Domain structure analysis of elongation factor-3 from Saccharomyces cerevisiae by limited proteolysis and differential scanning calorimetry

Elongation-factor-3 (EF-3) is an essential factor of the fungal protein synthesis machinery. In this communication the structure of EF-3 from Saccharomyces cerevisiae is characterized by differential scanning calorimetry (DSC), ultracentrifugation, and limited tryptic digestion. DSC shows a major transition at a relatively low temperature of 39 degrees C, and a minor transition at 58 degrees C. Ultracentrifugation shows that EF-3 is a monomer; thus, these transitions could not reflect the unfolding or dissociation of a multimeric structure. EF-3 forms small aggregates, however, when incubated at room temperature for an extended period of time. Limited proteolysis of EF-3 with trypsin produced the first cleavage at the N-side of Gln775, generating a 90-kDa N-terminal fragment and a 33-kDa C-terminal fragment. The N-terminal fragment slowly undergoes further digestion generating two major bands, one at approximately 75 kDa and the other at approximately 55 kDa. The latter was unusually resistant to further tryptic digestion. The 33-kDa C-terminal fragment was highly sensitive to tryptic digestion. A 30-min tryptic digest showed that the N-terminal 60% of EF-3 was relatively inaccessible to trypsin, whereas the C-terminal 40% was readily digested. These results suggest a tight structure of the N-terminus, which may give rise to the 58 degrees C transition, and a loose structure of the C-terminus, giving rise to the 39 degrees C transition. Three potentially functional domains of the protein were relatively resistant to proteolysis: the supposed S5-homologous domain (Lys102-Ile368), the N-terminal ATP-binding cassette (Gly463-Lys622), and the aminoacyl-tRNA-synthase homologous domain (Glu820-Gly865). Both the basal and ribosome-stimulated ATPase activities were inactivated by trypsin, but the ribosome-stimulated activity was inactivated faster.     

Proteolysis as a measure of the free energy difference between cytochrome c and its derivatives

Limited cleavage of oxidized and reduced horse heart cytochrome c (Cyt c) and the azide complex of Cyt c by proteinase K at room temperature yields a single cut within the central loop (36-60 in the sequence). Using an assay that allows spectroscopic evaluation of the fraction of intact protein as a function of time, together with a simple kinetic model for proteolysis, fluctuation opening of the loop can be related to the free energy of the corresponding protein. This allows us to estimate quantitatively the free energy difference between the oxidized form of Cyt c and other states using proteolysis as a probe. The results we obtain indicate that oxidized Cyt c is 2.0 kcal mol(-1) less stable than the reduced form, and 0.07 kcal mol(-1) is more stable than the Cyt c: azide complex at 25 degrees C. These values agree in magnitude with results from hydrogen exchange and unfolding studies, suggesting that the stability of a protein can be directly related to its structural dynamics.     

Protein-protein interactions on weak-cation-exchange sorbent surfaces during chromatographic separations

This paper examines the nature of chromatographic separations on a weak cation-exchange material in which immobilized protein coats 24% or less of the sorbent surface. It was found that columns on which proteins were immobilized still behaved as a cation-exchange chromatography sorbents, but their selectivity was different from the parent weak cation-exchange column. This was interpreted to mean that in addition to the normal electrostatic interactions expected in ion- exchange chromatography, protein analytes interact with immobilized protein on the sorbent surface. Anionic proteins were not adsorbed, indicating that immobilized proteins were acting synergistically with ionic stationary phase groups to enhance retention. It is concluded that these protein-protein interactions occur after proteins are captured by the primary interaction mechanism of the column, in this case, electrostatic interaction. Protein-protein interaction is a secondary, lateral process. These lateral interactions were observed between 4% and 24% surface saturation. The significance of this observation is that in preparative chromatography and the case of "fouled" columns, strongly adsorbed proteins could alter the elution characteristics of sample proteins being target for analysis or purification.     

A structural model of human erythrocyte spectrin. Alignment of chemical and functional domains

Proteolytic susceptibility has been used to probe the structure of human erythrocyte spectrin. Nine unique polypeptide segments have been defined by mild trypsin digestion (0 degrees C) and analyzed by two-dimensional peptide mapping techniques. These peptide segments, referred to operationally as chemical domains, exhibited varying degrees of sensitivity to further proteolytic cleavage. One region (beta I) which contained the phosphorylated amino acids of the beta subunit was quite sensitive to proteolysis and was rapidly degraded to numerous small peptides. Overlap peptides produced by enzymatic and chemical cleavages were used to align each domain in the appropriate spectrin subunit. The molecular weights of the largest unique peptides from both subunits sum to the approximate weight of the intact molecule. Similarly, summation of the two-dimensional peptide maps of the intermediate sized peptides approximates the two-dimensional maps of the intact spectrin subunits, indicating that most or all of the molecule is represented. These results suggest that spectrin is composed of multiple, ordered, largely alpha-helical domains that are connected by small protease-sensitive segments. A comprehensive structural model is presented.     

Cell-surface display of a Pseudomonas aeruginosa strain K pilin peptide within the paracrystalline S-layer of Caulobacter crescentus

The paracrystalline surface (S)-layer of Caulobacter crescentus is composed of a single secreted protein (RsaA) that interlocks in a hexagonal pattern to completely envelop the bacterium. Using a genetic approach, we inserted a 12 amino acid peptide from Pseudomonas aeruginosa strain K pilin at numerous semirandom positions in RsaA. We then used an immunological screen to identify those sites that presented the inserted pilin peptide on the C. crescentus cell surface as a part of the S-layer. Eleven such sites (widely separated in the primary sequence) were identified, demonstrating for the first time that S-layers can be readily exploited as carrier proteins to display 'epitope-size' heterologous peptides on bacterial cell surfaces. Whereas intact RsaA molecules carrying a pilin peptide could always be found on the surface of C. crescentus regardless of the particular insertion site, introduction of the pilin peptide at 9 of the 11 sites resulted in some proteolytic cleavage of RsaA. Two types of proteolytic phenomena were observed. The first was characterized by a single cleavage within the pilin peptide insert with both fragments of the S-layer protein remaining anchored to the outer membrane. The other proteolytic phenomenon was characterized by cleavage of the S-layer protein at a point distant from the site of the pilin peptide insertion. This cleavage always occurred at the same location in RsaA regardless of the particular insertion site, yielding a surface-anchored 26 kDa proteolytic fragment bearing the RsaA N-terminus; the C-terminal cleavage product carrying the pilin peptide was released into the growth medium. When the results of this work were combined with the results of a previous study, the RsaA primary sequence could be divided into three regions with respect to the location of a peptide insertion and its effect on S-layer biogenesis: (i) insertions in the extreme N-terminus of RsaA either produce no apparent effect on S-layer biogenesis or disrupt surface-anchoring of the protein; (ii) insertions in the extreme C-terminus either produce no apparent effect on S-layer biogenesis or disrupt protein secretion; and (iii) insertions more centrally located in the protein either have no apparent effect on S-layer biogenesis or result in proteolytic cleavage of RsaA. These data are discussed in relation to our previous assignment of the RsaA N- and C-terminus as regions that are important for surface anchoring and secretion respectively.     

Efficient autoproteolytic processing of the MHV-A59 3C-like proteinase from the flanking hydrophobic domains requires membranes

The replicase gene of the coronavirus MHV-A59 encodes a serine-like proteinase similar to the 3C proteinases of picornaviruses. This proteinase domain is flanked on both sides by hydrophobic, potentially membrane-spanning, regions. Cell-free expression of a plasmid encoding only the 3C-like proteinase (3CLpro) resulted in the synthesis of a 29-kDa protein that was specifically recognized by an antibody directed against the carboxy-terminal region of the proteinase. A protein of identical mobility was detected in MHV-A59-infected cell lysates. In vitro expression of a plasmid encoding the 3CLpro and portions of the two flanking hydrophobic regions resulted in inefficient processing of the 29-kDa protein. However, the efficiency of this processing event was enhanced by the addition of canine pancreatic microsomes to the translation reaction, or removal of one of the flanking hydrophobic domains. Proteolysis was inhibited in the presence of N-ethylmaleimide (NEM) or by mutagenesis of the catalytic cysteine residue of the proteinase, indicating that the 3CLpro is responsible for its autoproteolytic cleavage from the flanking domains. Microsomal membranes were unable to enhance the trans processing of a precursor containing the inactive proteinase domain and both hydrophobic regions by a recombinant 3CLpro expressed from Escherichia coli. Membrane association assays demonstrated that the 29-kDa 3CLpro was present in the soluble fraction of the reticulocyte lysates, while polypeptides containing the hydrophobic domains associated with the membrane pelletes. With the help of a viral epitope tag, we identified a 22-kDa membrane-associated polypeptide as the proteolytic product containing the amino-terminal hydrophobic domain.     

Plasma protein adsorption on biodegradable microspheres consisting of poly(D,L-lactide-co-glycolide), poly(L-lactide) or ABA triblock copolymers containing poly(oxyethylene). Influence of production method and polymer composition

Biodegradable particulate systems have been considered as parenteral drug delivery systems. The adsorption of plasma proteins on micro- and nanoparticles is determined by the surface properties and may, in turn, strongly influence the biocompatibility and biodistribution of both carriers. In the present study the influence of the polymer composition and the production method of microspheres on the in vitro plasma protein adsorption were investigated using two-dimensional electrophoresis (2-DE). Microparticles were prepared from poly(l-lactide) (l-PLA), poly(d,l-lactide-co-glycolide) (PLGA), and ABA triblock copolymers containing hydrophilic poly(oxyethylene) (B-blocks) domains connected to hydrophobic polyesters (A-blocks). Two different microencapsulation methods were employed, namely the w/o/w emulsion solvent evaporation method and the spray-drying technique. It could be demonstrated that the polymer composition and, especially, the encapsulation technique, influenced the interactions with plasma proteins significantly. For example, the percentages of several apolipoproteins in the plasma protein adsorption patterns of spray-dried PLGA- and l-PLA-particles were distinctly higher when compared to the adsorption patterns of the particles produced by the w/o/w-technique. Some adsorbed proteins were found to be characteristic or even specific for particles produced by the same method or consisting of identical polymers. Polyvinyl alcohol used as stabilizer in the w/o/w-technique may decisively influence the surface properties relevant for protein adsorption. The plasma protein adsorption on particles composed of ABA copolymers was drastically reduced when compared to microspheres made from pure polyesters. The adsorption patterns of ABA-particles were dominated by albumin. The plasma protein adsorption patterns detected on the different microspheres are likely to affect their in vivo performance as parenteral drug delivery systems.     

Bax directly induces release of cytochrome c from isolated mitochondria

Bax is a pro-apoptotic member of the Bcl-2 protein family that resides in the outer mitochondrial membrane. It is controversial whether Bax promotes cell death directly through its putative function as a channel protein versus indirectly by inhibiting cellular regulators of the cell death proteases (caspases). We show here that addition of submicromolar amounts of recombinant Bax protein to isolated mitochondria can induce cytochrome c (Cyt c) release, whereas a peptide representing the Bax BH3 domain was inactive. When placed into purified cytosol, neither mitochondria nor Bax individually induced proteolytic processing and activation of caspases. In contrast, the combination of Bax and mitochondria triggered release of Cyt c from mitochondria and induced caspase activation in cytosols. Supernatants from Bax-treated mitochondria also induced caspase processing and activation. Recombinant Bcl-XL protein abrogated Bax-induced release of Cyt c from isolated mitochondria and prevented caspase activation. In contrast, the broad-specificity caspase inhibitor benzyloxycarbonyl-valinyl-alaninyl-aspartyl-(0-methyl)- fluoromethylketone (zVAD-fmk) and the caspase-inhibiting protein X-IAP had no effect on Bax-induced release of Cyt c from mitochondria in vitro but prevented the subsequent activation of caspases in cytosolic extracts. Unlike Ca2+, a classical inducer of mitochondrial permeability transition, Bax did not induce swelling of mitochondria in vitro. Because the organellar swelling caused by permeability transition causes outer membrane rupture, the findings, therefore, dissociate these two events, implying that Bax uses an alternative mechanism for triggering release of Cyt c from mitochondria.     

Roles of different hydrophobic constituents in the adsorption of pulmonary surfactant

Surface tension-time adsorption isotherms were measured at 37 degrees C for calf lung surfactant extract (CLSE) and subfractions of its constituents: the complete mix of surfactant phospholipids (PPL), phospholipids depleted in anionic phospholipids (mPPL), hydrophobic surfactant proteins plus phospholipids (SP&PL, SP&mPL), and neutral lipids plus phospholipids (N&PL). Adsorption experiments were done using a static bubble surfactometer where diffusion resistance was present, and in a Teflon dish where diffusion was minimized by subphase stirring. The contribution of diffusion to bubble adsorption measurements decreased as phospholipid concentration increased, and was small at 0.25 mM phospholipid. At this phospholipid concentration, PPL, mPPL, and N&PL all adsorbed more rapidly and to lower final surface tensions than dipalmitoyl phosphatidylcholine (DPPC) on the bubble. However, none of these phospholipid mixtures adsorbed to surface tensions below 46 mN/m after 20 min, behavior that was significantly worse than CLSE, SP&PL, and SP&mPL which additionally contained hydrophobic SP. Both CLSE and SP&PL rapidly adsorbed to surface tensions below 25 mN/m at 0.25 mM phospholipid concentration on the bubble, as did SP&mPL at a somewhat reduced rate. Further experiments defining the influence of hydrophobic protein content showed that addition of even 0.13% SP (by wt) to PPL improved adsorption substantially, and that mixtures of PPL combined with 1% SP had adsorption very similar to CLSE. Mixtures of SP combined with mPPL had faster adsorption than corresponding mixtures of SP:DPPC, and neither fully matched the adsorption rates of CLSE and SP&PL even at high SP levels (4% in SP:mPPL and 5.2% in SP:DPPC). These results demonstrate that although the secondary zwitterionic and anionic phospholipids and neutral lipids in lung surfactant enhance adsorption relative to DPPC, the hydrophobic SP have a much more pronounced effect in promoting the rapid entry of pulmonary surfactant into the air-water interface.     

Identification of plasma proteins facilitated by enrichment on particulate surfaces: analysis by two-dimensional electrophoresis and N-terminal microsequencing

Plasma protein adsorption on intravenously injectable drug carriers is regarded as an important factor for the fate of the particles in the body after their administration. Therefore, the plasma protein adsorption patterns on a number of different carrier systems were analyzed in vitro employing two-dimensional electrophoresis (2-DE). The particulate systems presented in this study were polystyrene (PS) model particles, PS nanoparticles surface-modified by adsorption of a surfactant, a commercial fat emulsion, and magnetic iron oxide particles used as contrast agents in magnetic resonance imaging. Most of the spots in the plasma protein adsorption patterns could be identified by matching the resulting 2-DE gels with a reference map of human plasma proteins. Several other proteins that indicated preferentially adsorbed proteins on the surface of the particles investigated have either not been identified on the reference map, or their identity was found to be ambiguous. The relevant proteins are all present in plasma in low abundance. Since these proteins were strongly enriched on the surface of the particles, the resulting spots on the 2-DE gels were successfully identified by N-terminal microsequencing. With this approach, two chains of spots, designated PLS:6 and PLS:8, were determined on a plasma reference map: inter-alpha-trypsin inhibitor family heavy chain-related protein (also named PK-120) and a dimer of fibrinogen gamma, respectively. Plasma gelsolin is presented in a 2-DE adsorption pattern of PS model particles. One of the main proteins adsorbed by droplets of a commercial fat emulsion was identified as apoliprotein H. Moreover, the positions of apolipoproteins apoC-II and apoC-III were also verified on the 2-DE protein map of human plasma. Thus, protein adsorption experiments of the kind presented in this study are increasing our insight into human plasma proteins.     

Effect of substance P and receptor antagonists on secretion of lingual lipase and amylase from rat von Ebner''s gland

Electron donor acceptor gels based on cyanocarbons have been tested for human serum protein adsorption in the absence of salt-promotion by water-structuring salt. This phenomenon was compared with a normal adsorption process in the presence of salt. The tricyanoaminopropene-divinyl sulfone-agarose displayed unusual protein adsorption properties as binding could occur both independently or dependently of the salt-promotion. The absence of hydrophobic or ionic character of the salt-independent interaction suggests an electron donor acceptor adsorption mechanism which is shown, for the first time, to occur independently of salt-promotion in aqueous solution. Study of the protein adsorption specificity showed similar protein selectivity for the fractions adsorbed in both conditions.     

Solubilization of hydrophobic peptides by reversible cysteine PEGylation

"PEG-a-Cys" reagent, synthesized by the esterification of monomethoxy-poly(ethylene glycol) (avg. MW = 5 kDa) to Ellman's reagent [5,5'-dithiobis(2-nitrobenzoic acid)], is shown to "PEGylate" reversibly the cysteine residue of a 25-residue synthetic hydrophobic peptide (H2N-REAAALAAAAALAAWAALCPARRRR-CO2H) designed to model a transmembrane segment of a membrane protein. A mixed disulfide bond was formed between the reagent and the peptide that was readily cleaved with the mild reducing agent tricarboxyethylphosphine hydrochloride (TCEP.HCl). Carboxypeptidase B digestion of the charged carboxyl terminus of the peptide through to the Ala residue--which mimics the enzymatic cleavage of a TM segment from a fusion protein--releases a highly hydrophobic peptide. A time-dependent decrease in the amplitude of the digested peptide circular dichroism (CD) spectra was attributed to the aggregation and/or precipitation of the peptide. While PEGylation of the peptide with PEG-a-Cys had a negligible effect on conformation, it inhibited the loss of CD amplitude in both intact and digested peptides, suggesting that it was effective in solubilization of hydrophobic peptides.     

Nucleotide-induced conformational changes in the ATPase and substrate binding domains of the DnaK chaperone provide evidence for interdomain communication

Interactions of the DnaK (Hsp70) chaperone from Escherichia coli with substrates are controlled by ATP. Nucleotide-induced changes in DnaK conformation were investigated by monitoring changes in tryptic digestion pattern and tryptophan fluorescence. Using nucleotide-free DnaK preparations, not only the known ATP-induced major changes in kinetics and pattern of proteolysis but also minor ADP-induced changes were detected. Similar ATP-induced conformational changes occurred in the DnaK-T199A mutant protein defective in ATPase activity, demonstrating that they result from binding, not hydrolysis, of ATP. N-terminal sequencing and immunological mapping of tryptic fragments of DnaK identified cleavage sites that, upon ATP addition, appeared within the proposed C-terminal substrate binding region and disappeared in the N-terminal ATPase domain. They hence reflect structural alterations in DnaK correlated to substrate release and indicate ATP-dependent domain interactions. Domain interactions are a prerequisite for efficient tryptic degradation as fragments of DnaK comprising the ATPase and C-terminal domains were highly protease-resistant. Fluorescence analysis of the N-terminally located single tryptophan residue of DnaK revealed that the known ATP-induced alteration of the emission spectrum, proposed to result directly from conformational changes in the ATPase domain, requires the presence of the C-terminal domain and therefore mainly results from altered domain interaction. Analyses of the C-terminally truncated DnaK163 mutant protein revealed that nucleotide-dependent interdomain communication requires a 15-kDa segment assumed to constitute the substrate binding site.     

Structure of the membrane-bound form of the pore-forming domain of colicin A: a partial proteolysis and mass spectrometry study

The ion-channel-forming thermolytic fragment (thA) of colicin A binds to negatively charged vesicles and provides an example of the insertion of a soluble protein into a lipid bilayer. The soluble structure is known and consists of a 10-helix bundle containing a hydrophobic helical hairpin. In this study, partial proteolysis and mass spectrometry were used to determine the accessible sites to proteolytic attack by trypsin and alpha-chymotrypsin in the thA fragment in its membrane-bound state. Electrospray mass spectrometry was quite an efficient method for the identification of the cleavage products, even with partially purified peptide mixtures and with only few controls by N-terminal sequencing. This work confirms that a major part of the peptide chain lies at the membrane surface and that even the hydrophobic hairpin is not protected by the lipid bilayer from proteolytic degradation. In the absence of a membrane potential, the hydrophobic hairpin in the colicin A membrane-bound form seems not fixed in a transmembrane orientation.     

Conversion of proparathyroid hormone to parathyroid hormone: studies in vitro with trypsin

The conversion of proparathyroid hormone to parathyroid hormone (PTH) was studied in vitro employing pancreatic trypsin as a prototype converting enzyme. Digestion of intact radiolabeled bovine prohormone with trypsin (0.1%) (w/w) resulted in release of a peptide comigrating with intact hormone marker in systems resolving both on the basis of charge (urea polyacrylamide gels, pH 4.4) and size (sodium dodecyl sulfate-urea polyacrylamide gels, pH 7.2). Tryptic digestions of a synthetic analogue of bovine prohormone, ProPTH-(-6 + 34), consisting of the prohormone hexapeptide covalently bonded to the NIH2 terminus of the active fragment of the hormone, released in high yield the hexapeptide and the intact active hormone fragment before any other smaller fragments. Analyses of digestions were by: (i) thin-layer chromatography and amino acid analysis of digestion products; (ii) comparison of the biological activity of the prophormone substrate and the products of digestion; and (iii) peptide end-group analysis by the Edman method during progressive tryptic hydrolysis over 22 h. The latter experiments demonstrated cleavage of more than 75% of the hexapeptide-hormone peptide bond before cleavage of other trypsin-sensitive sites within the molecule. It is concluded that the specificity of cleavage at the hexapeptide-hormone bond in the process of intracellular hydrolysis of proparathyroid hormone resides primarily in the sequence and/or conformation of the precursor molecule; inasmuch as conversion of prohormone to hormone can be efficiently accomplished by pancreatic trypsin in vitro, there is, therefore, no need to postulate the existence of an intracellular converting enzyme within the parathyroid cell that possesses unique hydrolytic specificity.     

Rapid analytical tryptic mapping of a recombinant chimeric monoclonal antibody and method validation challenges

A rapid and reproducible analytical tryptic mapping method was developed as an identity test for a recombinant chimeric monoclonal antibody for lot release testing. The unfolding, reduction, carboxymethylation, trypsin digestion, and reversed-phase (RP) HPLC steps were optimized to provide a reproducible method. The optimized method requires 30 min for unfolding the protein, 30 min for carboxymethylation, 4 h for digestion with TPCK-trypsin and 140 min for RPHPLC analysis. The total time required is less than 8 h compared to conventional procedures, which must be performed over several days. The optimized method was validated for its precision, recovery, specificity, and robustness. The precision of the method was determined by repeatability and intermediate precision experiments. Relative standard deviation (RSD) values were < or = 10% for the relative peak areas of marker peaks. The mean recovery of these marker peaks was 88.4%. The specificity was demonstrated by the unique tryptic mapping patterns obtained compared with several other monoclonal antibodies. Robustness was demonstrated by the relative insensitivity of the tryptic map to small deliberate changes in key method parameters. Excessive relative peak area variability observed for one peak (RSD 52%) was traced to adsorption to glass autosampler vials. This variability was substantially reduced (RSD 11%) by substituting polypropylene autosampler vials. The data demonstrate that this method may be applicable to a wide range of pharmaceutically relevant monoclonal antibodies.     

125I labelling of human serum albumin and fibrinogen and a study of protein adsorption properties on the surface of titanium oxide film

In order to detect the surface concentration of proteins adsorbed on a solid surface for selecting blood compatible materials, a gentle iodination reagent, Iodogen, was used to label human serum albumin and fibrinogen, and has been applied to the study of protein adsorption properties on a plate of titanium oxide film. The yields of the labelled albumin and fibrinogen are 69.7% and 49.6%. The results of adsorption show that [125I]HSA and [125I]HFG are efficacious at the surface concentration detection and can be used to investigate the protein adsorption properties of a solid material.     

Purification and characterization of a recombinant hepatitis E protein vaccine candidate by liquid chromatography-mass spectrometry

A protein with a molecular mass of approximately 62.10(3), derived from open reading frame 2 (ORF-2) of the hepatitis E virus (HEV: Burma strain), was expressed in a baculovirus expression vector and purified to homogeneity. The recombinant 62 kDa protein appeared to be a doublet, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Tryptic digestion in conjunction with laser desorption mass spectrometry (LD-MS) and sequence analysis of the tryptic peptides indicated that the amino terminus was blocked, although no proteolytic degradation occurred. The determined internal sequences of peptides were in agreement with the predicted ORF-2 protein. Reversed-phase liquid chromatography coupled to electrospray mass spectrometry (LC-MS) resolved the doublet proteins into two major components with molecular masses of 56548.5 and 58161.4. Confirmation of the amino terminus of the molecule by LD-MS post-ion decay enabled us to tentatively assign the carboxyl terminus of each species at residues 540 and 525. Sequencing of the intact protein by automated carboxyl terminal sequencing confirmed that the carboxyl terminus was truncated and that the sequence assignment predicted by LC-MS was correct.     

Detection of a variant of protein 3, the major transmembrane protein of the human erythrocyte

A variant of the major transmembrane protein of the human erythrocyte has been detected following proteolytic digestion of intact erythrocytes. Pronase digestion of normal erythrocytes gives rise to a 60,000 molecular weight fragment of Protein 3, while digestion of erythrocytes with the variant protein produces two fragments of 60,000 and 63,000 molecular weight when peptides are separated by sodium dodecyl sulfate-acrylamide gel electrophoresis using the discontinuous buffer system of Laemmli (Laemmli, U. K. (1970) Nature 227, 680-685). The two fragments cannot be resolved if electrophoresis is conducted using the continuous phosphate or Tris/acetate buffer systems. This increased molecular weight of the variant fragment does not appear to be due to increased glycosylation, since neither sialic acid residues nor terminal galactose units can be detected. Furthermore, the transmembrane segment of Protein 3 can be detected after proteolytic digestion at both the external and cytoplasmic membrane surfaces. These transmembrane segments of both the normal and the variant peptide have identical molecular weights of 20,000 to 21,000. These results suggest that the increased molecular weight of the variant peptide is due to the incorporation of an additional segment into that region of the molecule which is exposed at the cytoplasmic side of the membrane.     

The effects of antioxidant supplementation during Percoll preparation on human sperm DNA integrity

Adenovirus DNA polymerase is one of three viral proteins and two cellular proteins required for replication of the adenovirus genome. During initiation of viral DNA synthesis the viral DNA polymerase transfers dCMP onto the adenovirus preterminal protein, to which it is tightly bound. The domain structure of the 140 kDa DNA polymerase has been probed by partial proteolysis and the sites of proteolytic cleavage determined by N-terminal sequencing. At least four domains can be recognised within the DNA polymerase. Adenovirus preterminal protein interacts with three of the four proteolytically derived domains. This was confirmed by cloning and expression of each of the individual domains. These data indicate that, like other members of the pol alpha family of DNA polymerases, the adenovirus DNA polymerase has a multidomain structure and that interaction with preterminal protein takes place with non-contiguous regions of the polypeptide chain over a large surface area of the viral DNA polymerase.