Tryptic Cleavage Sites Research Articles

A Ca2+-dependent Tryptic Cleavage Site and a Protein Kinase A Phosphorylation Site Are Present in the Ca2+ Regulatory Domain of Scallop Muscle Na+-Ca2+ Exchanger

Digestion of scallop muscle membrane fractions with trypsin led to release of soluble polypeptides derived from the large cytoplasmic domain of a Na(+)-Ca(2+) exchanger. In the presence of 1 mm Ca(2+), the major product was a peptide of approximately 37 kDa, with an N terminus corresponding to residue 401 of the NCX1 exchanger. In the presence of 10 mm EGTA, approximately 16- and approximately 19-kDa peptides were the major products. Polyclonal rabbit IgG raised against the 37-kDa peptide also bound to the 16- and 19-kDa soluble tryptic peptides and to a 105-110-kDa polypeptide in the undigested membrane preparation. The 16-kDa fragment corresponded to the N-terminal part of the 37-kDa peptide. The conformation of the precursor polypeptide chain in the region of the C terminus of the 16-kDa tryptic peptide was thus altered by the binding of Ca(2+). Phosphorylation of the parent membranes with the catalytic subunit of protein kinase A and [gamma-(32)P]ATP led to incorporation of (32)P into the 16- and 37-kDa soluble fragments. A site may exist within the Ca(2+) regulatory domain of a scallop muscle Na(+)-Ca(2+) exchanger that mediates direct modulation of secondary Ca(2+) regulation by cAMP.

Limited Proteolysis of Yeast Elongation Factor 3: SEQUENCE AND LOCATION OF THE SUBDOMAINS

Elongation factor 3 (EF-3) is an ATPase essential for polypeptide chain synthesis in a variety of yeasts and fungi. We used limited proteolysis to study the organization of the subdomains of EF-3. Trypsinolysis of EF-3 at 30 degrees C resulted in the formation of three fragments with estimated molecular masses of 90, 70, and 50 kDa. Yeast ribosomes protected EF-3 and the large fragments from further degradation. ATP exposed a new tryptic cleavage site and stabilized the 70- and 50-kDa fragments. The conformation of EF-3 as measured by fluorescence spectroscopy did not change upon ATP binding. Poly(G) stimulated proteolysis and quenched the intrinsic fluorescence of EF-3. Using gel mobility shift, we demonstrated a direct interaction between EF-3 and tRNA. Neither tRNA nor rRNA altered the tryptic cleavage pattern. The proteolytic products were sequenced by mass spectrometric analysis. EF-3 is blocked NH(2)-terminally by an acetylated serine. The 90-, 70-, and 50-kDa fragments are also blocked NH(2)-terminally, confirming their origin. The 50-kDa fragment (Ser(2)-Lys(443)) is the most stable domain in EF-3 with no known function. The 70-kDa fragment (Ser(2)-Lys(668)) containing the first nucleotide-binding sequence motif forms the core ATP binding subdomain within the 90-kDa domain. The primary ribosome binding site is located near the loosely structured carboxyl-terminal end.

Evidence That the NH2 Terminus of Vph1p, an Integral Subunit of the V0 Sector of the Yeast V-ATPase, Interacts Directly with the Vma1p and Vma13p Subunits of the V1Sector

The vacuolar-type H(+)-ATPase (V-ATPase) is composed of a peripherally bound (V(1)) and a membrane-associated (V(0)) complex. V(1) ATP hydrolysis is thought to rotate a central stalk, which in turn, is hypothesized to drive V(0) proton translocation. Transduction of torque exerted by the rotating stalk on V(0) requires a fixed structural link (stator) between the complexes to prevent energy loss through futile rotation of V(1) relative to V(0); this work sought to identify stator components. The 95-kDa V-ATPase subunit, Vph1p, has a cytosolic NH(2) terminus (Nt-Vph1p) and a membrane-associated COOH terminus. Two-hybrid assays demonstrated that Nt-Vph1p interacts with the catalytic V(1) subunit, Vma1p. Co-immunoprecipitation of Vma1p with Nt-Vph1p confirmed the interaction. Expression of Nt-Vph1p in a Deltavph1 mutant was necessary to recruit Vma13p to V(1). Vma13p bound to Nt-Vph1p in vitro demonstrating direct interaction. Limited trypsin digests cleaves both Nt-Vph1p and Vma13p. The same tryptic treatment results in a loss of proton translocation while not reducing bafilomycin A(1)-sensitive ATP hydrolysis. Trypsin cleaved Vph1p at arginine 53. Elimination of the tryptic cleavage site by substitution of arginine 53 to serine partially protected vacuolar acidification from trypsin digestion. These results suggest that Vph1p may function as a component of a fixed structural link, or stator, coupling V(1) ATP hydrolysis to V(0) proton translocation.

Trypsin Sheds Light on the Singular Case of Seminal RNase, a Dimer with Two Quaternary Conformations

Dimeric seminal RNase presents the singular case of a dimer with access at equilibrium to two conformations: one in which the subunits exchange, or swap, their NH(2)-terminal arms; the other with no exchange. Thus a continuous unfolding/refolding of structural elements into two alternative conformations takes place in the native protein at equilibrium. The phenomenon was investigated by kinetic and mass spectrometric analyses of the effects of trypsin on the native protein, on its isolated quaternary forms, as well as on a monomeric derivative of the protein and on homologous dimeric RNase A. The kinetics of tryptic action on the protein forms and on the protein derivatives, as well as the location of the tryptic cleavage sites, and their chronological sequence, led to the identification of relevant interconversion intermediates, to the description of a model for the interconversion process, and to a hypothesis for the unique phenomenon of the dual quaternary conformation of seminal RNase.

Homing in on the Role of Transition Metals in the HNH Motif of Colicin Endonucleases

The cytotoxic domain of the bacteriocin colicin E9 (the E9 DNase) is a nonspecific endonuclease that must traverse two membranes to reach its cellular target, bacterial DNA. Recent structural studies revealed that the active site of colicin DNases encompasses the HNH motif found in homing endonucleases, and bound within this motif a single transition metal ion (either Zn(2+) or Ni(2+)) the role of which is unknown. In the present work we find that neither Zn(2+) nor Ni(2+) is required for DNase activity, which instead requires Mg(2+) ions, but binding transition metals to the E9 DNase causes subtle changes to both secondary and tertiary structure. Spectroscopic, proteolytic, and calorimetric data show that, accompanying the binding of 1 eq of Zn(2+), Ni(2+), or Co(2+), the thermodynamic stability of the domain increased substantially, and that the equilibrium dissociation constant for Zn(2+) was less than or equal to nanomolar, while that for Co(2+) and Ni (2+) was micromolar. Our data demonstrate that the transition metal is not essential for colicin DNase activity but rather serves a structural role. We speculate that the HNH motif has been adapted for use by endonuclease colicins because of its involvement in DNA recognition and because removal of the bound metal ion destabilizes the DNase domain, a likely prerequisite for its translocation across bacterial membranes.

Molecular Cloning and Tissue Expression of the Murine Analog to Human Stratum Corneum Chymotryptic Enzyme

Human stratum corneum chymotryptic enzyme (SCCE) may play a central part in epidermal homeostasis. Its proposed function is to catalyze the degradation of intercellular structures, including desmosomes, in the stratum corneum as part of the desquamation process. In order to facilitate physiologic and pathophysiologic studies on SCCE we have looked for the corresponding murine enzyme. A cDNA obtained by reverse transcription-polymerase chain reaction with total RNA prepared from mouse tails as starting material was cloned, and the expression of the corresponding mRNA studied. The murine cDNA showed 77% homology to human SCCE cDNA. It had an open-reading frame encoding a protein comprising 249 amino acids with 82% amino acid sequence homology to human SCCE including the conserved sequences of the catalytic traid of mammalian serine proteases. The murine protein was deduced to have a 21 amino acid signal peptide and a four amino acid propeptide ending with a tryptic cleavage site, followed by a sequence motif identical to the N-terminal amino acid sequence of native active human SCCE. As in human SCCE the P2 position of the propeptide was occupied by an acidic amino acid residue, and the position corresponding to the suggested bottom of the primary substrate specificity pouch occupied by an asparagine residue. Analyses of mouse tissues by reverse transcriptase-polymerase chain reaction showed high expression in the skin, low expression in lung, kidney, brain, heart, and spleen, and no expression in liver or skeletal muscle. In situ hybridization of mouse skin showed expression in high suprabasal keratinocytes and in the luminal parts of hair follicles. Our results strongly suggest that we have cloned the murine analog of human SCCE cDNA.

Sequence determinants directing conversion of cysteine to formylglycine in eukaryotic sulfatases.

Sulfatases carry at their catalytic site a unique post-translational modification, an alpha-formylglycine residue that is essential for enzyme activity. Formylglycine is generated by oxidation of a conserved cysteine or, in some prokaryotic sulfatases, serine residue. In eukaryotes, this oxidation occurs in the endoplasmic reticulum during or shortly after import of the nascent sulfatase polypeptide. The modification of arylsulfatase A was studied in vitro and was found to be directed by a short linear sequence, CTPSR, starting with the cysteine to be modified. Mutational analyses showed that the cysteine, proline and arginine are the key residues within this motif, whereas formylglycine formation tolerated the individual, but not the simultaneous substitution of the threonine or serine. The CTPSR motif was transferred to a heterologous protein leading to low-efficient formylglycine formation. The efficiency reached control values when seven additional residues (AALLTGR) directly following the CTPSR motif in arylsulfatase A were present. Mutating up to four residues simultaneously within this heptamer sequence inhibited the modification only moderately. AALLTGR may, therefore, have an auxiliary function in presenting the core motif to the modifying enzyme. Within the two motifs, the key residues are fully, and other residues are highly conserved among all known members of the sulfatase family.

Conformational and metal-binding properties of androcam, a testis-specific, calmodulin-related protein from Drosophila.

Androcam is a testis-specific protein of Drosophila melanogaster, with 67% sequence identity to calmodulin and four potential EF-hand calcium-binding sites. Spectroscopic monitoring of the thermal unfolding of recombinant calcium-free androcam shows a biphasic process characteristic of a two-domain protein, with the apo-N-domain less stable than the apo-C-domain. The two EF hands of the C-domain of androcam bind calcium cooperatively with 40-fold higher average affinity than the corresponding calmodulin sites. Magnesium competes with calcium binding [Ka(Mg) approximately 3 x 10(3) M(-1)]. Weak calcium binding is also detected at one or more N-domain sites. Compared to apo-calmodulin, apo-androcam has a smaller conformational response to calcium and a lower alpha-helical content over a range of experimental conditions. Unlike calmodulin, a tryptic cleavage site in the N-domain of apo-androcam remains trypsin sensitive in the presence of calcium, suggesting an altered calcium-dependent conformational change in this domain. The affinity of model target peptides for androcam is 10(3)-10(5) times lower than for calmodulin, and interaction of the N-domain of androcam with these peptides is significantly reduced. Thus, androcam shows calcium-induced conformational responses typical of a calcium sensor, but its properties indicate calcium sensitivity and target interactions significantly different from those of calmodulin. From the sequence differences and the altered calcium-binding properties it is likely that androcam differs from calmodulin in the conformation of residues in the second calcium-binding loop. Molecular modeling supports the deduction that there are significant conformational differences in the N-domain of androcam compared to calmodulin, and that these could affect the surface, conferring a different specificity on androcam in target interactions related to testis-specific calcium signaling functions.

Interaction of the Globular Domains of pIII Protein of Filamentous Bacteriophage fd with the F-Pilus ofEscherichia coli

Gene 3 protein (pIII), a minor coat protein at one end of the filamentous bacteriophage fd, is involved in initiating the infection by the virus ofEscherichia colicells that display an F-pilus. Infection is thought to start with the adsorption of the D2 domain of pIII to the tip of the pilus, retraction of the pilus, and penetration of theE. colicell membrane mediated by an interaction between the D1 domain of pIII and the Tol protein complex in the membrane. A subgene encoding the pIII-D1D2 di-domain was created, and the subgene was successfully overexpressed inE. colicells. Domains D1 and D2 were separated after limited proteolysis of a modified pIII-D1D2 (designated pIII-D1D2.trp) into which two tryptic cleavage sites were introduced at appropriate points. The purified pIII-D1D2 di-domain and pIII-D2 domain were able to bind to the F-pilus, competing with the wild-type pIII and delaying infection by the intact filamentous phage. The pIII-D1 domain was unable to bind to the F-pilus by this criterion. This provides conclusive evidence that the pIII-D2 domain is responsible for the adsorption to the tip of the F-pilus and can achieve this in the absence of domain D1, opening the way to identifying the molecular basis of the interaction of pIII-D2 with the pilus.

Functional Symmetry of UhpT, the Sugar Phosphate Transporter ofEscherichia coli

UhpT, the sugar phosphate transporter of Escherichia coli, acts to exchange internal inorganic phosphate for external hexose 6-phosphate. Because of this operational asymmetry, we studied variants in which right-side-out (RSO) or inside-out (ISO) orientations could be analyzed independently to ask whether the inward- and outward-facing UhpT surfaces have different substrate specificities. To study the RSO orientation, we constructed a histidine-tagged derivative, His10K291C/K294N, in which the sole external tryptic cleavage site (Lys294) had been removed. Functional assay as well as immunoblot analysis showed that trypsin treatment of proteoliposomes containing His10K291C/K294N led to loss of about 50% of the original population, reflecting retention of only the RSO population. To study the ISO orientation, we used a His10V284C derivative, in which a newly inserted external cysteine (Cys284) conferred sensitivity to the thiol-reactive agent, 3-(N-maleimidylpropionyl)biocytin. In this case, 3-(N-maleimidylpropionyl)biocytin treatment of proteoliposomes containing His10V284C gave about a 60% loss of activity, and immunodetection of biotin showed parallel modification of an equivalent fraction of the original population. Together, such findings indicate that the UhpT RSO and ISO orientations are in about equal proportion in proteoliposomes and that a single population can be generated by exposure of these derivatives to the appropriate agent. This allowed us to study proteoliposomes with UhpT functioning in RSO orientation (His10K291C/K294N) or ISO orientation (His10V284C) with respect to the kinetics of glucose 6-phosphate transport by phosphate-loaded proteoliposomes and also the inhibitions found with 2-deoxy-glucose 6-phosphate, mannose 6-phosphate, galactose 6-phosphate, fructose 6-phosphate, and inorganic phosphate. We found no significant differences in the behavior of UhpT in its different orientations, indicating that the transporter possesses an overall functional symmetry.

Distinct biochemical and topological properties of the 31- and 27-kilodalton plasma membrane intrinsic protein subgroups from red beet.

Plasma membrane vesicles from red beet (Beta vulgaris L.) storage tissue contain two prominent major intrinsic protein species of 31 and 27 kD (X. Qi, C.Y Tai, B.P. Wasserman [1995] Plant Physiol 108: 387-392). In this study affinity-purified antibodies were used to investigate their localization and biochemical properties. Both plasma membrane intrinsic protein (PMIP) subgroups partitioned identically in sucrose gradients; however, each exhibited distinct properties when probed for multimer formation, and by limited proteolysis. The tendency of each PMIP species to form disulfide-linked aggregates was studied by inclusion of various sulfhydryl agents during tissue homogenization and vesicle isolation. In the absence of dithiothreitol and sulfhydryl reagents, PMIP27 yielded a mixture of monomeric and aggregated species. In contrast, generation of a monomeric species of PMIP31 required the addition of dithiothreitol, iodoacetic acid, or N-ethylmaleimide. Mixed disulfide-linked heterodimers between the PMIP31 and PMIP27 subgroups were not detected. Based on vectorial proteolysis of right-side-out vesicles with trypsin and hydropathy analysis of the predicted amino acid sequence derived from the gene encoding PMIP27, a topological model for a PMIP27 was established. Two exposed tryptic cleavage sites were identified from proteolysis of PMIP27, and each was distinct from the single exposed site previously identified in surface loop C of a PMIP31. Although the PMIP31 and PMIP27 species both contain integral proteins that appear to occur within a single vesicle population, these results demonstrate that each PMIP subgroup responds differently to perturbations of the membrane.

Preferential sites of tryptic cleavage on the major bovine caseins within the micelle

Caseins, major proteins in bovine milk, are structured in micelles within which the detailed casein organisation remains unclear. We have used limited proteolysis to find the most exposed regions of the caseins within the micelle. Under hydrolysis conditions, only 9% of colloidal calcium and 15.6% of the colloidal inorganic phosphate were removed from the micelle after 4 h. The released peptides during tryptic hydrolysis were separated from the residual micelle by ultracentri- fugation. They were subsequently analysed and characterised by reversed-phase HPLC coupled on- line with electrospray ion source mass spectrometry (ESI-MS). After 4 h of hydrolysis, the p-casein had completely disappeared while about 40% of us,-casein and 37% of x-casein remained undiges- ted. The initial distribution, i.e., 91 % caseins and 9% peptides prior to hydrolysis, was modified after hydrolysis to give 64% peptides remaining within the micelles together with 13% caseins and 22% of the peptides produced were released from the micelles. Among the 61 peptides identified, 34 arose from p-casein, 16 from us1-casein, II from us2-casein and none from x-casein. For the lat- ter casein, the failure ta detect peptides was due to its lower concentration in the large micelles corn- pared with that of the Pand us1-caseins. The peptides derived from p-casein were mainly released from the micelle, while peptides from bath us1- and us2-caseins were preferentially retained within the resi- dual micelles, suggesting that ail the caseins are accessible ta trypsin but are differentially involved in the micellar framework. Our results suggest that us-caseins play a significant role in the micellar cohesion via salt binding with colloidal calcium phosphate and hydrophobie interaction, with itself and with the other caseins. © Inra/Elsevier, Paris.

Probing the conformation of NhaA, a Na+/H+ antiporter from Escherichia coli, with trypsin.

One of the most striking features of NhaA, an Escherichia coli Na+/H+ antiporter, is its extreme sensitivity to pH. The activity of NhaA increases 2000-fold between pH 6.5 and 8.5. In this work, we investigated whether the activation of NhaA by pH is accompanied by conformational changes which can be detected using trypsin as a probe. We have found that NhaA is susceptible to proteolytic digestion at the pH range where it is activated, suggesting that these two events may be related; at alkaline pH, the protein becomes active and adopts an "open" conformational state in which more domains are exposed to the enzyme. This idea was further supported by results from two mutants of NhaA in which His-225, a residue involved in pH sensing, has been replaced by either Arg or Asp. The mutant H225R is activated at more acidic pH values, while H225D at more alkaline pH. In accordance with the results described for the wild-type protein, H225R was susceptible to digestion by trypsin at the pH at which it undergoes main activation. NhaA has many potential tryptic cleavage sites. However, analysis of the tryptic digestion fragments suggests that at alkaline pH, the protein is exposed to cleavage mainly at hydrophilic loops 6, 7, and 8. Thus, upon activation, NhaA appears to undergo a change in conformation that is reflected in specific regions of the protein.

Autoantibodies to IA-2 and IA-2 beta in insulin-dependent diabetes mellitus recognize conformational epitopes: location of the 37- and 40-kDa fragments determined.

IA-2 and IA-2 beta are major autoantigens in insulin-dependent diabetes mellitus (IDDM) and the precursors, respectively, of a 40-and 37-kDa tryptic fragment that reacts with IDDM sera. In the present study, by amino acid sequencing of recombinant IA-2 and IA-2 beta, we determined the tryptic cleavage sites involved in the generation of these fragments. Both cleavage sites are immediately after an arginine residue at position 653 for IA-2 and position 679 for IA-2 beta. The resulting tryptic fragments are 326 and 307 amino acids in length and retain their ability to react with IDDM sera. In contrast to IA-2 and IA-2 beta, other members of the protein tyrosine phosphatase (PTP) family (i.e., RPTP kappa, RPTPmu, NU-3, SHP, and 3CH134) are completely susceptible to digestion by trypsin. Sequence analysis revealed five conserved cysteine residues in IA-2 and IA-2 beta that are not present in other PTPs. Reduction and alkylation of IA-2 and IA-2 beta recombinant proteins resulted in loss of both resistance to digestion by trypsin and reactivity with autoantibodies in IDDM sera. It is concluded that disulfide bond formation plays a critical role in the maintenance of antigenic structure and that the autoantibodies to IA-2/IA-2 beta in IDDM sera recognize conformational epitopes.

Reduced Cell Attachment and Phosphorylation of Focal Adhesion Kinase Associated with Expression of a Mutant Insulin Receptor

Insulin signaling results in rapid changes to the cell cytoskeleton, and it has recently been shown that insulin stimulates the dephosphorylation of the cytoskeletal-associated tyrosine kinase, focal adhesion kinase (pp125(FAK)). We report here that mutation of two tryptic cleavage sites (Lys164 and Lys582 --> Asn; 2N) in the insulin receptor alpha-subunit results in a cell-line (CHO.2N-10) with altered morphology associated with an increase in cell size, a decrease in cell adhesiveness, and a decrease in pp125(FAK) tyrosine phosphorylation in the absence of insulin (45.2 +/- 9.7% compared to nontransfected Chinese hamster ovary (CHO) cells). In contrast to pp125(FAK), paxillin phosphorylation was similar in all cell lines despite lower levels (61.0 +/- 10.4% compared to CHO cells) of paxillin protein in CHO.2N-10 cells. We observed comparable protein levels of pp125(FAK) and the structural focal adhesion protein, vinculin, in all cell lines. Despite underphosphorylation of pp125(FAK) in the basal state, insulin stimulation of CHO.2N-10 cells still resulted in dephosphorylation of pp125(FAK). CHO.2N-10 and CHO.T (overexpress wild-type insulin receptor) cells have similar insulin binding characteristics, insulin-stimulated autokinase and peptide phosphorylation, and insulin-stimulated pp185/IRS-1 phosphorylation. Our results suggest that the insulin receptor may play an important role in cell-matrix interactions, such as modulating cell adhesion and inducing cell architecture changes.

Zipper Protein, a B-G Protein with the Ability to Regulate Actin/Myosin 1 Interactions in the Intestinal Brush Border

We recently identified a 28-kDa protein in the intestinal brush border that resembled tropomyosin in terms of size, homology, and alpha helical content. This protein contained 27 heptad repeats, nearly all of which began with leucine, leading to its name zipper protein. Subsequent analysis, however, indicated that both a 49-kDa and a 28-kDa immunoreactive protein existed in intestinal brush-border extracts. Using 5'-rapid amplification of cDNA ends analysis, we extended the N-terminal sequence of zipper protein to the apparent translation start site. This additional sequence contained a putative transmembrane domain and two potential tryptic cleavage sites C-terminal to the transmembrane domain which would release a 28-kDa cytoplasmic protein if utilized. The additional sequence was highly homologous to members of the B-G protein family, a family with no known function. Immunoelectron microscopy showed that zipper protein was confined to the membrane of the microvillus where it was in close association with brush-border myosin 1 (BBM1). Recombinant zipper protein (28-kDa cytoplasmic portion) blocked the binding of actin to BBM1 and inhibited actin-stimulated BBM1 ATPase activity. In contrast, zipper protein had no effect on endogenous or K/EDTA-stimulated BBM1 ATPase activity. Furthermore, zipper protein displaced tropomyosin from binding to actin, suggesting that these homologous proteins bind to the same sites on the actin molecule. We conclude that zipper protein is a transmembrane protein of the B-G family localized to the intestinal epithelial cell microvillus. The extended cytoplasmic tail either in the intact molecule or after tryptic cleavage may participate in regulating the binding and, thus, activation of BBM1 by actin in a manner similar to tropomyosin.

Proteolytic cleavage within a regulatory region of the gamma subunit of chloroplast coupling factor 1.

The gamma subunit of chloroplast coupling factor 1 (CF1) is susceptible to selective proteolysis when the enzyme is in solution and the epsilon subunit is removed [CF1(-epsilon)]. In spinach thylakoid membranes, rapid cleavage of gamma is dependent on the generation of an electrochemical proton potential. The tryptic cleavage sites within the gamma of oxidized CF1 in illuminated thylakoids as well as of reduced CF1(-epsilon) in solution were determined by N-terminal amino acid sequencing. Two large gamma fragments of 27 000 (gamma27) and 10 000 (gammma10) molecular weight were generated by trypsin treatment of membrane-bound CF1. THe N-terminal gamma27 contains amino acids 1-215, and the C-terminal gamma10 contains 232-323. These polypeptides were tightly associated with the trypsin-resistant core of the enzyme. In contrast, three large gamma fragments were produced by trypsinolysis of reduced CF1(-epsilon). These polypeptides, which were also tightly associated with the trypsin-resistant core, have molecular weights of 7 900(gamma8), 14 850 (gamma15), and 10 000 (gamma10). These fragments contain residues 1-70, 71-204, and 232-323, respectively. The C-terminal gamma10 fragment generated by trypsin treatment of membrane-bound and soluble CF1 are identical. These results suggest that the gamma subunit of CF1 in illuminated thylakoids resembles that of CF1(-epsilon) with respect to accessibility to proteolytic cleavage. Cleavage of gamma between residues 215 and 232 is sufficient to fully activate the ATPase activity of the enzyme without reduction of the gamma disulfide. In addition, cutting within this region is responsible for loss of affinity for the inhibitory epsilon subunit.

Analysis by limited proteolysis of domain organization and GSH-site arrangement of bacterial glutathione transferase B1-1

Limited proteolysis method has been used to study the structure—function relationship of bacterial glutathione transferase (GSTB1-1). In absence of three-dimensional structural data of prokaryote GST, the results represent the first information concerning the G-site and domains organization of GSTB1-1. The tryptic cleavages occur mainly at the peptide bonds Lys35-Lys36 and Phe43-Leu44, generating two major molecular species of 20-kDa, 3-kDa and traces of 10-kDa. 1-chloro-2,4-dinitrobenzene favoured the proteolysis of the 20-kDa fragment markedly enhancing the production of the 10-kDa peptide by cleaving the chemical bonds Lys87-Ala88 and Arg91-Tyr92. The tryptic cleavage sites of GSTB1-1 was found to be located close to those previously found for the mammalian GSTP1-1 isozyme. It was concluded that despite their low sequence homology (18%), GSTB1-1 and GSTP1-1 displayed similar structural features in their G-site regions and probably a common organization in structural domains.

Serine 209, Not Serine 53, Is the Major Site of Phosphorylation in Initiation Factor eIF-4E in Serum-treated Chinese Hamster Ovary Cells

Ser-53 has previously been considered the major phosphorylation site in eukaryotic initiation factor (eIF)-4E, and this appeared to be supported by studies using a S53A mutant. Recently, however, several lines of evidence have indicated that Ser-53 might not be the true phosphorylation site. This prompted us to re-examine the phosphorylation site in eIF-4E using factor purified from 32P-labeled, serum-treated Chinese hamster ovary cells. Isoelectric focusing and phosphoamino acid analysis indicated the existence of a single phosphorylated serine. Edman degradation of the major radiolabeled tryptic product from 32P-labeled eIF-4E showed that the phosphorylated site was positioned three residues from the N terminus of this peptide. There are three serines in the sequence of eIF-4E that are three residues away from a tryptic cleavage site (i.e. lysine or arginine). 32P-Labeled eIF-4E was digested with trypsin, Lys-C, or trypsin followed by Glu-C and subjected to two-dimensional mapping; the data obtained eliminated two of these potential sites, leaving Ser-209. Comigration of the synthetic peptide SGS(P)209TTK with the radiolabeled tryptic product on (i) reverse-phase chromatography and (ii) two-dimensional mapping at different pH values confirmed that Ser-209 is the major phosphorylation site in eIF-4E in serum-stimulated Chinese hamster ovary cells.

Structural Organisation of Human Bile-salt-activated Lipase Probed by Limited Proteolysis and Expression of a Recombinant Truncated Variant

Bile-salt-activated lipase belongs to the cholinesterase alpha/beta-hydrolase-fold family of proteins. Here, we have investigated the structural organisation of the human isoform by mapping tryptic cleavage sites using limited proteolysis and by expression studies using a recombinant truncated variant. Two accessible regions in the tertiary structure were identified. The first is defined by a tryptic cleavage at Lys429 and lies within the alpha/beta-hydrolase fold in bile-salt-activated lipase between a central beta-sheet and an active-site histidine residue, as deduced from sequence similarity across the cholinesterases and known structural properties. This region exhibits a proteolytic and topological similarity to the lid region in pancreatic lipase. The other accessible region in the tertiary structure is defined by a tryptic cleavage at Arg520 and occurs within a catalytically non-essential segment Leu519-Gln535, as identified by expression of a truncated variant which lacks the C-terminus starting from Leu519. This region is consistent with an interdomain region between the cholinesterase-related part of the protein structure and the unique proline-rich C-terminal repeats. Both protease-sensitive regions appear to occur at domain borders, and, therefore, are consistent with a multi-domain structure. The truncated variant was fully functional as a lipase and as a bile-salt-stimulated esterase. However, compared to the full-length enzyme, the truncated variant showed an increased susceptibility to limited proteolysis, suggesting that the C-terminal repeats may regulate proteolytic degradation of the protein.