Comparison of tryptic peptide maps of two isoenzymes of 6-phosphogluconate dehydrogenase from tobacco tissue cultures

In a previous study by the authors, two isoenzymes of 6-phosphogluconate dehydrogenase were isolated from cultures of tobacco tissue Nicotiana tabacum W-38 and shown to be similar in their pH optima and MWs and in their affinities toward 6-phosphogluconate or NADP⁺. In an attempt to clarify the structural relationships between these two isoenzymes, peptide mapping of trypsin digests of the purified isoenzymes was performed. The maps were found to be similar, with at least 29 peptide groups from the trypsin digestion of each isoenzyme being alike. There were, however, definite minor differences in the peptide maps of the two isoenzymes.     

Kinetic properties of human liver alcohol dehydrogenase: oxidation of alcohols by class I isoenzymes

Class I isoenzymes of alcohol dehydrogenase (ADH) were isolated by chromatography of human liver homogenates on DEAE-cellulose, 4-[3-[N-(6-aminocaproyl)-amino]propyl]pyrazole--Sepharose and CM-cellulose. Eight isoenzymes of different subunit composition (alpha gamma 2, gamma 2 gamma 2, alpha gamma 1, alpha beta 1, beta 1 gamma 2, gamma 1 gamma 1, beta 1 gamma 1, and beta 1 beta 1) were purified, and their activities were measured at pH 10.0 by using ethanol, ethylene glycol, methanol, benzyl alcohol, octanol, cyclohexanol, and 16-hydroxyhexadecanoic acid as substrates. Values of Km and kcat for all the isoenzymes, except beta 1 beta 1-ADH, were similar for the oxidation of ethanol but varied markedly for other alcohols. The kcat values for beta 1 beta 1-ADH were invariant (approximately 10 min-1) and much lower (5-15-fold) than those for any other class I isoenzyme studied. Km values for methanol and ethylene glycol were from 5- to 100-fold greater than those for ethanol, depending on the isoenzyme, while those for benzyl alcohol, octanol, and 16-hydroxyhexadecanoic acid were usually 100-1000-fold lower than those for ethanol. The homodimer beta 1 beta 1 had the lowest kcat/Km value for all alcohols studied except methanol and ethylene glycol; kcat values were relatively constant for all isoenzymes acting on all alcohols, and, hence, specificity was manifested principally in the value of Km. Values of Km and kcat/Km revealed for all enzymes examined that the short chain alcohols are the poorest while alcohols with bulky substituents are much better substrates. The experimental values of the kinetic parameters for heterodimers deviate from the calculated average of those of their parent homodimers and, hence, cannot be predicted from the behavior of the latter. Thus, the specificities of both the hetero- and homodimeric isoenzymes of ADH toward a given substrate are characteristics of each. Ethanol proved to be one of the "poorest" substrates examined for all class I isoenzymes which are the predominant forms of the human enzyme. On the basis of kinetic criteria, none of the isoenzymes of class I studied oxidized ethanol in a manner that would indicate an enzymatic preference for that alcohol.     

Comparison of ATPase activities and heavy chains of rabbit atrial and thyrotoxic ventricular myosin subfragment-1

Summary Subfragment-1 of rabbit atrial and thyrotoxic ventricular myosin (V₁ isomyosin) has been prepared and purified by DEAF-cellulose column chromatography. Pyrophosphate-polyacrylamide gel electrophoretic patterns and column chromatographic profile of the atrial subfragment differ from those of thyrotoxic ventricular myosin subfragment-1. On the other hand, Ca²⁺, Mg²⁺ and actin-activated ATPase activities of these subfragments are identical. Comparison of the peptide mapping by limited proteolysis in the presence of sodium dodecyl sulfate of the heavy and the light subunits of these subfragments reveals that the patterns for the heavy chain peptides of these subfragments are substantially similar but their light chain peptide patterns differ. The results suggest that the enzymatic and structural similarities that have been recognized between these isoenzymes using intact myosin hold true for the myosin subfragment-1.The differences between these subfragments are due to the differences in the light chains associated with them.Abbreviations EDTA Ethylene Diamine Tetra-acetic Acid - SDS Sodium Dodecyl Sulfate - S₁ myosin subfragment-1 - HC Heavy Chain - LC Light Chain     

A model for the common control of enzymes of ethanolamine catabolism in Escherichia coli

By varying the composition of the growth medium and the genotype of the bacterial strain, five isoenzymes of CoA-dependent aldehyde dehydrogenase could be detected in Escherichia coli. Two isoenzymes (A, mol. wt 520 000; and B, mol. wt 370 000) were produced only in the presence of ethanolamine and vitamin (or coenzyme) B12 ('inducible isoenzymes'), whereas the other three isoenzymes (C, mol. wt 900 0000; D, mol. wt 120 000; and E, mol. wt 720 000) were produced only in the absence of ethanolamine and vitamin B12 ('repressible isoenzymes'). Partial purification and characterization of these isoenzymes revealed strong similarities, with respect to pH optima and substrate affinities, between isoenzymes within either of the two classes, but significant differences between the two classes. Mutant studies demonstrated that the relationships between the isoenzymes and between CoA-dependent aldehyde dehydrogenase and ethanolamine ammonia-lyase are both structural and regulatory in nature, and a two-operon model is proposed to account for the common control of the enzymes of ethanolamine catabolism in E. coli.     

Studies on muscular dystrophy: a comparison by physical and chemical means of the normal human ATP-creatine transphosphorylases (creatine kinases) with those from tissues of Duchenne muscular dystrophy.

The four human Duchenne dystrophic isoenzymes (M-M, M-B, B-B, from the muscle and B-B from the brain) of ATP-creatine transphosphorylase (S. A. Kuby, H. J. Keutel, K. Okabe, H. K. Jacobs, F. Ziter, D. Gerber, and F. H. Tyler, 1977, J. Biol. Chem.252, 8382–8390) have now been compared physically and chemically with their normal human counterparts (viz., with the three isoenzymes, M-M, M-B, B-B, 2). All isoenzymes proved to be composed of two noncovalently linked polypeptide chains, by sedimentation equilibrium analyses in the presence and absence of disruptive agents. In the presence of 2-mercaptoethanol at 0.16(Γ/2), pH 7.8, the two native muscle types yielded identical values for s_20,w, concentration dependencies, and molecular weight, and similarly for the brain types (from the brain). But the human brain type proved to be slightly heavier than the muscle type (viz. 88,400 vs 85,900). All of the isoenzymes showed similar electrophoretic behavior between their several counterparts between pH 5–8, except perhaps between pH 8–10, where small differences appeared. The three native normal human isoenzymes, as well as the dystrophic human isoenzymes (M-M from the muscle and B-B from the brain) all contain 2 reactive sulfhydryl groups per mole or 1 per polypeptide chain of these two-chain proteins, which may be titrated with 5,5′-dithiobis(2-nitrobenzoic acid) (Nbs₂); and under acidic conditions, quantitative titrations with 4,4′-dithiodipyridine yield a total of 10 -SH groups per mole of each brain type and 8 -SH groups per mole of muscle type, in the case of man, dystrophic man, calf, and rabbit. The kinetics of reactions between Nbs₂ and the sulfhydryl groups of all three normal human isoenzymes and two dystrophic human isoenzymes have been measured under several sets of denaturing conditions. A comparison of their reactive calculated second-order velocity constants reveal significant differences between these three normal human isoenzymes, but the k_{second order} values for the reactions of the sulfhydryl groups of the dystrophic M-M and B-B with Nbs₂, when compared with their normal counterparts, gave identical values in the presence of 7.3 m urea or 1.8% laurylsulfate, from which it may be inferred that very similar, if not identical, environments surround these two sets of sulfhydryl groups. A comparison of the amino acid compositions of the normal human muscle type and brain type with the human dystrophic M-M and B-B (from the brain) reveal essentially identical values for the muscle types but nearly identical values for the brain types, with a few differences. Their respective tryptic peptide maps have been compared of the S-carboxy-methylated proteins (alkylated with iodo[2-¹⁴C]acetic acid at the two exposed -SH groups per mole). Thus, the muscle types, normal and dystrophic, yield identical maps, but the brain types nearly identical maps, with a few significant differences. Isolation of the tryptic tridecapeptide from the S-carboxymethylated normal human and dystrophic human dimeric muscle-type ATP-creatine transphosphorylases, labeled at the single exposed SH group per polypeptide chain with iodo[2-¹⁴C]acetate, yielded the following sequence for both proteins: ValLeuThrCys(CH₂COOH)ProSerAsnLeuGlyThr GlyLeuArg [where Cys(CH₂COOH) is S-carboxymethyl cysteine]. This sequence showed remarkable homology with a few other equivalent peptides reported to be derived from the exposed SH group of other ATP-creatine transphosphorylases. In conclusion, there does not appear to be a mutation in the structural genes for the muscle-type creatine kinases detectable by the analyses presented here. However, the brain types warrant further investigation.     

Ca-asp bound X-ray structure and inhibition of Bacillus anthracis dihydroorotase (DHOase)

Dihydroorotase (DHOase) is the third enzyme in the de novo pyrimidine synthesis pathway and is responsible for the reversible cyclization of carbamyl-aspartate (Ca-asp) to dihydroorotate (DHO). DHOase is further divided into two classes based on several structural characteristics, one of which is the length of the flexible catalytic loop that interacts with the substrate, Ca-asp, regulating the enzyme activity. Here, we present the crystal structure of Class I Bacillus anthracis DHOase with Ca-asp in the active site, which shows the peptide backbone of glycine in the shorter loop forming the necessary hydrogen bonds with the substrate, in place of the two threonines found in Class II DHOases. Despite the differences in the catalytic loop, the structure confirms that the key interactions between the substrate and active site residues are similar between Class I and Class II DHOase enzymes, which we further validated by mutagenesis studies. B. anthracis DHOase is also a potential antibacterial drug target. In order to identify prospective inhibitors, we performed high-throughput screening against several libraries using a colorimetric enzymatic assay and an orthogonal fluorescence thermal binding assay. Surface plasmon resonance was used for determining binding affinity (K_D) and competition analysis with Ca-asp. Our results highlight that the primary difference between Class I and Class II DHOase is the catalytic loop. We also identify several compounds that can potentially be further optimized as potential B. anthracis inhibitors.     

Class II alcohol dehydrogenase (ADH2)--adding the structure

Class II alcohol dehydrogenase (ADH2) represents a highly divergent class of alcohol dehydrogenases predominantly found in liver. Several species variants of ADH2 have been described, and the rodent enzymes form a functionally distinct subgroup with interesting catalytic properties. First, as compared with other ADHs, the catalytic efficiency is low for this subgroup. Second, the substrate repertoire is unique, e.g. rodent ADH2s are not saturated with ethanol as substrate, and while omega-hydroxy fatty acids are common substrates for the human ADH1-ADH4 isoenzymes, including ADH2, these compounds function as inhibitors rather than substrates. The recently determined structure of mouse ADH2 reveals a novel substrate-pocket topography that accounts for the observed substrate specificity and may, therefore, be important for the exploration of orphan substrates of ADH2. It is possible to improve the catalytic efficiency of mouse ADH2 by an array of mutations at position 47. Residue Pro47 of the wild type ADH2 enzyme seems to strain the binding of coenzyme, which prevents a close approach between the coenzyme and substrate for efficient hydrogen transfer. Based on crystallographic and mechanistic investigations, the effects of residue replacements at position 47 are multiple, affecting the distance for hydride transfer, the pK(a) of the bound alcohol substrate as well as the affinity for coenzyme.     

Bovine cytochrome c oxidases,purified from heart,skeletal muscle,liver and kidney,differ in the small subunits but show the same reaction kinetics with cytochrome c

(1) Polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate of purified cytochrome c oxidase preparations revealed that bovine kidney, skeletal muscle and heart contain different cytochrome c oxidase isoenzymes, which show differences in mobility of the subunits encoded by the nuclear genome. No differences in subunit pattern were observed between the oxidase preparations isolated from kidney and liver. (2) The kinetics of the steady-state reactions between bovine ferrocytochrome c and the four types of bovine cytochrome c oxidase preparation were compared under conditions of both high- and low-ionic strength. Also the pre-steady-state kinetics were studied. Only minor differences were observed in the electron-transfer activity of the isoenzymes. Thus, our experiments do not support the notion that the subunits encoded by the nuclear genome act as modulators conferring different activities to the isoenzymes of cytochrome c oxidase. (3) The cytochrome c oxidase preparation from bovine skeletal muscle was found to consist mainly of dimers, whereas the enzymes isolated from bovine kidney, liver and heart were monomeric.     

Predicting Class II MHC-Peptide binding: a kernel based approach using similarity scores

Background  

Modelling the interaction between potentially antigenic peptides and Major Histocompatibility Complex (MHC) molecules is a key step in identifying potential T-cell epitopes. For Class II MHC alleles, the binding groove is open at both ends, causing ambiguity in the positional alignment between the groove and peptide, as well as creating uncertainty as to what parts of the peptide interact with the MHC. Moreover, the antigenic peptides have variable lengths, making naive modelling methods difficult to apply. This paper introduces a kernel method that can handle variable length peptides effectively by quantifying similarities between peptide sequences and integrating these into the kernel.     

The transphosphatidylation potential of a membrane-bound phospholipase D from poppy seedlings

Plant phospholipases D (PLDs) occur in a large variety of isoenzymes, which differ in Ca(2+) ion requirement, phosphatidylinositol-4,5-bisphosphate (PIP(2)) activation and substrate selectivity. In the present study a membrane-bound PLD has been identified in the microsomal fractions of poppy seedlings (Papaver somniferum). The maximum PLD activity is found after 2 days of germination in endosperms and after 3 days in developing seedlings. In contrast to the four poppy PLD isoenzymes described hitherto, the membrane-bound form is active at lower Ca(2+) ion concentrations (in the micromolar instead of millimolar range) and needs PIP(2) for hydrolytic activity. Remarkable differences are also observed in head group exchange reactions. The reaction rates of the transphosphatidylation of phosphatidylcholine by various acceptor alcohols follow the sequence glycerol>serine>myo-inositol>ethanolamine, whereas ethanolamine is preferred by most other PLDs. Despite the biocatalytic differences, the membrane-bound PLD interacts with polyclonal antibodies raised against α-type PLD, which reveals some structural similarities between these two enzymes.     

Triosephosphate isomerases and aldolases from light- and dark-grown Euglena gracilis

Euglena gracilis synthesizes two distinct types of triosephosphate isomerase which can be resolved by isoelectric focusing. The more acidic Type A isomerase (pI = 4.4) predominates when cells are grown photoautotrophically and is localized in the chloroplasts. The Type B isoenzyme exhibits a more basic isoelectric pH (pI = 4.8), predominates under heterotrophic growth conditions and is of cytoplasmic origin. The two isoenzymes exhibit similar molecular weights (56,000–60,000) and catalytic properties but can be distinguished by their pH activity profiles. The situation parallels that of fructose diphosphate aldolase where a chloroplastic Class I enzyme (pI = 4.6, M_r 120,000) found in autotrophically grown cells can be resolved from the cytoplasmic Class II (pI = 5.7, M_r 88,000) enzyme which predominates under heterotrophic conditions. Inhibition of chloroplastic 70S ribosomal synthesis by chloramphenicol blocks the formation of the Type A triosephosphate isomerase and the Class I aldolase.     

Regulation of enzyme activity of alcohol dehydrogenase through its interactions with pyruvate-ferredoxin oxidoreductase in Thermoanaerobacter tengcongensis

Alcohol dehydrogenases (ADHs) from thermophilic microorganisms are interesting enzymes that have their potential applications in biotechnology and potentially provide insight into the mechanisms of action of thermo-tolerant proteins. The molecular mechanisms of ADHs under thermal stress in vivo have yet to be explored. Herein, we employed a proteomic strategy to survey the possible interactions of secondary-ADH (2-ADH) with other proteins in Thermoanaerobacter tengcongensis (T. tengcongensis) cultured at 75°C and found that 2-ADH, pyruvate-ferredoxin oxidoreductase (PFOR) and several glycolytic enzymes coexisted in a protein complex. Using anion exchange chromatography, the elution profile indicated that the native 2-ADH was present in two forms, PFOR-bound and PFOR-free. Immuno-precipitation and pull down analysis further validated the interactions between 2-ADH and PFOR. The kinetic behaviours of 2-ADH either in the recombinant or native form were evaluated with different substrates. The enzyme activity of 2-ADH was inhibited in a non-competitive mode by PFOR, implying the interaction of 2-ADH and PFOR negatively regulated alcohol formation. In T. tengcongensis, PFOR is an enzyme complex located at the upstream of 2-ADH in the alcohol generation pathway. These findings, therefore, offered a plausible mechanism for how alcohol metabolism is regulated by hetero-interactions between 2-ADH and PFOR, especially in anaerobic thermophiles.     

Incorporation of carbohydrate residues into peroxidase isoenzymes in horseradish roots

Sliced root tissue of the horseradish plant (Armoracia rusticana), when incubated with mannose-U-¹⁴C, incorporated radioactivity into peroxidase isoenzymes. Over 90% of the radioactivity in the highly purified peroxidase isoenzymes was present in the neutral sugar residues of the molecule, i.e. fucose, arabinose, xylose, mannose. When the root slices were incubated simultaneously with leucine-4,5-³H and mannose-U-¹⁴C, cycloheximide strongly inhibited leucine incorporation into the peptide portion of peroxidase isoenzymes but had little effect on the incorporation of ¹⁴C into the neutral sugars. These results indicated that synthesis of the peptide portion of peroxidase was completed before the monosaccharide residues were attached to the molecule. This temporal relationship between the synthesis of protein and the attachment of carbohydrate residues in the plant glycoprotein, horseradish peroxidase, appears to be similar to that reported for glycoprotein biosynthesis in many mammalian systems.     

The C-terminal Extension Peptide of Non-photoconvertible Water-Soluble Chlorophyll-Binding Proteins (Class II WSCPs) Affects Their Solubility and Stability: Comparative Analyses of the Biochemical and Chlorophyll-Binding Properties of Recombinant Brassica, Raphanus and Lepidium WSCPs with or Without Their C-terminal Extension Peptides

Numerous members of the Brassicaceae possess non-photoconvertible water-soluble chlorophyll (Chl)-binding proteins (Class II WSCPs), which function as Chl scavengers during cell disruption caused by wounding, pest/pathogen attacks, and/or environmental stress. Class II WSCPs have two extension peptides, one at the N-terminus and one at the C-terminus. The N-terminal peptide acts as a signal peptide, targeting the protein to the endoplasmic reticulum body, a unique defensive organelle found only in the Brassicaceae. However, the physiological and biochemical functions of the C-terminal extension peptide had not been characterized previously. To investigate the function of the C-terminal extension peptide, we produced expression constructs of recombinant WSCPs with or without the C-terminal extension peptide. The WSCPs used were of Brussels sprouts (Brassica oleracea), Japanese wild radish (Raphanus sativus) and Virginia pepperweed (Lepidium virginicum). The solubility of all of the WSCPs with the C-terminal extension peptide was drastically lower than that of the recombinant WSCPs without the C-terminal extension peptide. In addition, the stability of the reconstituted WSCPs complexes with the C-terminal extension peptide was altered compared with that of the proteins without the C-terminal extension peptide. These finding indicate that the C-terminal extension peptide affects not only the solubility, but also the stability of Class II WSCP. Furthermore, we characterized the Chl-binding properties of the recombinant WSCP from Japanese wild radish (RshWSCP-His) in a 40 % methanol solution. An electrophoretic mobility shift assay revealed that RshWSCP-His required a half-molar ratio of Chls to form a tetramer.     

Comparative Structural Modeling of Six Old Yellow Enzymes (OYEs) from the Necrotrophic Fungus Ascochyta rabiei : Insight into Novel OYE Classes with Differences in Cofactor Binding,Organization of Active Site Residues and Stereopreferences

Old Yellow Enzyme (OYE1) was the first flavin-dependent enzyme identified and characterized in detail by the entire range of physical techniques. Irrespective of this scrutiny, true physiological role of the enzyme remains a mystery. In a recent study, we systematically identified OYE proteins from various fungi and classified them into three classes viz. Class I, II and III. However, there is no information about the structural organization of Class III OYEs, eukaryotic Class II OYEs and Class I OYEs of filamentous fungi. Ascochyta rabiei, a filamentous phytopathogen which causes Ascochyta blight (AB) in chickpea possesses six OYEs (ArOYE1-6) belonging to the three OYE classes. Here we carried out comparative homology modeling of six ArOYEs representing all the three classes to get an in depth idea of structural and functional aspects of fungal OYEs. The predicted 3D structures of A. rabiei OYEs were refined and evaluated using various validation tools for their structural integrity. Analysis of FMN binding environment of Class III OYE revealed novel residues involved in interaction. The ligand para-hydroxybenzaldehyde (PHB) was docked into the active site of the enzymes and interacting residues were analyzed. We observed a unique active site organization of Class III OYE in comparison to Class I and II OYEs. Subsequently, analysis of stereopreference through structural features of ArOYEs was carried out, suggesting differences in R/S selectivity of these proteins. Therefore, our comparative modeling study provides insights into the FMN binding, active site organization and stereopreference of different classes of ArOYEs and indicates towards functional differences of these enzymes. This study provides the basis for future investigations towards the biochemical and functional characterization of these enigmatic enzymes.     

Difference between uracilylalanine synthases and cysteine synthases in Pisum sativum

Purification of cysteine synthase from seedlings of pea (Pisum sativum) reveals the presence of three forms of this enzyme, separated by chromatography on DEAE-Sephadex A-50, and also differences between the cysteine- and uracilylalanine-synthases. Isoenzymes A and B of pea cysteine synthase were purified about 1200-fold and had specific activities of 933 U/mg protein and 892 U/mg protein, respectively. Both isoenzymes were found to have the same M_r (52 000) and to dissociate into two identical subunits (M_r 26 000). The K_m value of isoenzyme A is 2.1 mM for O-acetyl-l-serine (OAS) and 36 μM for sulphide, while that of isoenzyme B is 2.3 mM for OAS and 38 μM for sulphide. None of the three isoenzymes from pea seedlings catalyses the formation of the uracilylalanines l-willardiine and l-isowillardiine from OAS and uracil, although isoenzyme A catalyses the formation of β-cyano-l-alanine, and isoenzyme C catalyses the formation of l-quisqualic acid and l-mimosine. Other significant differences occur in the substrate specificity of the three isoenzymes. Several properties, including the amino acid composition of the purified cysteine synthase isoenzymes, are also described.     

Use of esterase isoenzymes revealed by gel isoelectric focusing as an aid in chemotaxonomical study of the genusAllium

Eleven species of the genusAllium belonging to the sectionCepa, Phyllodolon, Rhizirideum, Melanocrommyum andAlliun ware investigated as to the presence of esterase isoenzymes by means of the gal isoelectric focusing which shows better resolution than polyaorylamide gel electrophoresis. In all examined species twenty six isoenzymes were found. The individual sections are well characterized by esterase isoenzymes revealed by this method, on the other hand, the differences, as shown by the Jaceard index, between the subganera are insignificant. Investigation of seven cultivars ofAllium cepa shows the isolated position of the cultivar Ka?tická. These results are in full agreement with those found with use of the polyacrylamide gel electrophoresis and show the usefulness of gel isoelectric focusing for the solution of the chemotaxonomical problems.     

Human ovarian tumor-associated trypsin. Its purification and characterization from mucinous cyst fluid and identification as an activator of pro-urokinase

In search of the target protease for the tumor-associated trypsin inhibitor TATI we recently identified a trypsin-like protease in cyst fluid of mucinous ovarian tumors (Stenman, U.-H., Koivunen, E., and Vuento, M. (1988) Biol. Chem. Hoppe-Seyler 369, 9-14). We have now purified this protease and demonstrate that it represents isoenzyme forms of trypsinogen, here called tumor-associated trypsin(ogen)s (TAT). The purification procedure comprised batchwise anion exchange chromatography, immunoaffinity chromatography with antibodies to trypsin, and separation of the two isoenzymes by reverse phase chromatography. In sodium dodecyl sulfate (SDS)-gel electrophoresis, the TAT-1 and TAT-2 isoenzymes have relative molecular weights (Mr) of 25,000 and 28,000, respectively, TAT-2 being the major component. The amino-terminal amino acid sequences correspond to those of pancreatic trypsinogen-1 and -2, respectively, and activation of the zymogens results in cleavage of a NH2-terminal activation peptide of 8 residues characteristic of trypsinogen. Isoelectric focusing in the presence of urea gives pI values of about 5 and 4 for TAT-1 and -2, respectively. The substrate specificities of the two TAT isoenzymes are very similar to, but not identical with, those of trypsin-1 and trypsin-2, respectively, suggesting slight differences in substrate binding site. TAT was found to be an efficient activator of pro-urokinase. Hence, TAT could take part in the protease cascade associated with tumor invasion.     

Exploring the ‘N-terminal arm’ & ‘Convex surface’ Binding Interfaces of the T3SS Chaperone-Translocator Complexes from P. Aeruginosa

One infection method widely used by many gram-negative bacteria involves a protein nanomachine called the Type Three Secretion System (T3SS). The T3SS enables the transportation of bacterial “toxins” via a proteinaceous channel that directly links the cytosol of the bacteria and host cell. The channel from the bacteria is completed by a translocon pore formed by two proteins named the major and minor translocators. Prior to pore formation, the translocator proteins are bound to a small chaperone within the bacterial cytoplasm. This interaction is crucial to effective secretion. Here we investigated the specificity of the binding interfaces of the translocator–chaperone complexes from Pseudomonas aeruginosa via the selection of peptide and protein libraries based on its chaperone PcrH. Five libraries encompassing PcrH’s N-terminal and central α-helices were panned, using ribosome display, against both the major (PopB) and minor (PopD) translocator. Both translocators were shown to significantly enrich a similar pattern of WT and non-WT sequences from the libraries. This highlighted key similarities/differences between the interactions of the major and minor translocators with their chaperone. Moreover, as the enriched non-WT sequences were specific to each translocator, it would suggest that PcrH can be adapted to bind each translocator individually. The ability to evolve such proteins indicates that these molecules may provide promising anti-bacterial candidates.     

Distribution of myosin isoenzymes among skeletal muscle fiber types.

Using an immunocytochemical approach, we have demonstrated a preferential distribution of myosin isoenzymes with respect to the pattern of fiber types in skeletal muscles of the rat. In an earlier study, we had shown that fluorescein-labeled antibody against "white" myosin from the chicken pectoralis stained all the white, intermediate and about half the red fibers of the rat diaphragm, a fast-twitch muscle (Gauthier and Lowey, 1977). We have now extended this study to include antibodies prepared against the "head" (S1) and "rod" portions of myosin, as well as the alkali- and 5,5'dithiobis (2-nitrobenzoic acid) (DTNB)-light chains. Antibodies capable of distinguishing between alkali 1 and alkali 2 type myosin were also used to localize these isoenzymes in the same fast muscle. We observed, by both direct and indirect immunofluorescence, that the same fibers which had reacted previously with antibodies against white myosin reacted with antibodies to the proteolytic subfragments and to the low molecular-weight subunits of myosin. These results confirm our earlier conclusion that the myosins of the reactive fibers in rat skeletal muscle are sufficiently similar to share antigenic determinants. The homology, furthermore, is not confined to a limited region of the myosin molecule, but includes the head and rod portions and all classes of light chains. Despite the similarities, some differences exist in the protein compositions of these fibers: antibodies to S1 did not stain the reactive (fast) red fiber as strongly as they did the white and intermediate fibers. Non-uniform staining was also observed with antibodies specific for A2 myosin; the fast red fiber again showed weaker fluorescence than did the other reactive fibers. These results could indicate a variable distribution of myosin isoenzymes according to their alkali-light chain composition among fiber types. Alternatively, there may exist yet another myosin isoenzyme which is localized in the fast red fiber. Those red fibers which did not react with any of the antibodies to pectoralis myosin, did react strongly with an antibody against myosin isolated from the anterior latissimus dorsi (ALD), a slow red muscle of the chicken. The myosin in these fibers (slow red fibers) is, therefore, distinct from the other myosin isoenzymes. In the rat soleus, a slow-twitch muscle, the majority of the fibers reacted only with antibody against ALD myosin. A minority, however, reacted with antiboddies to pectoralis as well as ALD myosin, which indicates that both fast and slow myosin can coexist within the same fiber of a normal adult muscle. These immunocytochemical studies have emphasized that a wide range of isoenzymes may contribute to the characteristic physiological properties of individual fiber types in a mixed muscle.