Role of the carbohydrate chain and two phosphate moieties in the heat-induced aggregation of hen ovalbumin

We investigated the effect of the carbohydrate chain and two phosphate moieties on heat-induced aggregation of hen ovalbumin. The dephosphorylated form of ovalbumin was obtained by treating the original protein with acid phosphatase. The single carbohydrate chain was removed by digestion of heat-denatured ovalbumin with glycopeptidase F, and the resulting polypeptide without this carbohydrate chain was correctly refolded to acquire protease-resistance. Thermal unfolding can be approximated by a mechanism involving a two-state transition between the folded and unfolded states with a midpoint temperature of 76 degrees C for the original form, of 74 degrees C for the dephosphorylated form, and of 71 degrees C for the carbohydrate-free form. The conformational stability of the original form was higher than that of the carbohydrate-free form. When the three forms of ovalbumin were heated to 80 degrees C and then cooled rapidly in an ice bath, the polypeptide chains were compactly collapsed to metastable intermediates with secondary structures whose properties were indistinguishable. Upon incubation at 60 degrees C, renaturation was possible for a large portion of the intermediates of the original form, but for only a small portion of those of the carbohydrate-free form. Light scattering experiments showed that in the presence of sulfate anions, the intermediates of the carbohydrate-free form aggregated to a greater extent than did those of the original form. The intermediates of the carbohydrate-free form bound to the chaperonin GroEL with about 10-fold higher affinity than those of the original form. It follows that the carbohydrate chain and the two phosphate moieties do not affect hydrophobic collapse in the kinetic refolding of hen ovalbumin but play an important role in the slow rearrangement. They block the off-pathway reaction that competes with correct refolding by effectively decreasing surface hydrophobicity.     

Comparative study of chicken ovalbumin subfractions having different carbohydrate chain from each other by high performance anion exchange chromatography

1. Ovalbumin was fractionated to six fractions according to their phosphate content by high performance anion exchange chromatography. 2. This method was applied to analyze the phosphate content of ovalbumin subfractions having different carbohydrate chain from each other which were prepared by concanavalin A/Sepharose chromatography from oviduct slices incubated with [2-3H]mannose. 3. Most biosynthetic intermediates bearing a carbohydrate chain of Man8 or 9 GlcNAc2 was not phosphorylated while other fractions bearing differently processed carbohydrate chains such as Man5 or 6 GlcNAc2 or hybrid type carbohydrate chain was phosphorylated at their peptide portion.     

Carbohydrate-free carboxypeptidase Y is transferred into the lysosome-like yeast vacuole

Carboxypeptidase Y, localized in the lysosome-like yeast vacuole, has been metabolically labeled with [2-³H]mannose. After immunoprecipitation the carbohydrate moieties were released by treatment with endo-β-N-acetyl-glucosaminidase H and separated by paper electrophoresis. Evidence for the presence of phospho-monoester and -diester groups in the molecule has been obtained. In the latter phosphate links C-1 of mannose or of mannosyl 1,3-mannose to C-6 of a mannose residue within a larger oligomannose moiety. In the presence of tunicamycin yeast cells synthesize a carbohydrate-free carboxypeptidase Y, which could be traced after metabolic labeling with [¹⁴C]-phenylalanine. The carbohydrate-free enzyme was segregated into the vacuoles to the same extent as the intact glycoprotein.     

The stability of yeast invertase is not significantly influenced by glycosylation

Yeast invertase exists in two different forms. The cytoplasmic enzyme is nonglycosylated, whereas the external invertase contains about 50% carbohydrate of the high mannose type. The protein moieties of both enzymes are identical. The two invertases have been used previously as a model system to investigate the influence of covalently linked carbohydrate chains on the stability of large glycoproteins, and controversial results were obtained. Here, we measured thermal and denaturant-induced unfolding by various probes, such as the loss of enzymatic activity, and by the changes in absorbance and fluorescence. The ranges of stability of the two invertases were found to be essentially identical, indicating that the presence of a high amount of carbohydrate does not significantly contribute to the stability of external invertase. Earlier findings that invertase is stabilized by glycosylation could not be confirmed. The stability of this glycoprotein is apparently determined by the specific interactions of the folded polypeptide chain. Unlike the glycosylated form, the carbohydrate-free invertase is prone to aggregation in the denatured state at high temperature and in a partially unfolded form in the presence of intermediate concentrations of guanidinium chloride.     

A novel method for the incorporation of glycoprotein-derived oligosaccharides into neoglycopeptides.

We describe a new method for the transfer of carbohydrate moieties to polypeptides in which complex carbohydrate, in the form of glycosyl amino acid, is removed from an available glycoprotein, derivatized, and reacted with a polypeptide via an iodoacetylated alpha-amino group. A family of oligomannose chains, N-linked to the side chain of Asn, was obtained from ovalbumin by pronase digestion and purified as previously described. A reactive sulfhydryl group was specifically placed on these molecules by reaction of 2-iminothiolane with the Asn alpha-amino group. Separately, the alpha-amino group of the peptide GGYR was specifically iodoacetylated by reaction with iodoacetic anhydride at pH 6. Reaction of the thiol-containing carbohydrate with iodoacetylated peptide at pH 8 gave in high yield the corresponding oligomannosyl-peptides, whose structures were confirmed by mass spectrometry. A peptide inhibitor of HIV protease was also oligomannosylated by this procedure. The principle advantage of this method is the efficiency of the reaction even when performed with stoichiometric amounts of the two molecules at low concentration. It should be feasible to extend this chemistry to larger polypeptides.     

The influence of operational parameters on lysozyme refolding using size-exclusion chromatography

The influence of several parameters on the gel filtration refolding of hen egg white lysozyme from a starting concentration of 40 mg/ml was investigated. Refolding was found to be unaffected by temperature between 30 and 50°C, giving 100% recovered specific activity. At 10°C a 20% reduction in refolding yield was observed. Refolding was carried out successfully with both acrylamide (Sephacryl S100)- and dextran (Superdex 75)-based gel media. At the isoelectric pH of lysozyme, aggregation was suppressed in the column method, whereas protein aggregates were formed during dilution-based refolding. A number of compounds (carboxymethyl cellulose, dextran, sucrose) were added to the mobile phase to reduce the relative viscosity between the sample and mobile phase. Only sucrose, up to 20% (wt), was found not to interfere with lysozyme refolding.     

Temperature-sensitive mutants blocked in the folding or subunit assembly of the bacteriophage P22 tail spike protein: III. Inactive polypeptide chains synthesized at 39 °C

Salmonella typhimurium cells infected by temperature-sensitive mutants in gene 9 of bacteriophage P22 at the restrictive temperature (39 °C) fail to accumulate functional tail spike protein. We report here studies of the inactive mutant tail spike polypeptide chains synthesized at 39 °C by temperature-sensitive mutants at 15 different sites of gene 9. For all 15 mutants, the gene 9 polypeptide chains were synthesized at 39 °C at rates similar to wild type. The mutant polypeptide chains were stable within the infected cells.The inactive polypeptide chains were tested for three functions displayed by the mature tail spike protein: irreversible binding to phage heads, endorhamnosidase activity, and reaction with anti-tail antibody. The 15 mutant proteins that accumulated at 39 °C lacked all three functions. Since the amino acid substitutions do not affect these functions of the mature protein, the mutant polypeptide chains synthesized at 39 °C have a conformation very different from the wild type, and different from the same proteins when matured at 30 °C. The fact that amino acid substitutions throughout the 76,000 M_r polypeptide chain prevent all three functions suggests that the mutations prevent the correct folding of the gene 9 polypeptide chain at restrictive temperature. Thus, these mutations identify sites in the polypeptide chain critical for protein maturation.Many of the mutant proteins could be activated in the absence of new protein synthesis by shifting infected cells from restrictive to permissive temperature before cell lysis. For these mutants, the immature chains accumulating at high temperature must be reversibly related to intermediates in protein folding or subunit assembly.     

Effects of urea and guanidine · HCl on the folding and unfolding of pancreatic trypsin inhibitor

Concentrated solutions of urea and of guanidine · HCl produced a random spectrum of single-disulphide forms of the polypeptide chain of the pancreatic trypsin inhibitor. Guanidine · HCl also unfolded completely, with accompanying interchange of disulphide bonds, the two-disulphide form of this protein in the native-like conformation; urea produced an equilibrium mixture in which one-quarter of the molecules had the native-like conformation and disulphide bonds. The unfolded forms of the protein in the denaturants were very flexible polypeptide chains. The observations suggest that urea and guanidine · HCl are denaturants because they produce essentially equally favourable solvation of all portions of a polypeptide.The energetics of the conformational transitions involved in folding and unfolding of the inhibitor were determined in urea and compared with those observed in its absence. The denaturant lowers the stability of the native, folded inhibitor relative to that of the reduced, unfolded state by 6.5 kilocalories per mole; the greatest part of this apparent free-energy difference was expressed at the two-disulphide stage of folding. The results are consistent with other indications that most of the favourable interactions stabilizing the native conformation of this protein are not encountered until the final stage of folding, when all may occur simultaneously.The unfolded one- and two-disulphide species produced in guanidine · HCl were trapped, and their rearrangement to the normal intermediates followed after removal of the denaturant. The random single-disulphide species, with one exception, reverted very rapidly to the non-random spectrum of intermediates normally observed during folding; this confirms that these species are normally rapidly interconverted and that normal refolding of the reduced protein is not dependent kinetically upon residual stable conformation in the reduced protein. The unfolded two-disulphide species refolded to the native-like conformation more slowly, but appeared to pass through the same intermediates normally observed during refolding from the fully reduced state.     

A Fourier-transform infrared spectroscopic study of the phosphoserine residues in hen egg phosvitin and ovalbumin

A Fourier-transform infrared spectroscopic study of hen egg phosvitin and ovalbumin has been carried out. Bands arising from monoanionic and dianionic phosphate monoester [Shimanouchi, T., Tsuboi, M., & Kyogoku, Y. (1964) Adv. Chem. Phys. 8, 435-498] can be identified easily in the 1300-930 cm-1 region in spectra of solutions of O-phosphoserine and phosvitin, a highly phosphorylated protein. On the other hand, spectra of ovalbumin show a relatively strong absorption above 1000 cm-1 arising from the protein moiety. Below 1000 cm-1, a single band at 979 cm-1 is observed; this band is not present in spectra of dephosphorylated ovalbumin, and therefore, it has been assigned to the symmetric stretching of the phosphorylated Ser-68 and Ser-344 in the dianionic ionization state. In addition, bands arising from symmetric and antisymmetric stretchings of the monoanionic ionization state, and from the antisymmetric stretching of the dianionic state, can be detected above 1000 cm-1 in difference spectra of ovalbumin minus dephosphorylated ovalbumin. The effect of pH on the infrared spectra of O-phosphoserine, phosvitin, and ovalbumin is consistent with the phosphoserine residues undergoing ionization with pK values about 6. This study demonstrates that Fourier-transform infrared spectroscopy can be a useful technique to assess the ionization state of phosphoserine residues in proteins in solution.     

The mechanism of folding of pancreatic ribonucleases is independent of the presence of covalently linked carbohydrate

The role of asparagine-linked oligosaccharides for the mechanism of protein folding was investigated. We compared the stability and folding kinetics for two sets of pancreatic ribonucleases (RNases) with identical amino acid sequences and differences in glycosylation. First the folding of RNases A (carbohydrate free) and B (a single N-linked oligosaccharide) from bovine pancreas was investigated. The kinetics of refolding were identical under a wide range of conditions. The rate of unfolding by guanidinium chloride was decreased in RNase B. In further experiments the folding of porcine RNase (three carbohydrate chains at Asn-21, -34, and -76) was compared with the corresponding data for the deglycosylated protein. Even for this RNase with almost 40% carbohydrate content the mechanism of refolding is independent of glycosylation. Although the folding mechanism is conserved, the rates of individual steps in folding are decreased about 2-fold upon deglycosylation. We interpret this to originate from a slight destabilization of folding intermediates by carbohydrate depletion. In control experiments with nonglycosylated bovine RNase A it was ascertained that treatment with HF (as used for deglycosylation) did not affect the folding kinetics. The in vitro folding mechanism of glycosylated RNases apparently does not depend on the presence of N-linked oligosaccharide chains. The information for the folding of glycoproteins is contained exclusively in the protein moiety, i.e. in the amino acid sequence. Carbohydrate chains are attached at chain positions which remain solvent exposed. This ensures that the presence of oligosaccharides does not interfere with correct folding of the polypeptide chain.     

Recombination of S-peptide with S-protein during folding of ribonuclease S. I. Folding pathways of the slow-folding and fast-folding classes of unfolded S-protein.

The refolding kinetics of ribonuclease S have been measured by tyrosine absorbance, by tyrosine fluorescence emission, and by rapid binding of the specific inhibitor 2′CMP ² to folded RNAase S. The S-protein is first unfolded at pH 1.7 and then either mixed with S-peptide as refolding is initiated by a stopped-flow pH jump to pH 6.8, or the same results are obtained if S-protein and S-peptide are present together before refolding is initiated. The refolding kinetics of RNAase S have been measured as a function of temperature (10 to 40 °C) and of protein concentration (10 to 120 μm). The results are compared to the folding kinetics of S-protein alone and to earlier studies of RNAase A. A thermal folding transition of S-protein has been found below 30 °C at pH 1.7; its effects on the refolding kinetics are described in the following paper (Labhardt &; Baldwin, 1979).In this paper we characterize the refolding kinetics of unfolded S-protein, as it is found above 30 °C at pH 1.7, together with the kinetics of combination between S-peptide and S-protein during folding at pH 6.8. Two classes of unfolded S-protein molecules are found, fast-folding and slow-folding molecules, in a 20: 80 ratio. This is the same result as that found earlier for RNAase A; it is expected if the slow-folding molecules are produced by the slow cis-trans isomerization of proline residues after unfolding, since S-protein contains all four proline residues of RNAase A.The refolding kinetics of the fast-folding molecules show clearly that combination between S-peptide and S-protein occurs before folding of S-protein is complete. If combination occurred only after complete folding, then the kinetics of formation of RNAase S should be rather slow (5 s and 100 s at 30 °C) and nearly independent of protein concentration, as shown by separate measurements of the folding kinetics of S-protein, and of the combination between S-peptide and folded S-protein. The observed folding kinetics are faster than predicted by this model and also the folding rate increases strongly with protein concentration (apparent 1.6 order kinetics). The fact that RNAase S is formed more rapidly than S-protein alone is sufficient by itself to show that combination with S-peptide precedes complete folding of S-protein. Computer simulation of a simple, parallel-pathway scheme is able to reproduce the folding kinetics of the fast-folding molecules. All three probes give the same folding kinetics.These results exclude the model for protein folding in which the rate-limiting step is an initial diffusion of the polypeptide chain into a restricted range of three-dimensional configurations (“nueleation”) followed by rapid folding (“propagation”). If this model were valid, one would expect comparable rates of folding for RNAase A and for S-protein and one would also expect to find no populated folding intermediates, so that combination between S-peptide and S-protein should occur after folding is complete. Instead, RNAase A folds 60 times more rapidly than S-protein and also combination with S-peptide occurs before folding of S-protein is complete. The results demonstrate that the folding rate of S-protein increases after the formation, or stabilization, of an intermediate which results from combination with S-peptide. They support a sequential model for protein folding in which the rates of successive steps in folding depend on the stabilities of preceding intermediates.The refolding kinetics of the slow-folding molecules are complex. Two results demonstrate the presence of folding intermediates: (1) the three probes show different kinetic progress curves, and (2) the folding kinetics are concentration-dependent, in contrast to the results expected if complete folding of S-protein precedes combination with S-peptide. A faster phase of the slow-refolding reaction is detected both by tyrosine absorbance and fluorescence emission but not by 2′CMP binding, indicating that native RNAase S is not formed in this phase. Comparison of the kinetic progress curves measured by different probes is made with the use of the kinetic ratio test, which is defined here.     

Interaction of melittin with dimyristoyl phosphatidylcholine liposomes. Evidence for boundary lipid by raman spectroscopy

The interaction of melittin, a polypeptide consisting of 26 amino acid residues, with dimyristoyl phosphatidylcholine bilayers was investigated by vibrational Raman spectroscopy. Spectral peak height intensity ratios, involving vibrational transitions in both the 3000 cm^?1 acyl chain methylene carbon-hydrogen stretching mode region and the 1100 cm^?1 acyl chain carbon-carbon skeletal stretching mode interval, served as temperature profile indices for monitoring the bilayer order-disorder processes. For a lipid : melittin molar ratio of 14 : 1 two order-disorder transitions were observed. In comparison to a gel to liquid crystalline phase transition of 22.5°C for the pure lipid, the lower transition, exhibiting a 2°C width, is centered at 17°C and is associated with a depression of the main lipid phase transition of dimyristoyl phosphatidylcholine. The second thermal transition, displaying a 7°C interval, occurs at approx. 29°C and is associated with the melting behavior of approximately seven immobilized boundary lipids which surround the inserted hydrophobic segment of the polypeptide. For a lipid : melittin molar ratio of 10 : 1 two thermal transitions are also observed at 11 and 30°C. As before, they represent, respectively, the main gel to liquid crystalline phase transition and the melting behavior of approximately four boundary lipids attached to melittin. From these data alternative schemes are suggested for disposing the immobilized lipids around the hydrophobic portion of the polypeptide within the bilayer.     

Immunochemical analysis of the conformational properties of intermediates trapped in the folding and unfolding of bovine pancreatic trypsin inhibitor.

Immunochemical methods have been used to examine the conformational properties of the entire polypeptide chain in the various trapped intermediate states which are kinetically important in the unfolding and refolding of pancreatic trypsin inhibitor. The interactions of each of the trapped intermediates, having their disulphide bonds frozen, with antibodies specific for either the native, folded or the reduced, unfolded states of the entire protein have been used to determine the probabilities of the various segments of the polypeptide chain adopting either conformation recognized by the antibodies.The results are considered with regard to the kinetic roles of the various species and to their conformational properties during folding and unfolding inferred from the observed propensities of each of the six cysteine residues to participate in disulphide bond formation, interchange, or breakage. It is concluded that no segment of the polypeptide chain adopts a stable native-like conformation until the entire polypeptide chain is able to do so simultaneously. The best correlation of conformation with the kinetic role in refolding of the intermediates is observed not with their propensity to adopt native-like conformation, but with their stability to full unfolding as measured by their interaction with antibodies against the reduced protein.     

Factors influencing the capacity for photosynthetic carbon assimilation in barley leaves at low temperatures

The aim of this work was to examine the effect upon photosynthetic capacity of short-term exposure (up to 10 h) to low temperatures (5° C) of darkened leaves of barley (Hordeum vulgare L.) plants. The carbohydrate content, metabolite status and the photosynthetic rate of leaves were measured at low temperature, high light and higher than ambient CO₂. Under these conditions we could detect whether previous exposure of leaves to low temperature overcame the limitation by phosphate which occurs in leaves of plants not previously exposed to low temperatures. The rates of CO₂ assimilation measured at 8° C differed by as much as twofold, depending upon the pretreatment. (i) Leaves from plants which had previously been darkened for 24 h had a low content of carbohydrate, had the lowest CO₂-assimilation rates at low temperature, and photosynthesis was limited by carbohydrate, as shown by a large stimulation of photosynthesis by feeding glucose, (ii) Leaves from plants which had previously been illuminated for 24 h and which contained large carbohydrate reserves showed an accumulation of phosphorylated intermediates and higher CO₂-assimilation rates at low temperature, but nevertheless remained limited by phosphate, (iii) Maximum rates of CO₂ assimilation at low temperature were observed in leaves which had intermediate reserves of carbohydrate or in leaves which were rich in carbohydrate and which were also fed phosphate. It is suggested that carbohydrate reserves potentiate the system for the achievement of high rates of photosynthesis at low temperatures by accumulation of photosynthetic intermediates such as hexose phosphates, but that this potential cannot be realised if, at the same time, carbohydrate accumulation is itself leading to feedback inhibition of photosynthesis. This work was supported by the Agricultural and Food Research Council, UK (Research grant PG50/67) and by the Science and Engineering Reserach Council, UK. C.A.L. was supported by the British Council, by an Overseas Research Student Award and by the Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (CNPq), Brazil.     

Refolding studies using pressure: the folding landscape of lysozyme in the pressure-temperature plane

Refolding of hen egg white lysozyme after pressure unfolding was measured by FTIR spectroscopy. The high-pressure treatment was found to be useful for unfolding/refolding studies because pressure acts against aggregation, and therefore no irreversible aggregation takes place during the pressure treatment. After the release of the pressure, folding intermediate structures were found which were formed during the decompression of the lysozyme. These were aggregation prone when heated, as indicated by their lower stability against aggregation. The intermediates were only formed if the protein was unfolded, subdenaturing pressures could not populate these intermediates. We introduced the notion of a superfunnel to describe the free energy landscape of interacting polypeptide chains. This can explain the propensity of folding intermediates to aggregate. A possible Gibbs-free energy landscape for lysozyme was constructed for the whole pressure-temperature plane.     

Nature and mode of action of NAD and ATP dephosphorylating enzyme from Aspergillus terreus DSM 826

NAD and ATP were dephosphorylated by Aspergillus terreus extracts optimally at pH 8 and 40 °C. The data obtained indicate that one phosphohydrolase was involved in the cleavage of all the phosphate linkages of these two energy-carrying molecules, and also indicate that this enzyme can be classified as a non-specific alkaline phosphatase. This is based on the following criteria: during fractionation of the enzymes of the extracts, using Sephadex G-200 column chromatography, the recorded elution diagram showed only one phosphohydrolase activity peak and this peak was the same with NAD, ATP, inorganic pyrophosphate and phenyl phosphate as substrates; the activity profiles with these four substrates were similar; and these four substrates were hydrolyzed at almost constant relative rates. Moreover, the activities of the pooled fractions with these different substrates responded similarly on changing some experimental conditions, such as addition of fluoride to the reaction mixtures or exposing the enzyme preparation to temperatures above 40 °C. Chromatographic detection of the intermediates and the products formed during the progression of NAD and ATP dephosphorylation by the most purified fraction of this enzyme was found to be consistent with the following mode of its action: This revised version was published online in June 2006 with corrections to the Cover Date.     

Cleaved serpin refolds into the relaxed state via a stressed conformer

Serine proteinase inhibitors (serpins) are believed to fold in vivo into a metastable "stressed" state with cleavage of their P1-P1' bond resulting in reactive center loop insertion and a thermostable "relaxed" state. To understand this unique folding mechanism, we investigated the refolding processes of the P1-P1'-cleaved forms of wild type ovalbumin (cl-OVA) and the R339T mutant (cl-R339T). In the native conditions, cl-OVA is trapped as the stressed conformer, whereas cl-R339T attains the relaxed structure. Under urea denaturing conditions, these cleaved proteins completely dissociated into the heavy (Gly(1)-Ala(352)) and light (Ser(353)-Pro(385)) chains. Upon refolding, the heavy chains of both proteins formed essentially the same initial burst refolding intermediates and then reassociated with the light chain counterparts. The reassociated intermediates both refolded into the native states with indistinguishable kinetics. The two refolded proteins, however, had a notable difference in thermostability. cl-OVA refolded into the stressed form with T(m) = 68.4 degrees C, whereas cl-R339T refolded into the relaxed form with T(m) = 85.5 degrees C. To determine whether cl-R339T refolds directly to the relaxed state or through the stressed state, conformational analyses by anion-exchange chromatography and fluorescence measurements were executed. The results showed that cl-R339T refolds first to the stressed conformation and then undergoes the loop insertion. This is the first demonstration that the P1-P1'-cleaved serpin peptide capable of loop insertion refolds to the stressed conformation. This highlights that the stressed conformation of serpins is an inevitable intermediate state on the folding pathway to the relaxed structure.     

Effect of Inhibition of Glycosylation on the Appearance of a 60 kD Membrane Glycopolypeptide in Endomembrane Fractions of Soybean Root

Endomembrane (endoplasmic reticulum, Golgi apparatus, plasma membrane) proteins of soybean (Glycine max) root cells are highly glycosylated. We investigated whether N-linked oligosaccharide moieties are essential for the correct intracellular transport of plant endomembrane glycoproteins. Excised roots were incubated with tunicamycin, to block cotranslational glycosylation of proteins, and dual labeled with [³H]glucosamine and [³⁵S] (methionine, cysteine). In the presence of tunicamycin, the incorporation of glucosamine into membrane proteins was inhibited by 60 to 90% while amino acid incorporation was only slightly affected. Autoradiograms of two-dimensionally separated polypeptides from each endomembrane fraction revealed the presence of at least one new polypeptide in tunicamycin-treated tissue. The new polypeptide was of the same isoelectric point but lower molecular weight than a preexisting polypeptide. The new polypeptide was unreactive to concanavalin A, as opposed to the preexisting polypeptide, suggesting the absence of the glycan portion. Trifluoromethanesulfonic acid and N-glycanase were used to cleave the carbohydrate from the preexisting concanavalin A binding polypeptide. In each case a deglycosylated polypeptide of the same isoelectric point and molecular weight as the new polypeptide from tunicamycin-treated tissue resulted. Since the absence of carbohydrate from the new endomembrane polypeptide did not prevent its appearance on autoradiograms of Golgi and plasma membrane, intracellular transport and intercalation of newly synthesized glycoproteins into plant cell membranes may not require the presence of polysaccharide moieties.     

Complete amino acid sequence of the catalytic chain of human complement subcomponent C1-r

The amino acid sequence of human C1-r b chain hs been determined, from sequence analysis performed on fragments obtained by CNBr cleavage, dilute acid hydrolysis, tryptic cleavage of the succinylated protein, and subcleavages by staphylococcal protease. The polypeptide chain contains 242 amino acids (Mr 27 096), and the sequence shows strong homology with other mammalian serine proteases. The histidine, aspartic acid, and serine residues of the active site (His-57, Asp-102, and Ser-195 in bovine chymotrypsinogen) are located at positions 39, 94, and 191, respectively. The chain which lacks the "histidine-loop" disulfide bridge, contains five half-cystine residues, of which four (positions 157-176 and 187-217) are homologous to residues involved in disulfide bonds generally conserved in serine proteases, whereas the half-cystine residue at position 114 is likely to be involved in the single disulfide bridge connecting the catalytic b chain to the n-terminal a chain. Two carbohydrate moieties are attached to the polypeptide chain, both via asparagine residues at positions 51 and 118.     

13C-n.m.r.-spectral study of galactosyltransferase specificity with regard to the carbohydrate side-chain of native ovalbumin

As a prelude to studies using bovine N-acetylglucosaminide-β-(1→4)-galactosyltransferase to label membrane-surface glycoproteins with isotopically enriched d-galactose, the structural specificity of the enzymic reaction with water-soluble, hen ovalbumin has been examined. The enzyme-catalyzed transfer of d-galactose from UDP-d-galactose requires a (nonreducing) terminal 2-acetamido-2-deoxy-d-glucosyl group and exhibits selectivity towards saccharide chains containing d-mannose. This study considers the structural specificity of the enzyme with regard to the anomeric linkage between 2-acetamido-2-deoxy-d-glucose and d-mannose in the carbohydrate chains of hen ovalbumin. Uniformly ¹³C-enriched d-galactose was enzymically attached to the ovalbumin carbohydrate chain (which exhibits microheterogeneity in its structure), the protein was hydrolyzed, and separate glycopeptide fractions were chromatographically isolated. The ¹³C-n.m.r. spectra (60.5 MHz) of the fractions revealed two peaks for the anomeric carbon atom of d-galactose. The two peaks, at 104.20 and 104.39 p.p.m., were ascribed to d-galactosyl groups attached to 2-acetamido-2-deoxy-d-glucose respectively linked β-(1→4) and β-(1→2), to d-mannose in the glycopeptide chains. Quantifying of the spectral data revealed no specificity of d-galactosyltransferase towards the linkage from the terminal 2-acetamido-2-deoxy-d-glucosyl group to the penultimate d-mannosyl residue.