Intermediates in bacteriophage Mu lysogenization of Escherichia coli him hosts.

Characterization of a putative intermediate in the Mu lysogenization pathway is possible in a variant Escherichia coli himD strain which exhibits greatly diminished lysogen formation. In this strain, most infecting Mu genomes form stable, transcribable, nonreplicating structures. Many of these genomes can be mobilized to form lysogens by a second Mu infection, which can be delayed by at least 100 min. This intermediate structure can be formed in the absence of Mu A or B function. We suggest that the inferred intermediate could be the previously reported protein-linked circular form of the Mu genome. Providing Mu B function from a plasmid enhances Mu lysogenization in this him strain, and the enhancement is much greater when both Mu A and B functions are provided.     

Detection and characterization of the intermediate on the folding pathway of human alpha-lactalbumin

To discuss the relation between the folding mechanism and the chemical structure of proteins, the reversible unfolding reactions of human alpha-lactalbumin by acidification and by guanidine hydrochloride at 25 degrees C are studied by means of circular dichroism, difference spectra and pH-jump measurements and are compared with those for bovine alpha-lactalbumin. As shown previously for bovine alpha-lactalbumin, the folding process at neutral pH is not explained by a simple two-state mechanism but involves an intermediate form that has the same amount of helical structures as the native form. The transition between the intermediate and the fully denatured states is too rapid to be measured and corresponds to the helix-coil transition of the backbone. One of the differences of human alpha-lactalbumin from the bovine protein is the remarkable stability of the intermediate at neutral pH, which can be explained by differences in the primary chemical structure. Another difference is the existence at acid pH of an additional helical form, which is more helical than the native form. The transition from this to the intermediate or to the fully denatured one also is shown to resemble the helix-coil transition. The following folding scheme of human alpha-lactalbumin is proposed: formula: (see text). Here N is the native form, and the intermediate is a macroscopic state distributed around the state A3 at neutral pH, while the distribution in the acid and fully denautured states shifts toward Am and A-n, respectively.     

Urea Induced Inactivation and Unfolding of Arginine Kinase from the Sea Cucumber Stichopus japonicus

Urea titration was used to study the inactivation and unfolding equilibrium of arginine kinase (AK) from the sea cucumber Stichopus japonicus. Both fluorescence spectral and circular dichroism spectral data indicated that an unfolding intermediate of AK existed in the presence of 1.0 to 2.0 M urea. This was further supported by the results of size exclusion chromatography. The spectral data suggested that this unfolding intermediate shared many structural characteristics with the native form of AK including its secondary structure, tertiary structure, as well as its quaternary structure. Furthermore, according to the residual activity curve, this unfolding intermediate form still retained its catalytic function although its activity was lower than that of native AK. Taken together, the results of our study give direct evidence that an intermediate with partial activity exists in unfolding equilibrium states of AK during titration with urea.     

Evidence for an aldehyde intermediate in the catalytic mechanism of thiamine oxidase

Thiamine oxidase catalyzes the four-electron oxidation of the 5-hydroxyethyl group of thiamine to form thiamine acetic acid via an aldehyde intermediate. Evidence for the formation of this intermediate is derived from a number of kinetic approaches. The rate of thiamine acetic acid formation, as monitored by the rate of proton release, is subject to substrate inhibition and to inhibition by the presence of semicarbazide while the rate of O2 consumption (due to thiamine oxidation to the aldehyde and subsequently to the carboxylic acid) is unaffected. The transient formation of an intermediate with a maximal absorption at 370 nm in stopped-flow turnover experiments is dependent on the pH and the substrate concentration, and is prevented by the presence of semicarbazide, thus suggesting this transient absorption intermediate to be a result of formation of the aldehyde intermediate. A similar spectral intermediate is observed when hydroxythiamine is the substrate but is not observed with pyrithiamine. In the presence of large concentrations of pyrithiamine, the enzyme undergoes an irreversible inactivation which is not reversed on removal of pyrithiamine or its oxidation products by gel filtration or dialysis. This inhibition is prevented by the presence of thiols or of semicarbazide and is suggested to be due to the release of the aldehyde form of pyrithiamine from the catalytic site, which then reacts with the enzyme in a nonspecific manner. The structure of the 370-nm-absorbing intermediate is currently unknown but is suggested not to be the "yellow form" of thiamine. This suggestion is due to observed differences in absorption spectral properties and to the fact that it can also be formed from hydroxythiamine, which does not form the "yellow form" of thiamine on alkaline treatment. Taken together, these data suggest that, at or below saturating concentrations, thiamine remains bound to the catalytic site during the two sequential two-electron transfer steps, with 2 mol O2 being reduced to 2 mol H2O2. At high concentrations (greater than 10 Km), the intermediate thiamine aldehyde can be displaced from the catalytic site by thiamine simply by a mass-action effect.     

Functional consequences of mutations in the beta-strand sector of the Ca2(+)-ATPase of sarcoplasmic reticulum

Kinetic studies of the phosphoenzyme intermediates of site-specific mutants were used to examine the role of Gly233 in the reaction mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase. When this glycine residue, which is highly conserved among cation-transporting ATPases, was replaced by valine, arginine, or glutamic acid, a complete loss of the ability to pump Ca2+ was observed. The mutant enzymes were able to form an ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP in the presence of Ca2+, but this intermediate decayed to the ADP-insensitive form (E2P) very slowly, relative to the wild-type enzyme. The mutant phosphoenzyme intermediate remained ADP-sensitive, even when phosphorylation from ATP was performed under conditions which permitted accumulation of the ADP-insensitive phosphoenzyme intermediate in the wild type. The mutants were also defective in their ability to form the ADP-insensitive phosphoenzyme intermediate by phosphorylation from inorganic phosphate. In addition, they displayed a higher affinity for Ca2+ and a lower cooperativity in Ca2+ binding than did the wild-type enzyme, as measured through the phosphorylation reaction with ATP. These findings can be rationalized either in terms of a parallel shift of E1 to E2 and E1P to E2P conformational equilibria toward the E1 and E1P forms, respectively, or in terms of destabilization of the phosphoryl-protein interaction in the E2P form. The roles of 7 other residues located in the vicinity of Gly233 were also examined by mutation. Although the side chains of these residues are potential Ca2+ ligands, their replacement did not affect the Ca2+ affinity of the enzyme, suggesting the lack of a role of this region of the peptide in formation of Ca2(+)-binding sites.     

Gap filling and not replication fork progression is the rate-limiting step in the replication of UV-damaged simian virus 40 DNA.

We have analyzed the structural characteristics of simian virus 40 replicative intermediate DNA produced after UV irradiation and the kinetics of conversion of this intermediate DNA into form I DNA. Replicative intermediate DNA isolated at 30 or 60 min after UV irradiation consists primarily of two species of molecules that sediment in neutral sucrose gradients as either Cairns theta structures or relaxed monomeric circles. Replication forks on the Cairns intermediate DNA are symmetrically located with respect to the origin of replication, ruling out the possibility of asymmetric pauses or blocks to replication fork progression at damage sites. The relaxed circles contain at least one randomly located discontinuity in the daughter strand. Pulse-chase experiments demonstrated that a UV fluence-dependent fraction of the Cairns intermediate DNA progresses through the relaxed circular intermediate before being converted to completed form I molecules. Disappearance of Cairns intermediate DNA occurs at the same rate in irradiated and unirradiated cells, whereas completion of the relaxed circular intermediate DNA occurs at a slow rate, relatively independent of UV fluence. These data support a model for replication of UV-damaged DNA in which replication rapidly continues past damage sites via a gap formation event.     

Detection of N-acetylated forms of beta-endorphin and nonacetylated alpha-MSH in the intermediate pituitary of the toad, Bufo marinus

Steady-state analysis of the acid extracts of the intermediate pituitary of the toad, Bufo marinus, revealed the presence of multiple forms of beta-endorphin and alpha-MSH. Approximately 98% of the immunoreactive beta-endorphin was N-acetylated. The major form of N-acetylated beta-endorphin, which represented 81.5% of the total beta-endorphin recovered from this tissue, had an apparent molecular weight of 1.2 kDa and a net charge of +1 at pH 2.75. Approximately 98% of the immunoreactive alpha-MSH present in the Bufo intermediate pituitary had reverse phase HPLC properties similar to the nonacetylated form of alpha-MSH, ACTH(1-13)amide. These observations are in agreement with studies on the intermediate pituitary of the frog, Xenopus laevis, which have shown that the N-acetylation of alpha-MSH in this species is a cosecretory processing event, whereas the N-acetylation of beta-endorphin is a posttranslational processing event (2, 5, 15). These observations indicate that the N-acetylation of beta-endorphin and alpha-MSH occurs at distinct subcellular sites in intermediate pituitary cells of anuran amphibians. The Bufo intermediate pituitary will serve as a good model system for studying these novel N-acetyltransferase reactions.     

The elusive intermediate on the folding pathway of the prion protein

A key molecular event in prion diseases is the conversion of the cellular conformation of the prion protein (PrP(C)) to an altered disease-associated form, generally denoted as scrapie isoform (PrP(Sc)). The molecular details of this conformational transition are not fully understood, but it has been suggested that an intermediate on the folding pathway of PrP(C) may be recruited to form PrP(Sc). In order to investigate the folding pathway of PrP we designed and expressed two mutants, each possessing a single strategically located tryptophan residue. The secondary structure and folding properties of the mutants were examined. Using conventional analyses of folding transition data determined by fluorescence and CD, and novel phase-diagram analyses, we present compelling evidence for the presence of an intermediate species on the folding pathway of PrP. The potential role of this intermediate in prion conversion is discussed.     

Structural characterization of the partially folded intermediates of an immunoglobulin light chain leading to amyloid fibrillation and amorphous aggregation

Immunoglobulin light chain deposition diseases involve various types of extracellular deposition of light chain variable domains, including amyloid fibrils and amorphous deposits. The decreased thermodynamic stability of the light chain is believed to be the major factor leading to fibrillation. However, the differences in the nature of the deposits among the light chain deposition diseases raise the question of whether the mechanisms leading to fibrillar or amorphous aggregation is different. In this study, we generated two partially folded intermediates of the light chain variable domain SMA in the presence of guanidine hydrochloride (GuHCl) and characterized their conformations. The more unfolded intermediate formed fibrils most rapidly, while the more native-like intermediate predominantly led to amorphous deposits. The results also show that the monomeric, rather than the dimeric state, was critical for fibrillation. The data also indicate that fibril elongation involves addition of a partially unfolded intermediate, rather than the native state. We postulate that a more highly unfolded intermediate is more suited to undergo the topological rearrangements necessary to form amyloid fibrils than a more structured one and that this also correlates with increased destabilization. In the case of light chain aggregation, it appears that more native-like intermediate conformations are more prone to form amorphous deposits.     

Oxygen-induced changes in the redox state of the cytochrome b559 in photosystem II depend on the integrity of the Mn cluster

The effect of oxygen and anaerobiosis on the redox properties of Cyt b ₅₅₉ was investigated in PSII preparations from spinach with different degree of disintegration of the donor side. Comparative studies were performed on intact PSII membranes and PSII membranes that were deprived of the 18-kDa peripheral subunit (0.25 NaCl washed), the 18- and 24-kDa peripheral subunits (1 M NaCl washed), the 18-, 24- and 33-kDa peripheral subunits (1.2 M CaCl₂ washed), Cl depleted and after complete depletion of the Mn cluster (Tris washed). In active PSII centers, about 75% of Cyt b ₅₅₉ was found in the high-potential form and the rest in the intermediate potential form. With decomposition of the donor side, the intermediate potential form started to dominate, reaching more than 90% after Tris treatment. The oxygen-dependent conversion of the intermediate potential form of Cyt b ₅₅₉ into the low-potential and high-potential forms was only observed after treatments that directly affect the Mn cluster. In PSII membranes, deprived of all three extrinsic subunits (CaCl₂ treatment), 21% of the intermediate potential form was converted into the low-potential form and 14% into the high-potential form by the removal of oxygen. In Tris-washed PSII membranes, completely lacking the Mn cluster, this conversion amounted to 60 and 33%, respectively. In intact PSII membranes, the oxygen-dependent conversion did not occur. The possible physiological role of this oxygen-dependent behavior of the Cyt b ₅₅₉ redox forms during the assembly/photoactivation cycle of PSII is discussed.     

Cryoenzymology and spectrophotometry of pea seedling diamine oxidase

Diamine oxidase follows bi-ter ping-pong kinetics, with an intermediate, "reduced" free-enzyme form being generated after the anaerobic conversion of amine to aldehyde. Visible spectra of diamine oxidase reacting at subzero temperatures provide evidence that this intermediate enzyme form is obtained via several other intermediates and that the environment of the Cu(II) changes dramatically during the course of the reaction [even though it is not reduced to Cu(I) during the catalytic cycle]. The spectrum of this form of diamine oxidase, which is obtained 0.5--2 h after the addition of amine at -5 to -15 degrees C, is independent of substrate, is identical with that obtained by anaerobic addition of substrate at room temperature, and provides evidence for a direct interaction of Cu(II) with the organic cofactor of the enzyme. This interaction is apparently charge transfer in nature. Upon removal of Cu(II) from the native enzyme, one obtains spectral evidence that the organic cofactor is still present. However, removal of the Cu(II) from the reduced (intermediate) enzyme form yields a featureless enzyme spectrum and a Cu(II)--chelate complex which contains a new ligand, which is presumably the second prosthetic group.     

Studies on the biosynthesis of TMV

Summary The replicative form and the replicative intermediate of TMV-RNA were isolated from synchronously infected tobacco leaves, labeled with H³-uridine for 1 hour. The replicative form is over 90% resistant to RNase and sediments slightly slower than the 16S ribosomal RNA. Sucrose gradient centrifugation of the thermally denatured replicative form revealed that it contains strands of the same size as single-stranded TMV-RNA. The replicative intermediate showed only partial resistance to RNase and heterogeneous sedimentation behavior in sucrose gradients. After mild RNase treatment the replicative intermediate sedimented homogeneously, and with an S value slightly lower than the replicative form.The following abbreviations are used RF replicative form - RI replicative intermediate - STE 0.1 M NaCl-1 mM Tris-HCl-1 mM EDTA, pH 7.4 - SSC 0.15 M NaCl-0.015 M sodium citrate, pH 7.0 - 10xSSC and 0.1xSSC tenfold concentrated and tenfold diluted SSC respectively - MAK methylated albumin coated kieselguhr     

Isolation and Characterization of Nonconverting Mutants of Bacteriophage ε34

epsilon(34) Is a converting bacteriophage which brings about a glucosylation of the O antigen in the E group salmonella, thus producing antigen 34. Uridine diphosphate glucose is the precursor of antigen 34 and a glucosyl-lipid is an essential intermediate in this process. Mutants of epsilon(34) which cannot produce antigen 34 have been isolated. Cells lysogenic for these mutants are of two types. Type 1 mutant lysogens cannot form the glucosyl-lipid intermediate; those of type 2 can form glucosyl-lipid but are unable to transfer glucose from the lipid to the O antigen.     

Transitional Forms of Corynebacterium acnes in Disease

A clear-cut triad of sequential Corynebacterium acnes transitional forms from disease has been discovered. This entity includes three major forms which are capable of stabilization in culture, the spherical, the intermediate, and the definitive C. acnes. During conversion or reversion among the three forms, a variety of forms with mixed characteristics was observed. The spherical form was gram-negative and osmotically fragile, but it possessed a vestigial cell wall and mesosomes which excluded it from the L forms. In lieu of the L-form designation, the term "transitional" was adopted for all forms leading up to the definitive C. acnes. Culture of the spherical form was successful only on Mycoplasma-type media. The intermediate form was gram-negative, had mixed spherical and filamentous morphology, and bore a striking resemblance to Streptobacillus moniliformis. Like the spherical form, it was nutritionally exacting. The definitive form of C. acnes was preceded by gram-positive transitional forms of C. acnes morphology. It lacked, however, the carbohydrases and proteinases of C. acnes and susceptibility to C. acnes bacteriophages. Reversion was often blocked at this stage. A series of blood cultures from a patient with endocarditis was studied. Postmortem stain sections of the heart-valve lesion included intracellular masses of gram-negative spherical organisms. Indirect fluorescent antibody staining of these masses was strongly positive with antiserum to the spherical form and weakly positive with antiserum to the intermediate form.     

Occurrence of two different pathways in the activation of porcine pepsinogen to pepsin

Activation of porcine pepsinogen at pH 2.0 was found to proceed simultaneously by two different pathways. One pathway is the direct conversion process of pepsinogen to pepsin, releasing the intact activation segment. The isolation of the released 44-residue segment was direct evidence of this one-step process. At pH 5.5 the segment bound tightly to pepsin to form a 1:1 pepsin-activation segment complex, which was chromatographically indistinguishable from pepsinogen. The other is a stepwise-activating or sequential pathway, in which pepsinogen is activated to pepsin through intermediate forms, releasing activation peptides stepwisely. These intermediate forms were isolated and characterized. The major intermediate form was shown to be generated by removal of the amino-terminal 16 residues from pepsinogen. The released peptide mixture was composed of two major peptides comprising residues 1-16 and 17-44, and hence the stepwise-activating process was deduced to be mainly a two-step process.     

ATP-dependent import of a lumenal protein by isolated thylakoid vesicles

The 33 kd protein of the photosynthetic oxygen-evolving complex is synthesized in the cytoplasm as a larger precursor and transported into the thylakoid lumen via a stromal intermediate form. In this report we describe a reconstituted system in which the later stages of this import pathway can be studied in isolation. We demonstrate import of the 33 kd protein, probably as the intermediate form, into isolated pea thylakoids by a mechanism which is stimulated by the addition of ATP. The imported protein is processed to the mature size and is resistant to digestion by proteases. The thylakoidal protein transport system is specific in that non-chloroplast proteins and precursors of stromal proteins are not imported.     

Glycine-extended processing intermediate of proVIP: a new form of VIP in the rat

The biosynthesis of many peptides including vasoactive intestinal polypeptide (VIP) requires enzymatic alpha-carboxyamidation via a glycine-extended intermediate form. In an effort to identify and quantify glycine-extended VIP in rat tissue extracts a radio-immunoassay specific for this peptide was developed. The concentrations of glycine-extended VIP ranged from 1.3 pmol/g in the brain to 83.9 pmol/g in the small intestine. The identity of the peptide was substantiated by cation-exchange HPLC. The ratio of glycine-extended VIP to amidated VIP varied considerably being highest (63%) in the small intestine. The natural occurrence of glycine-extended VIP in connection with our recent demonstration of its biological activity suggest a physiological role for this biosynthetic intermediate VIP form.     

Aminotransferase activity and bioinformatic analysis of 1-aminocyclopropane-1-carboxylate synthase

The mechanistic fate of pyridoxal phosphate (PLP)-dependent enzymes diverges after the quinonoid intermediate. 1-Aminocyclopropane-1-carboxylate (ACC) synthase, a member of the alpha family of PLP-dependent enzymes, is optimized to direct electrons from the quinonoid intermediate to the gamma-carbon of its substrate, S-adenosyl-L-methionine (SAM), to yield ACC and 5'-methylthioadenosine. The data presented show that this quinonoid may also accept a proton at C(4)' of the cofactor to yield alpha-keto acids and the pyridoxamine phosphate (PMP) form of the enzyme when other amino acids are presented as alternative substrates. Addition of excess pyruvate converts the PMP form of the enzyme back to the PLP form. C(alpha)-deprotonation from L-Ala is shown by NMR-monitored solvent exchange to be reversible with a rate that is less than 25-fold slower than that of deprotonation of SAM. The rate-determining step for transamination follows the formation of the quinonoid intermediate. The rate-determining step for alpha, gamma-elimination from enzyme-bound SAM is likewise shown to occur after C(alpha)-deprotonation, and the quinonoid intermediate accumulates during this reaction. BLAST searches, sequence alignments, and structural comparisons indicate that ACC synthases are evolutionarily related to the aminotransferases. In agreement with previously published reports, an absence of homology was found between the alpha and beta families of the PLP-dependent enzyme superfamily.     

Multiple pathways of the reaction of 2,2-diphenyl-1-picrylhydrazyl radical (DPPH·) with (+)-catechin: evidence for the formation of a covalent adduct between DPPH· and the oxidized form of the polyphenol

The pathways of the reaction of 2,2-diphenyl picrylhydrazyl radicals (DPPH·) with (+)-catechin were studied in alcoholic solvents. The reaction mixtures were analysed by using reversed-phase liquid chromatography (HPLC) and electrospray ionization mass spectrometry (ESI-MS). The intermediate o-quinone of catechin, yellow dimers, trimers and, interestingly, an adduct of the oxidized form of catechin with DPPH radicals were identified. The mass of this adduct was 681 Da, suggesting that one molecule of the DPPH radical complexes with the oxidized form of catechin. It is concluded that once the intermediate o-quinone is formed, the reaction proceeds in two pathways, either the o-quinone reacts with catechin to form a hydrophilic dimer (type B), which is further oxidized to hydrophobic dimers (type A) and consequently to oligomers of higher molecular weights; or the A-ring of the o-quinone is further oxidized by a DPPH radical and that this oxidized intermediate then reacts with another DPPH radical to form the observed adduct. The identification of the latter mechanism could explain the contradictory results reported in the literature for the reaction of polyphenols with DPPH radicals.