Sodium Dodecyl Sulfate Denaturation Research Articles

Biochemical characterization of the human diabetes‐associated HLA‐DQ8 allelic product: Similarity to the major histocompatibility complex class II I‐Ag7 protein of non‐obese diabetic mice

The human HLA-DQ8 (A1*0301/B1*0302) allelic product manifests a strong association with insulin-dependent diabetes mellitus (IDDM). Previous biochemical studies of the major histocompatibility complex (MHC) class II I-A(g)7 protein of IDDM-prone non-obese diabetic mice produced controversial results. To better define the biochemical properties of IDDM-associated MHC class II molecules, we analyzed DQ8 proteins, in comparison to other DQ allelic products, by partially denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We now report that DQ8 proteins have a normal peptide occupancy and lifespan in cells. Similar to I-A(g)7, DQ8 proteins formed only a minor fraction of SDS-stable complexes with peptides. Although this phenotype was not unique to DQ8, some DQ allelic products such as IDDM-protective DQ6 proteins were SDS resistant. The DQ9 allelic product, differing from DQ8 only at position (P) beta 57, was SDS stable, suggesting that non-Asp residues at beta 57 might decrease the SDS stability of DQ proteins. We identified a single peptide which specifically induced an SDS-stable conformation in DQ8 as well as in I-A(g)7 molecules. The residues at anchor P1 in this peptide were found to influence the SDS stability of both molecules. Together with our previous observation of similar binding motifs of I-A(g)7 and DQ8, these results demonstrate an overall biochemical similarity of mouse and human diabetes-associated MHC class II molecules. This similarity might contribute to a common immunological mechanism of IDDM in both species.

Effect of Mutations in Vesicle-Associated Membrane Protein (VAMP) on the Assembly of Multimeric Protein Complexes

The assembly of multimeric protein complexes that include vesicle-associated membrane protein 2 (VAMP-2) and the plasma membrane proteins syntaxin 1A and synaptosome-associated protein of 25 kDa (SNAP-25) are thought to reflect the biochemical correlates of synaptic vesicle targeting, priming, or fusion. Using a variety of protein-protein interaction assays and a series of deletion and point mutations, we have investigated the domains of VAMP-2 required for the formation of binary complexes with either syntaxin 1A or SNAP-25 and ternary complexes with both syntaxin 1A and SNAP-25. Deletions within the central conserved domain of VAMP-2 eliminated binding to either syntaxin 1A or both syntaxin 1A and SNAP-25. Although all of the deletion mutants were able to form ternary complexes, only some of these complexes were resistant to denaturation in sodium dodecyl sulfate. These results demonstrate that cooperative interactions result in the formation of at least two biochemically distinct classes of ternary complex. Two point mutations previously shown to have effects on the intracellular trafficking of VAMP-2 (M46A, reduced endocytosis and sorting to synaptic vesicles; N49A, enhanced sorting to synaptic vesicles) lie within a domain required for both syntaxin 1A and SNAP-25 binding. Syntaxin 1A and SNAP-25 binding was reduced by the M46A mutation and enhanced by the N49A mutation, suggesting that a correlation exists between the membrane-trafficking phenotype of the two VAMP-2 point mutants and their competence to form complexes with either syntaxin 1A or SNAP-25.

Purification and characterization of N-acetylneuraminate synthase from Escherichia coli K1-M12.

N-Acetylneuraminate (NeuAc) synthase, which catalyzes NeuAc synthesis by condensation of N-acetyl-D-mannosamine (ManNAc) and phosphoenolpyruvate (PEP), was purified from a cell extract of Escherichia coli K1-M12 to electrophoretically homogeneity by serial column chromatographies. The molecular weight of native enzyme was estimated to be 106,000 by gel filtration. After denaturation in sodium dodecyl sulfate, the molecular weight was reduced to 52,000, indicating the existence of 2 identical subunits. The optimum pH was 7.5 and the stable pH range was 7.0 to 10.0. The enzyme was thermostable up to 30 degrees C. No metal ion was required for the enzyme activity. SH-inhibitors such as p-chloromercuribenzoic acid and mercury chloride were potent inhibitors. The K(m) for ManNAc and PEP were 5.6 mM and 0.04 mM, respectively.

Cloning, nucleotide sequence, and expression of the Brucella melitensis omp31 gene coding for an immunogenic major outer membrane protein.

The gene coding for the major outer membrane protein (OMP) of 31 to 34 kDa, now designated Omp31, of Brucella melitensis 16M was cloned and sequenced. A B. melitensis 16M genomic library was constructed in lambda GEM-12 XhoI half-site arms, and recombinant phages expressing omp31 were identified by using the anti-Omp31 monoclonal antibody (MAb) A59/10F09/G10. Subcloning of insert DNA from a positive phage into pGEM-7Zf allowed the selection of a plasmid bearing a 4.4-kb EcoRI fragment that seemed to contain the entire omp31 gene under control of its own promoter. omp31 was localized within a region of the EcoRI insert of approximately 1.1 kb. Sequencing of this region revealed an open reading frame of 720 bp encoding a protein of 240 amino acids and a predicted molecular mass of 25,307 Da. Cleavage of the first 19 amino acids, showing typical features of signal peptides for protein export, leaves a mature protein of 221 amino acids with a predicted molecular mass of 23,412 Da. The predicted amino acid sequence of B. melitensis 16M Omp31 showed 35.2% identity with the RopB OMP of Rhizobium leguminosarum bv. viciae 248 and 34.3% identity with Omp25 of B. abortus 544. As in Brucella spp., Omp31 was located in the outer membrane of recombinant Escherichia coli, but its reported peptidoglycan association in Brucella cells was not detected in E. coli. The ability of Omp31 to form oligomers resistant to sodium dodecyl sulfate denaturation at low temperatures, a characteristic described for several bacterial porins, was observed in both B. melitensis and recombinant E. coli. The epitope recognized by the anti-Omp31 MAb A59/10F09/G10, for which a protective activity has been suggested, has been delimited to a region of 36 amino acids of Omp31 covering the most hydrophilic part of the protein. The availability of recombinant Omp31 and the identification of the antigenic determinant recognized by MAb A59/10F09/G10 will allow the evaluation of their potential protective activity and their potential for the development of subcellular vaccines against brucellosis.

THE FUCUS (PHAEOPHYCEAE) SPERM RECEPTOR FOR EGGS. II. ISOLATION OF A BINDING PROTEIN WHICH PARTIALLY ACTIVATES EGGS1

ABSTRACT An in vitro binding assay involving egg plasma membrane vesicles (PMVs) of Fucus serratus L. and proteins contained in a KCl extract of sperm has been used to identify a sperm protein involved in egg binding. High‐performance gel filtration (HPGF) separated the sperm KCl extract into several major fractions, and a protein (apparent M, 60 kDa) was identified as being involved in binding to the egg PMVs. This protein ran on denaturing sodium dodecyl sulfate (SDS)gels with an apparent molecular weight of 27 kDa. This suggests that either the native form of the protein is a dimer or the molecular weight on HPGF is an artifact caused by high ionic strength buffer promoting hydrophobic interactions. When KCl‐sol‐uble proteins were separated by SDS‐polyacrylamide gel electrophoresis (PAGE), blotted onto nitrocellulose, and incubated with biotinylated egg PMVs, these bound to a band at 27 kDa, confirming the role of this protein. Addition of the Fucus sperm extract or HPGF fractions containing the binding protein to eggs in the absence of sperm induced the release of polysaccharides onto the egg cell surface. This labeling was patchy, in contrast to the uniform release of polysaccharides observed when sperm were added to eggs. The monoclonal antibody (MAb) FS17 was raised against the 27‐kDa sperm protein. It labeled the sperm body and both flagella by immunofluorescence, though the sperm had to he permeabilized to observe labeling, suggesting that the epitope recognized is not exposed at the cell surface. Addition of FS17 to the KCl extract in the binding assay reduced subsequent binding of egg PMVs. Removal of the 27‐kDa protein recognized by FS17 from the sperm extract prevented the binding of egg PMVs in the binding assay and the triggering of the patchy release of polysaccharides when added to eggs. Overall the results suggest that the 27‐kDa sperm protein is involved in binding to the egg plasma membrane and can trigger partial activation of the egg.

Molecular cloning and nucleotide sequence of the gene encoding the major peptidoglycan hydrolase of Lactococcus lactis, a muramidase needed for cell separation.

A gene of Lactococcus lactis subsp. cremoris MG1363 encoding a peptidoglycan hydrolase was identified in a genomic library of the strain in pUC19 by screening Escherichia coli transformants for cell wall lysis activity on a medium containing autoclaved, lyophilized Micrococcus lysodeikticus cells. In cell extracts of L. lactis MG1363 and several halo-producing E. coli transformants, lytic bands of similar sizes were identified by denaturing sodium dodecyl sulfate (SDS)-polyacrylamide gels containing L. lactis or M. lysodeikticus cell walls. Of these clearing bands, corresponding to the presence of lytic enzymes with sizes of 46 and 41 kDa, the 41-kDa band was also present in the supernatant of an L. lactis culture. Deletion analysis of one of the recombinant plasmids showed that the information specifying lytic activity was contained within a 2,428-bp EcoRV-Sau3A fragment. Sequencing of part of this fragment revealed a gene (acmA) that could encode a polypeptide of 437 amino acid residues. The calculated molecular mass of AcmA (46,564 Da) corresponded to that of one of the lytic activities detected. Presumably, the enzyme is synthesized as a precursor protein which is processed by cleavage after the Ala at position 57, thus producing a mature protein with a size of 40,264 Da, which would correspond to the size of the enzyme whose lytic activity was present in culture supernatants of L. lactis. The N-terminal region of the mature protein showed 60% identity with the N-terminal region of the mature muramidase-2 of Enterococcus hirae and the autolysin of Streptococcus faecalis. Like the latter two enzymes, AcmA contains C-terminal repeated regions. In AcmA, these three repeats are separated by nonhomologous intervening sequences highly enriched in serine, threonine, and asparagine. Genes specifying identical activities were detected in various strains of L. lactis subsp. lactis and L. lactis subsp. cremoris by the SDS-polyacrylamide gel electrophoresis detection assay and PCR experiments. By replacement recombination, an acmA deletion mutant which grew as long chains was constructed, indicating that AcmA is required for cell separation.

Creatine kinase compactness and thiol accessibility during sodium dodecyl sulfate denaturation estimated by resonance energy transfer and 2-nitro-5-thiocyanobenzoic acid cleavage

We have investigated the effect of increasing sodium dodecyl sulfate (SDS) concentrations on rabbit muscle cytosolic creatine kinase structure by two methods. We have first determined the variation of accessibility of the thiol groups of the enzyme during SDS denaturation by a technique which involves an irreversible chemical modification of CK accessible thiol groups, followed by NTCB cleavage before the unmodified cysteines in 8 M urea (pH 9) and analysis of the peptides obtained by resolutive gel electrophoresis, without sequencing. We have determined that the order of accessibility of CK MM cysteine residues during SDS denaturation is Cys-282, Cys-145 and then Cys-253. The fourth cysteine residue, Cys-73, is never titrated even at high SDS/CK molar ratio. In contrast, the three last residues are simultaneously titrated when CK is denatured in guanidinium chloride. Thus, SDS-denatured CK seems to retain some residual organized structure. In order to confirm this hypothesis, compactness of the molecule was estimated by fluorescence energy transfer between CK tryptophans and AEDANS, an extrinsic fluorophore. The location of this fluorophore on the accessible thiol of Cys-282 was verified by the previous technique. The results of these experiments do indicate that SDS-denatured CK is more compact than CK completely unfolded in guanidinium chloride.

Purification and partial characterization of a glutamyl-tRNA synthetase from the unicellular green alga Scenedesmus obliquus, mutant C-2A?

The synthesis of 5-aminolevulinic acid commences with the ligation of glutamate to a specific tRNAGlu by a glutamyl-tRNA synthetase (E.C. 6.1.1.17) (Huang et al., 1984, Science 225, 1482–1484). The synthetase from the yellow pigment mutant C-2A′ of the unicellular green alga Scenedesmus obliquus was purified by sequential column chromatography on Sephacryl S-300, Blue Sepharose, phosphocellulose P11 and by fast protein liquid chromatography (FPLC) on Mono Q. After denaturing sodium dodecylsulfate (SDS)-gel electrophoresis the purified enzyme preparation revealed a single protein band with a molecular mass of 55 kDa, proving the apparent homogeneity of the glutamyl-tRNA synthetase. A molecular mass of 105 ± 10 kDa was determined for the native protein by chromatography on Sephadex G-150. From these data it can be concluded that the glutamyl-tRNA synthetase from S. obliquus is a homodimer. The purified protein is active within a pH range from 7.0 to 9.0 with a maximum activity at pH 8.0. Kinetics for the binding of glutamate to the tRNA, performed with highly purified enzyme preparations, showed a Km value of 2.3 μM ± 0.3 for glutamate.

Appearance of type 1, 2, and 3 light-harvesting complex II and light-harvesting complex I proteins during light-induced greening of barley (Hordeum vulgare) etioplasts.

Monospecific antibodies directed against typical domains of type 1, 2, and 3 light-harvesting complex (LHC) II apoproteins have been used (a) to identify these apoproteins on denaturing sodium dodecyl sulfate gels of barley (Hordeum vulgare) thylakoids, (b) to determine their distribution between grana and stroma membranes, and (c) to follow their accumulation during light-induced greening of etioplasts. In addition, we have studied the light-induced assembly of chlorophyll-protein complexes with a native green gel system (K.D. Allen, L.A. Staehelin [1991] Anal Biochem 194: 214-222). Western blot analysis of the three major LHCII apoprotein bands has identified the highest molecular mass band at 27.5 kD as containing the type 2 LHCII apoproteins, the middle band at 26.9 kD as containing the type 1 LHCII apoproteins, and the lowest band at 26.0 kD as containing the type 3 LHCII apoproteins. During light-induced greening of 6-d-old etiolated barley seedlings, the type 1, 2, and 3 LHCII apoproteins accumulate simultaneously and at similar rates but appear somewhat sooner (< 4 h) in thylakoids from apical than from basal (4-8 h) leaf segments. LHCI polypeptides accrue with similar kinetics, whereas the 33-kD oxygen-evolving complex polypeptides can be detected already in the 0-h light samples. During the most rapid phase of thylakoid development (8-24 h), two slightly larger (28.3 and 28.7 kD) type 2 LHCII apoproteins (precursor intermediates?) also accumulate in the thylakoids. No corresponding higher molecular mass forms of type 1 and 3 LHCII apoproteins could be detected. It is interesting that differences are still apparent in the composition of chlorophyll-protein complexes of light-control plants and those of etiolated plants greened for 8 d.

Antichymotrypsin interaction with chymotrypsin. Partitioning of the complex.

The interactions of bovine pancreatic chymotrypsin (Chtr) and recombinant alpha 1-antichymotrypsin (rACT) and rACT variants were studied by kinetic and gel electrophoretic analyses, leading to the formulation of a general kinetic scheme that accounts for all known results concerning this serpin-protease pair, as well as for results obtained with other such pairs. Incubation of rACT and Chtr leads rapidly to the formation of an inhibited complex, Chtr.rACT*, that is stable toward sodium dodecyl sulfate denaturation and boiling. The extent of release of active Chtr from this complex increases markedly as ionic strength, mu, is raised. The kinetic scheme quantitatively accounts for this effect on the basis of a partitioning of Chtr.rACT* between dissociation of the complex to yield active enzyme and cleaved rACT, and Chtr-catalyzed conversion of the complex to a form that is much more resistant to release of active enzyme. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analyses of reaction mixtures of rACT and Chtr are consistent with the scheme. Also consistent are the results of experiments measuring the effects of 1) Chtr.rACT* concentration, 2) uncomplexed Chtr, and 3) added alpha 2-macroglobulin on active Chtr release from Chtr.rACT*. Proteolysis of Chtr.rACT* to give a resistant complex is also catalyzed by human neutrophil elastase, a process with potential physiological relevance. Comparison of the rates of Chtr dissociation from the complexes formed with rACT and with rACT variants mutated at the P1 site suggests that such rates are more sensitive to P1 substitution at low mu than at high mu. Several equivalents of the L358R-rACT variant are required for full inhibition of Chtr. This observation is also quantitatively accounted for by the proposed kinetic scheme, on the basis of another partitioning step between L358R-rACT acting as a substrate or as an inhibitor toward Chtr.

Acetolactate synthase from Brassica napus: Immunological characterization and quaternary structure of the native enzyme

Acetolactate synthase (ALS, AHAS; EC 4. 1. 3. 18) from Brassica napus has been partially purified and characterized using polyclonal antibodies. Following denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis and western blot analysis, 65 and 66 kDa ALS subunit polypeptides were immunologically detected, along with a novel 36 kDa polypeptide which cross‐reacted with the anti‐ALS antibody. Partial peptide sequencing of the 36 kDa peptide revealed significant similarity to plant aldolase proteins. ALS activity from stromal extracts fractionated by gel filtration chromatography as a single species of estimated molecular mass of 124 kDa, while comparative sedimentation coefficient in glycerol gradients indicated a corresponding molecular mass of 132 kDa. The results suggest that the native enzyme is a dimer of 65 and/or 66 kDa subunits. Anion exchange chromatography resolved two classes of ALS activity of equal native molecular weight, but which exhibited different properties with respect to subunit structure, sensitivity to inhibition by chlorsulfuron and feedback inhibition by branched chain amino acids.

Secondary structural changes of large and small fragments of bovine serum albumin in thermal denaturation and in sodium dodecyl sulfate denaturation.

The helicities in various fragments of bovine serum albumin (BSA) were examined in the thermal denaturation and in sodium dodecyl sulfate (SDS) denaturation. The thermal denaturation was examined in a temperature range between 2 and 65 degrees C. The helicity decreased with a rise of temperature and it recovered to some degree upon cooling temperature. A rather high reversibility was observed in the BSA fragments, which were located in the N-terminal of the parent protein and then contained the first large loop with no disulfide bridge. The high reversibility was available also for the helicity in the first large loop of the fragment, disulfide bridges of which were reduced. The fragments, which were smaller than one domain, became unstable in the SDS denaturation. The helicities of such fragments decreased in lower SDS concentrations compared with those of the intact BSA and the large fragments, which contained one or more domains. A resistance to the SDS denaturation appeared in the helices of every large loop even after the fragmentation. On the other hand, helicities of the fragments decreased to 20-25% upon the reduction of disulfide bridges. However, the helicities of these fragments increased to 35-40% in the SDS denaturation.

Reformation of the Helical Structure of Bovine Serum Albumin by the Addition of Small Amounts of Sodium Dodecyl Sulfate after the Disruption of the Structure by Urea

Upon the addition of small amounts of sodium dodecyl sulfate (SDS), the helicity of bovine serum albumin (BSA), lost in the urea denaturation, was mostly recovered. The profile of the recovery differed depending on the urea concentration. The urea concentrations were then divided into three ranges. (1) A range below 3 M where the helicity only decreased, as in the absence of urea (the helicity decreased down to 47% in the SDS solution). (2) A range between 4 and 8 M where the helicity initially increased up to 62% and then sharply decreased below 2 m M SDS. (3) A range above 9 M where the helicity only increased with an increase in SDS concentration. In each case, the helicity finally reached a value of around 47%, attained in the SDS solution without urea. In the middle concentration range, profiles of the structural change were rather complicated. It is worth noting that the helicity is recovered upon the addition of SDS of less than 0.3 m M in this range except for in the 8 M urea case. At 8 M urea, the helicity is recovered upon the addition of 1.0 m M SDS. These results were obtained upon the addition of SDS to BSA denatured by urea. However, the same helicity was obtained at each SDS concentration when the surfactant was added prior to the urea denaturation. It appears likely that the SDS denaturation finally predominates over the urea denaturation. The same competition was observed in the absorbance change in the aromatic region.

Characterization of the human Ah receptor

Using [ 3 H]2,3,7,8- tetrachlorodibenzo-p- dioxin ([ 3H]TCDD) or 7-[ 125 I]- iodo-2,3- dibromodibenzo-p- dioxin ([ 125I]DBDD) as high affinity photoaffinity ligands, the photoaffinity-labeled nuclear Ah receptor complexes from human cell lines have been analyzed by denaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis. The results show that the apparent molecular mass for the Ah receptor from several of these cell lines was 110-kDa and no significant variations were observed. However, there were significant differences in the apparent molecular weights of the nuclear Ah receptor complexes. For example, in the estrogen receptor (ER)-positive (MCF-7) and ER-negative (MDA-MB-231) human breast cancer cell lines, the apparent molecular weights of the nuclear receptor complexes were 171,000 and 192,000, respectively. These results indicate that there are differences in the molecular weights of the human nuclear Ah receptor complexes and the intraspecies heterogeneity may be associated with the non-ligand binding protein and not the Ah receptor.

Broad Bean Leaf Polyphenol Oxidase Is a 60-Kilodalton Protein Susceptible to Proteolytic Cleavage

Polyphenol oxidase (PPO) in leaves is generally considered to be a 45-kilodalton protein. PPO purified to homogeneity from broad bean (Vicia faba L.) leaves in the presence of protease inhibitors had an apparent molecular mass of 60 kD determined by denaturing sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Under partially denaturing conditions, the protein had an apparent molecular mass of 43 kilodaltons, but this was shifted to 60 kilodaltons in the presence of sulfhydryl reductants, suggesting the presence of disulfide bonding. The purified protein was totally latent; i.e. PPO activity was only detected when assayed with sodium dodecyl sulfate. Treatment with proteases in the presence of 0.1% SDS inhibited enzyme activity, but in the absence of SDS the 60-kilodalton PPO was proteolytically cleaved with no loss of PPO activity. This yielded a 42-kilodalton peptide that had PPO activity and inactive peptides of 12 to 18 kilodaltons. Amino acid sequencing established that the 42-kilodalton protein was derived from the N-terminal end of the 60-kilodalton form of PPO. By comparison with the sequence of a cDNA clone for broad bean leaf PPO, the 18-kilodalton peptide was located at the C-terminal end of the 60-kilodalton protein. Northern analysis of mRNA from broad bean leaves probed with a cDNA clone of PPO identified a transcript of 2.2 kilobase pairs, which is more than sufficient to encode the 60-kilodalton protein. It is concluded that PPO is a 60-kilodalton protein in broad bean leaves but that it is susceptible to proteolysis during extraction, removing a peptide of 15 to 18 kilodaltons from the C-terminal end without decreasing enzyme activity.

Structure-function studies of bacteriorhodopsin XV. Effects of deletions in loops B-C and E-F on bacteriorhodopsin chromophore and structure

Bacteriorhodopsin mutants containing deletions in loop B-C, delta Thr67-Glu74 or delta Gly65-Gln75 or a deletion in the loop E-F, delta Glu161-Ala168, were prepared. Following their expression in Escherichia coli, the mutant proteins were purified to homogeneity and refolded with retinal in detergent-phospholipid mixtures. The mutants containing deletions in the loop B-C were normal at 4 degrees C but showed the following changes at 20 degrees C. 1) The lambda max shifted from 540 to below 510 nm; 2) the rates of bleaching by hydroxylamine in the dark increased; and 3) the rate and steady state of proton pumping decreased. Deletion of the eight amino acids in loop E-F did not affect wild-type behavior. However, all the mutant proteins were more prone to thermal and sodium dodecyl sulfate denaturation than the wild-type bacteriorhodopsin. These observations show that the structures of the B-C and E-F loops are not essential for correct folding of bacteriorhodopsin, but they contribute to the stability of the folded protein.

Trypsin inhibitor paradoxically stabilizes trypsin activity in sodium dodecyl sulfate, facilitating proteolytic fingerprinting

Normally trypsin has negligible activity after being dissolved in sodium dodecyl sulfate (SDS), and so it has had little utility for proteolytic fingerprinting during gel electrophoresis. Here it is demonstrated that trypsin retained activity in SDS if it was first complexed to either of two soybean-derived protease inhibitors: trypsin inhibitor (Kunitz) or trypsin-chymotrypsin inhibitor (Bowman-Birk). The inhibitors alone did not cause proteolysis. Heating or acidification in SDS inactivated the inhibitor-dependent tryptic activity, as did prior treatment with tosyl lysine chloromethyl ketone, a covalent affinity reagent for trypsin. Quenching of samples with acid at intervals prior to gel electrophoresis revealed that proteolysis did not occur in sample buffer (pH 6.8), but only at higher pH and during gel electrophoresis. Exposure of trypsin to SDS prior to addition of trypsin inhibitor resulted in an irreversible loss of activity with a half-life of about 10 s. It is proposed that the trypsin inhibitors stabilize trypsin by retarding its denaturation in SDS. The substrate for these experiments was the α subunit of the Na,K-ATPase. The same pattern of Na,K-ATPase fragments was obtained with bovine and porcine trypsin and with rat and porcine Na,K-ATPases. Different fragments resulted when chymotrypsin or elastase were substituted for trypsin; these proteases were active in the absence of an inhibitor, and were not markedly stabilized by interaction with soybean trypsin-chymotrypsin inhibitor (Bowman-Birk).

Extracellular Hemoglobins of Hydrothermal Vent Annelids: Structural and Functional Characteristics in Three Alvinellid Species.

The polychaete annelids Alvinella pompejana, Alvinella caudata, and Paralvinella grasslei are strictly associated with deep sea hydrothermal vents. Each species possesses an extracellular hemoglobin, Hb, which has been studied and compared to that of a common intertidal polychaete, the lugworm Arenicola marina. The four Hbs exhibit very similar quaternary structures and spectral properties, and only small differences appeared in the gross polypeptide compositions after reduction and sodium dodecyl sulfate denaturation of the native molecules. Conversely, by a comparison of the effects of pH (6.6-7.6) and temperature (10-40{deg}C) on their intrinsic O affinities, Bohr factors, cooperativities, and apparent heats of oxygenation, lugworm Hb can be differentiated from that of the alvinellids, and the Hb of A. pompejana from that of A. caudata. The known biology of the lugworm and a further analysis of the data suggest several hypotheses concerning the in vivo O2 transport function of the alvinellid Hbs, the in vivo blood pH value in the two alvinellid species, their respective range of optimal temperature, and their ability to create a differentiated and stable external microenvironment.

Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus

Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98 degrees C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons [kDa]) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98 degrees C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% beta-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.

THE RESOLUTION OF CHLOROPHYLL a/b BINDING PROTEINS BY A PREPARATIVE METHOD BASED ON FLAT BED ISOELECTRIC FOCUSING.

We describe a new fractionation method for intrinsic membrane proteins based on flat bed isoelectric focusing (IEF) in granulated gel. The characteristics of the separation in the presence of the non-ionic detergent dodecylmaltoside are considered. The method has been applied to the fractionation of chlorophyll a/b binding proteins from chloroplast grana membranes. Several Light Harvesting Complexes II (LHC II) have been resolved showing differences in their polypeptide composition. Probing with monoclonal and polyclonal antibodies showed that polypeptides belonging to different [EF fractions with the same mobility in denaturing sodium dodecyl sulphate polyacrylamide gel electrophoresis, are immunologically distinct polypeptides. This is the first report of the presence in the thylakoid membrane of a number of LHCII polypeptides that may reflect the genetic complexity of the Cab genes. Moreover preparative amounts have been obtained of the minor chlorophyll a/b proteins CP 29, CP 26 and CP 24 that have been recently described. The analysis of a currently used LHCII preparation by the present method shows that this fraction is actually contaminated by two minor chlorophyll a/b proteins.