AP) have been chemically synthesized and physically characterized. We report on the relative stabilities of base pairs between AP and each of the common DNA bases, as determined from heptamer duplex melts at 275 and 330 nm. Base pairs are ranked in order of decreasing stability: AP.T greater than AP.A greater than AP.C greater than AP.G. It is of interest that AP.A is more stable than AP.C even though DNA polymerase strongly favors the formation of AP.C over AP.A base pairs. Comparisons of melting profiles at 330 nm and 275 nm indicate that AP.T, AP.A, and AP.C base pairs are annealed in heptamer duplexes and melt 2-3 degrees prior to surrounding base pairs, whereas AP.G appears not to be annealed.     

Analyses of absorption and fluorescence spectra of water-soluble chlorophyll proteins, pigment system II particles and chlorophyll a in diethylether solution by the curve-fitting method.

Absorption and fluorescence spectra in the red region of water-soluble chlorophyll proteins, Lepidium CP661, CP663 and Brassica CP673, pigment System II particles of spinach chloroplasts and chlorophyll a in diethylether solution at 25 degrees C were analyzed by the curve-fitting method (French, C.S., Brown, J.S. and Lawrence, M.C. (1972) Plant Physiol 49, 421--429). It was found that each of the chlorophyll forms of the chlorophyll proteins and the pigment System II particles had a corresponding fluorescence band with the Stokes shift ranging from 0.6 to 4.0 nm. The absorption spectrum of chlorophyll a in diethylether solution was analyzed to one major band with a peak at 660.5 nm and some minor bands, while the fluorescence spectrum was analyzed to one major band with a peak at 664.9 nm and some minor bands. A mirror image was clearly demonstrated between the resolved spectra of absorption and fluorescence. The absorption spectrum of Lepidium CP661 was composed of a chlorophyll b form with a peak at 652.8 nm and two chlorophyll a forms with peaks at 662.6 and 671.9 nm. The fluorescence spectrum was analyzed to five component bands. Three of them with peaks at 654.8, 664.6 and 674.6 nm were attributed to emissions of the three chlorophyll forms with the Stokes shift of 2.0--2.7 nm. The absorption spectrum of Brassica CP673 had a chlorophyll b form with a peak at 653.7 nm and four chlorophyll a forms with peaks at 662.7, 671.3, 676.9 and 684.2 nm. The fluorescence spectrum was resolved into seven component bands. Four of them with peaks at 666.7, 673.1, 677.5 and 686.2 nm corresponded to the four chlorophyll a forms with the Stokes shift of 0.6--4.0 nm. The absorption spectrum of the pigment System II particles had a chlorophyll b form with a peak at 652.4 nm and three chlorophyll a forms with peaks at 662.9, 672.1 and 681.6 nm. The fluorescence spectrum was analyzed to four major component bands with peaks at 674.1, 682.8, 692.0 and 706.7 nm and some minor bands. The former two bands corresponded to the chlorophyll a forms with peaks at 672.1 and 681.6 nm with the Stokes shift of 2.0 and 1.2 nm, respectively. Absorption spectra at 25 degrees C and at --196 degrees C of the water-soluble chlorophyll proteins were compared by the curve-fitting methods. The component bands at --196 degrees C were blue-shifted by 0.8--4.1 nm and narrower in half widths as compared to those at 25 degrees C.     

Conformational intermediates in the folding of a coiled-coil model peptide of the N-terminus of tropomyosin and alpha alpha-tropomyosin.

Circular dichroism was used to study the folding of alpha alpha-tropomyosin and AcTM43, a 43-residue peptide designed to serve as a model for the N-terminal domain of tropomyosin. The sequence of the peptide is AcMDAIKKKMQMLKLDVENLLDRLEQLEADLKALEDRYKQLEGGC. The peptide appeared to form a coiled coil at low temperatures (< 25 degrees C) in buffers with physiological ionic strength and pH. The folding and unfolding of the peptide, however, were noncooperative. When CD spectra were examined as a function of temperature, the apparent degree of folding differed when the ellipticity was followed at 222, 208, and 280 nm. Deconvolution of the spectra suggested that at least three component curves contributed to the CD in the far UV. One component curve was similar to the CD spectrum of the coiled-coil alpha-helix of native alpha alpha-tropomyosin. The second curve resembled the spectrum of single-stranded short alpha-helical segments found in globular proteins. The third was similar to that of polypeptides in the random coil conformation. These results suggested that as the peptide folded, the alpha-helical content increased before most of the coiled coil was formed. When the CD spectrum of striated muscle alpha alpha-tropomyosin was examined as a function of temperature, the unfolding was also not totally cooperative. As the temperature was raised from 0 to 25 degrees C, there was a decrease in the coiled coil and an increase in the conventional alpha-helix type spectrum without formation of random coil. The major transition, occurring at 40 degrees C, was a cooperative transition characterized by the loss of all of the remaining coiled coil and a concomitant increase in random coil.     

Simultaneous peptide and oligonucleotide formation in mixtures of amino acid, nucleoside triphosphate, imidazole, and magnesium ion.

Simultaneous peptide and oligonucleotide formation was observed in reaction mixtures of amino acid, nucleoside triphosphate, imidazole, and MgCl2. At 70 degrees C in solutions that were evaporated to dryness the formation of peptide for phe and pro was greatest with CTP relative to ATP, GTP, and UTP. Lysine exhibited a preference for GTP and glycine for UTP. At ambient temperature insolution at pH 7.8, CTP was preferred by glycine, but at pH 8.7 UTP was preferred. The glycine nucleotide phosphoramidates were also detected and characterized in reactions at 40 degrees C. The glycine-reaction preference for CTP at pH 7.8 and UTP at 8.7 suggested that the basicity of the nucleoside triphosphate was involved in increasing the peptide yield. CTP near neutrality is the most basic nucleoside triphosphate and the basic anionic form UTP could facilitate peptide formation at pH 8.7. These data, together with information on the complexing of poly(C) by GTP, led to the experimentally approchable hypothesis that GTP, by forming a basic triplex between the cytosine residues adjacent to the peptidyl adenosine and aminoacyl adenosine at the termini of two proto-tRNAs, would promote peptide bond synthesis between the aminoacyl residue and peptidyl residue.     

Molecular architecture of the neurofilament. II. Reassembly process of neurofilament L protein in vitro

Reassembly of the neurofilament (NF) in vitro was studied by low-angle rotary shadowing electron microscopy. Various intermediate stages of the reassembly were reconstructed from the smallest molecular mass subunit (NF-L) under controlled reassembly conditions. NF-L in 6 M-urea took the form of spherical particles with a diameter of about 12 nm. NF-L aggregated into rodlets of 70 to 80 nm long in a low-salt solution at alkaline pH. By reducing the pH of the dialyzing solution to 6.6, a pair of rods was formed by association side-by-side. Increasing the temperature of low-salt solutions from 4 degrees C to 35 degrees C did not produce intermediate-sized filaments. The addition of Mg2+ to the dialyzing solution resulted in the formation of short intermediate-sized filaments even at 4 degrees C. Further dialysis of the short intermediate-sized filaments against reassembly solution containing both NaCl and MgCl2 at 37 degrees C failed to elongate them into longer filaments, suggesting that annealing does not contribute to the elongation of neurofilaments. Different roles for Mg+ and NaCl in neurofilament reassembly were indicated. While Mg2+ strengthened the lateral association between 70 to 80 nm rods, NaCl appeared to promote the end-to-end association of filaments preferentially. Longer filaments were formed by increasing the NaCl concentration. By dialyzing NF-L against a buffer containing 50 mM-NaCl in the absence of Mg2+, unraveled filaments were formed. The many unraveled filaments were composed of four 8 nm wide filaments, which have been called the subfilament or the protofibril. Time-course experiments of the reassembly were performed in the absence of Mg2+, in which condition the rate of neurofilament reassembly appeared to be reduced. Star-like clusters, about four protofibrils joined together at one end, were suggested to be the initial stage of the intermediate-sized filament formation. The following two-step elongation mechanism of neurofilaments was deduced from these results. The pairs of rods were added to the ends of the protofibrils of neurofilaments, and after all four protofibrils were elongated they were then packed into neurofilaments. Distribution of larger molecular mass subunits, NF-M and NF-H, was studied. Addition of NF-M or NF-H to NF-L did not change the assembly properties of neurofilaments. Unraveled filaments reconstituted from NF-L plus either NF-M or NF-H indicated that NF-M and NF-H are incorporated evenly into each protofibril.     

Phage tf-1: a filamentous bacteriophage specific for bacteria harbouring the IncT plasmid pIN25

Phage tf-1 is a filamentous phage which is about 800 nm in length, 10 nm in width and has slightly tapered ends. The phage was isolated from sewage and formed plaques or propagated only on Escherichia coli, Salmonella typhimurium and Klebsiella oxytoca strains harbouring the IncT plasmid pIN25 at 30 degrees C. It adsorbed in large numbers to pIN25-encoded long thick flexible conjugative pili formed at 30 degrees C and also to the short form of these pili synthesized at 37 degrees C. The reason for the failure to form plaques at 37 degrees C is not known. The adsorption site is a short length of the pilus shaft extending 100-200 nm back from the distal tip. Efficient phage tf-1 adsorption to the same site was found for pili determined by other IncT plasmids in spite of the fact that phage tf-1 did not plate or propagate on strains harbouring them. However, areas of specific partial clearing on lawns of these plasmid-containing bacteria were produced by phage in high concentrations. Lack of plaque-formation could be due to inefficient intracellular assembly coupled to avid adsorption of any liberated phage to pili. The phage differs from all but one other filamentous phage by being sensitive to diethyl ether.     

Reversible surface aggregation in pore formation by pardaxin.

The mechanism of leakage induced by surface active peptides is not yet fully understood. To gain insight into the molecular events underlying this process, the leakage induced by the peptide pardaxin from phosphatidylcholine/ phosphatidylserine/cholesterol large unilamellar vesicles was studied by monitoring the rate and extent of dye release and by theoretical modeling. The leakage occurred by an all-or-none mechanism: vesicles either leaked or retained all of their contents. We further developed a mathematical model that includes the assumption that certain peptides become incorporated into the vesicle bilayer and aggregate to form a pore. The current experimental results can be explained by the model only if the surface aggregation of the peptide is reversible. Considering this reversibility, the model can explain the final extents of calcein leakage for lipid/peptide ratios of > 2000:1 to 25:1 by assuming that only a fraction of the bound peptide forms pores consisting of M = 6 +/- 3 peptides. Interestingly, less leakage occurred at 43 degrees C, than at 30 degrees C, although peptide partitioning into the bilayer was enhanced upon elevation of the temperature. We deduced that the increased leakage at 30 degrees C was due to an increase in the extent of reversible surface aggregation at the lower temperature. Experiments employing fluorescein-labeled pardaxin demonstrated reversible aggregation of the peptide in suspension and within the membrane, and exchange of the peptide between liposomes. In summary, our experimental and theoretical results support reversible surface aggregation as the mechanism of pore formation by pardaxin.     

Low-temperature phase behaviour of the major plant leaf lipid monogalactosyldiacylglycerol.

Heating and cooling thermograms of unsaturated MGDG samples isolated from the leaves of Vicia faba are surprisingly featureless. This reflects the low enthalpies associated with phase transitions in highly unsaturated lipids and the fact that these transitions, in the case of MGDG, are to a large extent masked by those associated with the freezing and melting of ice. Careful choice of thermal heating/cooling regimes, combined with the use of real-time X-ray diffraction and freeze-fracture measurements, permits a detailed analysis of the phase behaviour of the system. The phase behaviour of unsaturated MGDG samples is shown to be basically similar to that seen in saturated MGDG samples. The lipid which exists in the inverted hexagonal (HexII) liquid crystal phase at room temperature forms a highly disordered lamellar gel (L beta) phase on cooling to temperatures below about -15 degrees C. On reheating, this first reorganizes at a temperature of about -10 degrees C to form a well-defined Lc1 phase. Above about -2 degrees C, this melts to re-form the HexII phase. Samples re-cooled from temperatures between -2 degrees C and 14 degrees C revert directly to the Lc1 phase while samples cooled from higher temperatures form the L beta phase. This reflects the fact that the former samples contain small amounts of unmelted Lc1 phase lipid. The implications of these observations are discussed in terms of the general problems associated with the measurement of low-temperature phase behaviour of membrane lipids.     

Thermodynamic characterization of an artificially designed amphiphilic alpha-helical peptide containing periodic prolines: observations of high thermal stability and cold denaturation.

To investigate the structural stability of proteins, we analyzed the thermodynamics of an artificially designed 30-residue peptide. The designed peptide, NH2-EELLPLAEALAPLLEALLPLAEALAPLLKK-COOH (PERI COIL-1), with prolines at i + 7 positions, forms a pentameric alpha-helical structure in aqueous solution. The thermal denaturation curves of the CD at 222 nm (pH 7.5) show an unusual cold denaturation occurring well above 0 degrees C and no thermal denaturation is observable under 90 degrees C. This conformational change is reversible and depends on peptide concentration. A 2-state model between the monomeric denatured state (5D) and the pentameric helical state (H5) was sufficient to analyze 5 thermal denaturation curves of PERI COIL-1 with concentrations between 23 and 286 microM. The analysis was carried out by a nonlinear least-squares method using 3 fitting parameters: the midpoint temperature, Tm, the enthalpy change, delta H(Tm), and the heat capacity change, delta Cp. The association number (n = 5) was determined by sedimentation equilibrium and was not used as a fitting parameter. The heat capacity change suggests that the hydrophobic residues are buried in the helical state and exposed in the denatured one, as it occurs normally for natural globular proteins. On the other hand, the enthalpy and the entropy changes have values close to those found for coiled-coils and are quite distinct from typical values reported for natural globular proteins. In particular, the enthalpy change extrapolated at 110 degrees C is about 3 kJ/mol per amino acid residue, i.e., half of the value found for globular proteins.(ABSTRACT TRUNCATED AT 250 WORDS)     

Competition between the 735 nm fluorescence and the photochemistry of Photosystem I in chloroplasts at low temperature

Fluorescence emission spectra of chloroplasts, initially frozen to--196 degrees C, were measured at various temperatures as the sample was allowed to warm. The 735 nm emission band attributed to fluorescence from Photosystem I was approx. 10-fold greater at--196 degrees C than at--78 degrees C. The initial rate of photooxidation of P-700 was also measured at--196 degrees C and--78 degrees C and was found to be approximately twice as large at the higher temperature. It is proposed that the 735 nm emission band is fluorescence from a long wavelength form of chlorophyll, C-705, which acts as a trap for excitation energy in the antenna chlorophyl system of Photosystem I. Furthermore, it is proposed that C-705 only forms on cooling to low temperatures and that the temperature dependence of the 735 nm emission is the temperature dependence for the formation of C-705. C-705 and P-700 compete to trap the excitation energy in Photosystem I. It is estimated from the data that at--78 degrees C P-700 traps approx. 20 times more energy than C-705 while, at--196 degrees C, the two traps are approximately equally effective. By analogy, the 695 nm fluorescence which also appears on cooling to--196 degrees C is attributed to traps in Photosystem II which form only on cooling to temperatures near--196 degrees C.     

Microstructures of an amelogenin gel matrix.

The thermo-reversible transition (clear <--> opaque) of the amelogenin gel matrix, which has been known for some three decades, has now been clarified by microstructural investigations. A mixed amelogenin preparation extracted from porcine developing enamel matrix (containing "25K," 7.4%; "23K," 10.7%; "20K," 49.5%; and smaller peptides, 32.4%) was dissolved in dilute formic acid and reprecipitated by adjusting the pH to 6.8 with NaOH solution. Amelogenin gels were formed in vitro by sedimenting the precipitate in microcentrifuge tubes. The gels were fixed with Karnovsky fixative at 4 and 24 degrees C, which was found to preserve their corresponding clear (4 degrees C) and opaque (24 degrees C) states. Scanning electron microscopy, atomic force microscopy, and transmission electron microscopy were employed for the microstructural characterization of the fixed clear and opaque gels. The amelogenin gel matrix was observed to possess a hierarchical structure of quasi-spherical amelogenin nanospheres and their assemblies. The nanospheres of diameters 8-20 nm assemble to form small spherical assemblies of diameters 40-70 nm that further aggregated to form large spherical assemblies of 70-300 nm in diameter. In the clear gel, most of the large assemblies are smaller than 150 nm, and the nanospheres and assemblies are uniformly dispersed, allowing an even fluid distribution among them. In the opaque gel, however, numerous spherical fluid-filled spaces ranging from 0.3 to 7 microm in diameter were observed with the majority of the large assemblies sized 150-200 nm in diameter. These spaces presumably result from enhanced hydrophobic interactions among nanospheres and/or assemblies as the temperature increased. The high opacity of the opaque (24 degrees C) gel apparently arises from the presence of the numerous fluid-filled spaces observed compared to the low-temperature (4 degrees C) preparation. These observations suggest that the hydrophobic interactions among nanospheres and different orders of amelogenin assemblies are important in determining the structural integrity of the dental enamel matrix.     

Domain specificity in metal binding to metallothionein. A circular dichroism and magnetic circular dichroism study of cadmium and zinc binding at temperature extremes

Rabbit liver Zn metallothionein-(MT) will bind cadmium readily between -26 degrees C and 70 degrees C. The binding reaction was monitored by recording the circular dichroism and magnetic circular dichroism spectra, in the region of the RS(-)----Cd2+ charge transfer transition at 250 nm, at intervals as aliquots of cadmium were added. For all temperatures, these data can be analyzed in terms of a distributed mechanism for cadmium binding when Zn-MT is used, and a domain-specific mechanism when apo-MT is used. The CD spectrum measured at -26 degrees C for Cd,Zn-MT, which was made by adding excess cadmium directly to Zn7-MT at -26 degrees C, is not the same as the CD spectrum of Cd-MT prepared at room temperature from the same Zn7-MT. Measurements of the stoichiometry of the cadmium and zinc bound to MT in the presence of excess cadmium at different temperatures indicates that below 5 degrees C at least one zinc atom remains bound to the protein. The mixed metal metallothionein, Cd/Zn-MT, that always forms below 5 degrees C, is characterized by a single maximum near 250 nm in the CD spectrum, rather than the derivative-shaped CD envelope that is diagnostic of the (Cd4-S11)alpha cluster, which indicates that the zinc occupies a site in the alpha domain. Rearrangement of the bound metals to the domain-specific distribution takes place if Cd,Zn-MT, prepared at subzero temperatures, is warmed above 30 degrees C.     

The biological activity of alpha-1-antichymotrypsin: the change of chymotrypsin-inhibitory and immunoenhancing activities by heat treatment

The relationship between chymotrypsin-inhibitory and immunoenhancing activity of alpha-1-antichymotrypsin was studied. alpha-1-Antichymotrypsin was treated at 50 degrees C, 55 degrees C or 60 degrees C for 15 min. It was found that antichymotryptic activity was reduced by half when alpha-1-antichymotrypsin was heated at 55 degrees C and was not detected at all when heating was carried out at 60 degrees C. alpha-1-Antichymotrypsin which was heated at 60 degrees C did not form a complex with chymotrypsin, but became a substrate for chymotrypsin. The effect of native and heated alpha-1-antichymotrypsin on antibody response was studied in mice. alpha-1-Antichymotrypsin increased the number of anti-sheep erythrocytes antibody producing cells even when it was heated at 60 degrees C. Circular dichroism and single radial immunodiffusion were used to detect conformational changes. Circular dichroism in the region of side chain absorption showed that the intensities of the spectra at 296, 284, and 265 nm decreased with a rise in temperature from 50 to 60 degrees C. In single radial immunodiffusion analysis, alpha-1-antichymotrypsin did not form a halo after being heated at 60 degrees C. In conclusion, when alpha-1-antichymotrypsin was heated at 60 degrees C, the immunoenhancing activity remained intact while the antichymotryptic activity was lost with the conformational change.     

Characterization of the rebinding of 125I-epidermal growth factor released from BALB/c-3T3 cells following accumulation in the presence of chloroquine

Lysosomotropic amines, such as chloroquine and methylamine, increase the intracellular accumulation of 125I-EGF by inhibiting lysosomal degradation. It has been shown previously that BALB/c-3T3 cells, prelabeled at 4 degrees C with 125I-EGF for 3 h and subsequently chased at 37 degrees C in the presence of chloroquine, internalized the surface bound 125I-EGF which was subsequently released into the extracellular medium in a high molecular weight form which co-migrated with native 125I-EGF. The secreted 125I-EGF rebound to the cells from which it was released more efficiently than does peptide in the extracellular media. We now show that when the BALB/c-3T3 cells were prelabeled at 37 degrees C for 2 h in the presence of chloroquine, the internalized 125I-EGF released into the medium was in a high molecular weight form which co-migrated with native 125I-EGF and did not rebind anymore efficiently than did peptide in the extracellular media. This lack of rebinding was not due to an alteration in the 125I-EGF molecule since it was still capable of rebinding to naive A431 cells, nor was it due to the exhaustion of EGF receptors on the BALB/c-3T3 cells. The inhibition of rebinding was observed only when the cells were treated with EGF in the presence of chloroquine, and was not due to a general down-regulation of membrane receptors. The differences between the rebinding of 125I-EGF at 4 degrees C and 37 degrees C suggest that EGF may be processed via different pathways in the cell.     

Interrelationships among biological activity, disulfide bonds, secondary structure, and metal ion binding for a chemically synthesized 34-amino-acid peptide derived from alpha-fetoprotein.

A 34-amino-acid peptide has been chemically synthesized based on a sequence from human alpha-fetoprotein. The purified peptide is active in anti-growth assays when freshly prepared in pH 7.4 buffer at 0.20 g/l, but this peptide slowly becomes inactive. This functional change is proven by mass spectrometry to be triggered by the formation of an intrapeptide disulfide bond between the two cysteine residues on the peptide. Interpeptide cross-linking does not occur. The active and inactive forms of the peptide have almost identical secondary structures as shown by circular dichroism (CD). Zinc ions bind to the active peptide and completely prevents formation of the inactive form. Cobalt(II) ions also bind to the peptide, and the UV-Vis absorption spectrum of the cobalt-peptide complex shows that: (1) a near-UV sulfur-to-metal-ion charge-transfer band had a molar extinction coefficient consistent with two thiolate bonds to Co(II); (2) the lowest-energy visible d-d transition maximum at 659 nm, also, demonstrated that the two cysteine residues are ligands for the metal ion; (3) the d-d molar extinction coefficient showed that the metal ion-ligand complex was in a distorted tetrahedral symmetry. The peptide has two cysteines, and it is speculated that the other two metal ion ligands might be the two histidines. The Zn(II)- and Co(II)-peptide complexes had similar peptide conformations as indicated by their ultraviolet CD spectra, which differed very slightly from that of the free peptide. Surprisingly, the cobalt ions acted in the reverse of the zinc ions in that, instead of stabilizing anti-growth form of the peptide, they catalyzed its loss. Metal ion control of peptide function is a saliently interesting concept. Calcium ions, in the conditions studied, apparently do not bind to the peptide. Trifluoroethanol and temperature (60 degrees C) affected the secondary structure of the peptide, and the peptide was found capable of assuming various conformations in solution. This conformational flexibility may possibly be related to the biological activity of the peptide.     

Sequence dependence of conformations of self-complementary duplex tetradeoxynucleotides containing cytosine and guanine

The four self-complementary tetradeoxynucleotides which contain only cytosine and guanine are 5'-d-(CpGpCpG)-3', 5'-d(CpCpGpG)-3', 5'-d(GpCpGpC)-3', and 5'-d(GpGpCpC)-3'. The Raman spectra of aqueous solutions (about 0.05 M in monomer) of these tetranucleotides at pH 7 and 2 degrees C show clearly that these self-complementary tetranucleotides form double-stranded duplex structures of the canonical B type when the NaCl concentration is 0.5 M NaCl. If the temperature is raised to 50 degrees C, the Raman spectra show that in each case the double-helical B form melts in a non-cooperative way to a disordered single-chain form. On the other hand, if the salt concentration is raised to saturation, the Raman spectrum of only one of these four tetranucleotide solutions at 2 degrees C is changed in any substantial way. The Raman spectrum of the tetranucleotide 5'-d(CpGpCpG)-3' at 2.2 degrees C and at 4 M or higher salt concentration strongly resembles that of double-helical Z-form poly(dC-dG) taken under similar conditions. We conclude that the tetramer 5'-d(CpGpCpG)-3' is the only self-complementary double-helical tetranucleotide containing only cytosine and guanine in which the B-Z transition can be induced by increasing the salt concentration. This tetramer has several types of stacking interactions which differ markedly from stacking interactions in the other tetramers and may account for the enhanced stabilization of its Z conformation.