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D Suciu

Publications and source records attributed to D Suciu.

At least 19 recordsLinked to original sources
PubMed2000

Complexes between protein export chaperone SecB and SecA. Evidence for separate sites on SecA providing binding energy and regulatory interactions.

During localization to the periplasmic space or to the outer membrane of Escherichia coli some proteins are dependent on binding to the cytosolic chaperone SecB, which in turn is targeted to the membrane by specific interaction with SecA, a peripheral component of the translocase. Five variant forms of SecB, previously demonstrated to be defective in mediating export in vivo (Gannon, P. M., and Kumamoto, C. A. (1993) J. Biol. Chem. 268, 1590-1595; Kimsey, H. K., Dagarag, M. D., and Kumamoto, C. A. (1995) J. Biol. Chem. 270, 22831-22835) were investigated with respect to their ability to bind SecA both in solution and at the membrane translocase. We present evidence that at least two regions of SecA are involved in the formation of active complexes with SecB. The variant forms of SecB were all capable of interacting with SecA in solution to form complexes with stability similar to that of complexes between SecA and wild-type SecB. However, the variant forms were defective in interaction with a separate region of SecA, which was shown to trigger a change that was correlated to activation of the complex. The region of SecA involved in activation of the complexes was defined as the extreme carboxyl-terminal 21 aminoacyl residues.

Adenosine Triphosphatases↗
PubMed1999

Mutational alterations in the homotetrameric chaperone SecB that implicate the structure as dimer of dimers.

Variant forms of SecB with substitutions of aminoacyl residues in the region from 74 to 80 were analyzed with respect to their ability to bind a physiological ligand, precursor galactose-binding protein, and to their oligomeric states. SecBL75Q and SecBE77K are tetramers with affinity for ligand indistinguishable from that of the wild-type SecB, and thus the export defect exhibited by strains producing these variants must result from an effect on interactions between SecB and other components. SecBF74I is tetrameric but binds ligand with a lower affinity. Substitutions at positions 76, 78, and 80 cause a shift in the equilibrium so that the SecB tetramer dissociates into dimers. We conclude that the tetramer is a dimer of dimers and that the residues Cys76, Val78, and Gln80 must be involved either directly or indirectly in forming the interface between dimers. These variant species are defective in binding ligand; however, because their oligomeric state is altered no conclusion can be drawn concerning the direct role of these residues in ligand binding.

Amino Acid Substitution↗
PubMed1998

Calorimetric analyses of the interaction between SecB and its ligands.

SecB is a chaperone in Escherichia coli dedicated to export of proteins from the cytoplasm to the periplasm and outer membrane. It functions to bind and deliver precursors of exported proteins to the translocation apparatus before they fold into their native structures, thus maintaining them in a competent state for translocation across the membrane. The natural ligands of SecB are precursor proteins containing leader sequences. There are numerous reports in the literature indicating that SecB does not specifically recognize the leader peptides. However, two published investigations have concluded that the leader peptide is the recognition element (Watanabe M, Blobel G. 1989. Cell 58:685-705; Watanabe M, Blobel G. 1995. Proc Natl Acad Sci USA 92:10133-10136). In this work we use titration calorimetry to show that SecB binds two physiological ligands, which contain leader sequences, with no higher affinity than the same molecules lacking their leader sequences. Indeed, for one ligand the presence of the leader sequence reduces the affinity. Therefore, it can be concluded that the leader sequence provides no positive contribution to the binding energy.

ATP-Binding Cassette Transporters↗
PubMed1997

Catalytic efficiency of signal peptidase I of Escherichia coli is comparable to that of members of the serine protease family.

A method for estimating the activity of bacterial signal peptidase I (SPase I) was used to determine its activation energy (E[act]). Pro-OmpA-nuclease A, a hybrid secretory precursor, was purified to homogeneity under denaturing conditions and used as a substrate. This substrate was used to determine the activity of SPase I at different temperatures. The results show that the conformation of the mature domain of the substrate pro-OmpA-nuclease A has no discernible effect on the activity of SPase I. The activity data at a range of temperatures were then used to determine the activation energy using the Arrhenius equation. We have estimated E(act) to be 10.4 +/- 0.6 kcal/mol. This work indicates that SPase I is as catalytically efficient as the His-Ser-Asp family of proteases.

Bacterial Outer Membrane Proteins↗
PubMed1996

The 19-residue pro-peptide of staphylococcal nuclease has a profound secretion-enhancing ability in Escherichia coli.

Staphylococcus aureus secretes two forms of extracellular nuclease, nuclease A and nuclease B. Nuclease A, consisting of 149 residues, is a proteolytic product of nuclease B, which is a processing intermediate that has a 19-residue N-terminal pro-peptide between the signal peptide and nuclease A. It has been shown that nuclease A can be secreted by Escherichia coli by fusing it to the OmpA signal peptide. We now demonstrate that the addition of the pro-peptide between the OmpA signal peptide and nuclease A leads to a significantly enhanced secretion rate in E. coli. The processing and secretion rates of nuclease B at 37 degrees C were at least 10 times faster than those of nuclease A. Nuclease B was also secreted efficiently under conditions which blocked the secretion of nuclease A, such as secA mutations and the addition of phenethyl alcohol or sodium azide. This enhancing effect of the pro-peptide was not as striking when it was attached to beta-lactamase, indicating that the pro-peptide acts as a specific secretion enhancer for nuclease A. Equilibrium circular dichroism on purified nuclease A and nuclease B indicated that the pro-peptide itself had no significant destabilizing effect on the mature protein. The existence of similar pro-peptides in Gram-positive bacterial secretory proteins indicates that they may also serve as secretion enhancers for individual proteins.

Adenosine Triphosphatases↗
PubMed1995

Characterization of a soluble, catalytically active form of Escherichia coli leader peptidase: requirement of detergent or phospholipid for optimal activity.

Leader peptidase is a novel serine protease in Escherichia coli, which functions to cleave leader sequences from exported proteins. Its catalytic domain extends into the periplasmic space and is anchored to the membrane by two transmembrane segments located at the N-terminal end of the protein. At present, there is no information on the structure of the catalytic domain. Here, we report on the properties of a soluble form of leader peptidase (delta 2-75), and we compare its properties to those of the wild-type enzyme. We find that the truncated leader peptidase has a kcat of 3.0 S-1 and a Km of 32 microM with a pro-OmpA nuclease A substrate. In contrast to the wild-type enzyme (pI of 6.8), delta 2-75 is water-soluble and has an acidic isoelectric point of 5.6. We also show with delta 2-75 that the replacement of serine 90 and lysine 145 with alanine residues results in a 500-fold reduction in activity, providing further evidence that leader peptidase employs a catalytic serine/lysine dyad. Finally, we find that the catalysis of delta 2-75 is accelerated by the presence of the detergent Triton X-100, regardless if the substrate is pro-OmpA nuclease A or a peptide substrate. Triton X-100 is required for optimal activity of delta 2-75 at a level far below the critical micelle concentration. Moreover, we find that E. coli phospholipids stimulate the activity of delta 2-75, suggesting that phospholipids may play an important physiological role in the catalytic mechanism of leader peptidase.

Amino Acid Sequence↗
PubMed1995

Determination of Km and kcat for signal peptidase I using a full length secretory precursor, pro-OmpA-nuclease A.

An effective method for the determination of the activity of signal peptidase I (SPase I) of Escherichia coli is established using the hybrid protein pro-OmpA-nuclease A as substrate. Pro-OmpA-nuclease A, a hybrid secretory precursor was purified to homogeneity under denaturing conditions. When this protein was refolded, it could be quantitatively processed by purified SPase I. The Km of signal peptidase I was 0.0165 mM. The kcat was 8.73 s-1. The Km is 50 to 100 times lower than that obtained with peptide substrates indicating that SPase I has a significantly greater affinity for the protein substrate. The turnover number, kcat, is two to four orders of magnitude greater as well. Thus, the specificity constant, kcat/Km is six orders of magnitude greater with pro-OmpA-nuclease A than with peptide substrates. This is the first determination of kinetics of SPase I with a protein substrate.

Amino Acid Sequence↗
PubMed1995

Early re-feeding in the management of acute diarrhoea in infants of 0-1 year of age.

We compared early versus late re-feeding in the management of acute diarrhoea in the first year of life. In the study group (73 groups) breast feeding was resumed or early re-feeding was performed in non-breast-fed infants, so that the feeding regimen used prior to the onset of the disease was reached within 2-3 days. In the control group (49 patients) late re-feeding was performed so that the infants returned to their original feeding pattern after 4-6 days. there were no significant differences between the two groups in the number of stools and stool output per day, or in the duration of the disease. No weight gain or loss during the diet was noted more frequently in the late re-feeding group (67.2% versus 2.3.4%). This study confirms the favourable effect on body weight of early re-feeding in the management of acute diarrhoea.

Acute Disease↗
PubMed1991

Amsacrine-induced lesions in DNA and their modulation by novobiocin and 2,4-dinitrophenol.

The cancer chemotherapeutic agent amsacrine, 4'-(9-acridinylamino)-methanesulfon-m-anisidide (mAMSA), is thought to effect cytotoxicity by inhibiting the ATP-dependent enzyme topoisomerase II in the act of its duplex strand-passing action. Upon protein denaturation, the arrested "cleavable complex" that results gives rise to double- and single-strand breaks (dsbs and ssbs) and DNA-protein cross-links (dpcs). Simultaneous cotreatments with 2,4-dinitrophenol (DNP) or novobiocin (novo) abrogates mAMSA cytotoxicity in Chinese hamster cells (H. Utsumi et al., Cancer Res., 50:2577-2581, 1990). Pulsed-field gel electrophoresis was used to estimate dsbs, velocity sedimentation in alkaline sucrose gradients for ssbs, and alkaline elution without protease digestion for dpcs. Although cotreatment with DNP or novo modulated somewhat the yield of DNA lesions due to mAMSA, quantitatively these changes did not correlate at all with, and therefore could not account for, the reduced lethality that resulted from cotreatments. For example, DNA cotreatment markedly increased the yields of dsbs, ssbs, and dpcs, even though cell killing was appreciably reduced. Furthermore, neither DNP nor novo cotreatment affected the rate, or the completeness of, the repair of mAMSA-induced DNA damage, and neither cotreatment lowered total cellular ATP. Hence, the arresting of the cleavable complex by mAMSA, made evident by lesions in DNA, did not correlate with cytotoxicity. However, cotreatment with either DNP or novo resulted in an enhanced recovery of the mAMSA-induced inhibition of replicative DNA synthesis. Because DNP and novo (transiently) slow down DNA synthesis, it is proposed that these compounds abrogate mAMSA killing of S phase cells by reducing the disorganization of the processing of replicated DNA by topoisomerase II.

2,4-Dinitrophenol↗
PubMed1991

Reproductive death of Chinese hamster V79 cells after exposure to chemical inhibitors of DNA synthesis.

1. The results of this study have contributed to the definition of three categories of chemical inhibitors of DNA replication in mammalian cells. 2. Inhibitors of replicon cluster initiation [4-nitroquinoline-N-oxide (4-NQO), etoposide (VP-16), teniposide (VM-26), amsacrine (m-AMSA), N-methyl-N'-nitro-N-nitrozoguanidine (MNNG), cis-Pt(II)diammine dichloride (cis-PDD)], which needed similar doses to produce a slow and persistent (up to 4 hr) inhibition of DNA synthesis, followed by significant cell killing. 3. Inhibitors of DNA replication by indirect action [3-aminobenzamide [correction of 3-aminobezamide] (3-AB), cycloheximide (CHX), puromycin (PRC), bisbenzimide Hoechst No. 33258 (H-33258]), that showed reduced cytotoxic effects, and caused a slow (60 min) and reversible inhibition of DNA synthesis. 4. Inhibitors of formation and/or polymerization of deoxyribonucleotides [5-aminouracil (5-AU), bisbenzimide Hoechst No. 33342 (H-33342)], which induced a fast (20 min) and reversible suppression of DNA replication, associated with limited cell killing.

4-Nitroquinoline-1-oxide↗
PubMed1990

Topological DNA target size model.

This study presents a model that explains the difference in radiosensitivity between dividing and resting mammalian non-lymphoid tissue cells (liver, kidney, respiratory tract, muscle cells, neurons), based on the topological organization of DNA. In dividing cells, the target for radiation might be identified in replicon clusters or domains (7 X 10(8)-5.8 X 10(9) Da of DNA), in contrast with resting cells, in which the target could be limited to the size of chromatin loops or replicons (10(7)-10(8) Da). Hence, the target theory, D37(cGy) = 0.58 X 10(12)/weight of DNA in Da, indicates that the D37 dose (low-LET radiation) needed to inactivate 63% of the replicon clusters contained by the genome is around 100-850 cGy, and the D37 doses that could damage 63% of chromatin loops increase to 5800-58,000 cGy, with a value of 10,000 cGy for medium size replicons (5.8 X 10(7) Da). Accordingly, most dividing cells have D37 doses of 35 to 650 cGy, and the D37 values for the interphase death of non-lymphoid resting cells increase to several tens of Gy or more. These data are consistent with the idea that killing of dividing cells is correlated with the inactivation of most replicon clusters (about 720-6000 domains per genome), induced mainly by DNA single-strand breaks (SSBs), associated with double-strand breaks (DSBs); while the death of resting cells occurs when the majority of replicons comprised by the cell nucleus (about 72,000 chromatin loops) are damaged by radiation (SSBs, DSBs), which might prevent the process of transcription.

Cell Division↗
PubMed1990

Inhibition of DNA synthesis and cytotoxic effects of some DNA topoisomerase II and gyrase inhibitors in Chinese hamster V79 cells.

In this study, some DNA topoisomerase II and gyrase inhibitors have been identified as inhibitors of polymerization of deoxyribonucleotides [novobiocin (NVB), nalidixic acid (NDA), oxolinic acid (OXA)], or inhibitors of replicon initiation and DNA-chain elongation [etoposide (VP-16), teniposide (VM-26), 4'-(9-acridinylamino)methansulfon-m-anisidine (m-AMSA), ellipticine (ELT)]. The inhibitors of deoxyribonucleotide polymerization produced a significant (greater than 85%) suppression of [3H]thymidine incorporation into V79 cells within 20 min of treatment, followed by a rapid recovery of DNA synthesis, and reduced cell killing. In contrast, the inhibitors of replicon initiation and DNA-chain elongation needed about 60 min to induce a partial, but irreversible inhibition of DNA replication, associated with extensive cell killing.

Animals↗
PubMed1985

Nuclear volume and chromatin organization in some radiosensitive and radioresistant mammalian cells.

Nuclear dimensions in mammalian cells appear as a determining factor of chromatin organization and cellular radiosensitivity. Most radioresistant interphase cells have a nuclear volume (Vn) of 75 to 2700 micron 3, that could allow both the topological organization of chromatin as loops attached to the inner surface of the nuclear envelope and the unfolding of condensed chromatin within the topological constraints existing along the DNA molecule. In contrast, the radiosensitive small lymphocytes, with Vn values of 20 to 65 micron 3, seem to comprise significant amounts of highly condensed chromatin and dispose of an uncompleted topological organization of DNA, which may cause their incapacity to perform replication and transcription of DNA as well as the repair of radiation damage at a cell level. The indications are that radiosensitivity (1/D37) of animal cells, containing a similar quantity of DNA, should be directly proportional to 1/nuclear volume (1/Vn). However, DNA is unevenly distributed within the nuclear space, according to a partial ordering of interphase chromosomes; and it appears that radiosensitivity increases in zones of high DNA or chromatin density.

Animals↗
PubMed1985

Morphometric study of the interphase nucleus in some radiosensitive and radioresistant mammalian cells.

The radiosensitive cell populations, such as resting lymphocytes from thymus, spleen, lymph node and blood, have much smaller nuclei (Vn (nuclear volume) approximately 20 to 70 microns3) compared to radioresistant G0 cells from non-lymphoid tissues (liver, kidney, brain, heart; Vn approximately 75 to 2700 microns3). It is suggested that radiation-induced disorganization of nuclear structures and cell pycnosis (interphase death) are promoted in G0 lymphocytes because in normal physiological conditions their nuclei assume a higher degree of chromatin condensation. In contrast, dispersion of chromatin into larger nuclear volumes, such as those of most non-lymphoid G0 cells, may hinder or delay radiation-induced cell death.

Animals↗
PubMed1983

Cellular death by apoptosis in some radiosensitive and radioresistant mammalian tissues.

A number of facts suggest that chromatin autodigestion, occurring in the early phase of apoptosis, is carried out by an enzymatic system, composed of an endonuclease and a protease, which yields oligonucleosomic chromatin fragments. Though this enzymatic system appears to be present in most mammalian cell nuclei, radiation-induced apoptosis takes place, with a high frequency, only in cell populations having less well-developed nuclear matrices, such as lymphoid cells. Moreover, apoptosis seems to occur in a different manner in cells with less well-developed nuclear matrices (radiosensitive cells) compared with cells that contain dense nuclear matrices (radioresistant cells). Thus, dying lymphocytes progressively release their degraded chromatin from nuclei, without displaying the cellular budding and formation of apoptotic bodies. Nevertheless, apoptosis remains the main cause of cell death and cell depletion in irradiated lymphoid tissues. In contrast, the process of cellular budding and formation of apoptotic bodies appears to be specific for cells having well-developed nuclear matrix, such as those from small intestine and liver. However, in these tissues the frequency of apoptosis is relatively low and cannot be considered as the main cause of radiation-induced tissue involution.

Animals↗
PubMed1981

Autodigestion of chromatin in some radiosensitive and radioresistant mouse cells. Role of proteolysis and endonucleolysis.

Evidence is presented indicating that mouse thymus, spleen, kidney, lung and heart contain a protease activity with relatively high specificity for histones. It is suggested that degradation of chromatin occurring in irradiated lymphoid tissues is produced by the action of alkaline endonuclease in association with this histone protease. The autodigestion of chromatin was assessed by determining the release of soluble chromatin from cells suspended in sucrose media of low ionic strength. It was found that the protease inhibitors, phenylmethylsulphonyl fluoride and especially NaHSO3, were also capable of depressing the activity of alkaline endonuclease, the fragmentation of chromatin, and the release of soluble chromatin. The results suggest that the release of histones from irradiated lymphoid tissues cannot be considered as a determinant step in the fragmentation of DNA in chromatin.

Animals↗
PubMed1981

Protein composition of cells, nuclei and chromatin from some radiosensitive and radioresistant mouse tissues.

The radioresistant tissues, heart, liver, lung and kidney, contain 2 to 15 times more protein per cell, nucleus and chromatin compared with the radiosensitive tissues, spleen and thymus. As determined by polyacrylamide gel electrophoresis, in the nuclear fraction and chromatin of radiosensitive tissues there is a deficit of high molecular weight (Mw greater than 20 000) non-histone proteins. It is suggested that the radiosensitivity of lymphoid cells, which die of interphase death a few hours after exposure to small doses of radiation, is closely correlated with this deficit in non-histone proteins.

Animals↗