[Clinical study on early loading restoration of superhydrophilic implants].

Objective: To study the clinical effect of early loading restoration a superhydrophilic implant after 1 year, so as to provide reference evidence for clinical practice. Methods: A total of 41 patients with dental defects, including 20 males and 21 females [age (52.3±13.1) years old], were enrolled in the Department of VIP, Hangzhou Dental Hospital (Pinghai Hospital) from July 2017 to January 2019. A total of 74 superhydrophilic implants were implanted, including 27 maxillary implants and 47 mandiolar implants. All patients without bone augmentation or soft tissue transplantation, the maxilla was taken impression 4 weeks after implant implantation, with 6 weeks of loading, the mandible was taken impression 2 weeks after implant implantation, with 4 weeks of loading. The stability of the implant was measured by resonance frequency analyzer before implant implantation, impression and loading. Periapical radiograph were taken immediately after mold removal, immediately after loading and reexamination 1 year after loading, to measure and record the changes in the bone level of the mesial and distal margins of the implant. Results: No biological complications occurred in all implants before loading restoration, and the implant survival rate was 100%(74/74). In 2 cases, the implant stability quotient (ISQ) value of the implant at the mandibular site was lower than 65 at 2 weeks after surgery, and the restoration was delayed. The ISQ values of the other 72 implants at the time of implantation (75.22±4.32) were not significantly different from those at the time of modeling (75.13±4.23) (P>0.05), but the ISQ values at the moment of weight loading (76.46±3.73) were significantly higher than those at modeling (P<0.05). All the early loading implants were reviewed 1 year after early loading, and none of them were loose or fell off, and the implant survival rate was 100%(72/72). X-ray measurement and evaluation showed that after 1 year of early loading restoration, the mean marginal bone absorption of 72 implants was (0.18±0.06) mm, among which the mandibular was (0.17±0.06) mm and the maxillary was (0.19±0.06) mm, showing no statistical difference (P>0.05). After 1 year of early loading restoration, the mean marginal bone absorption of 72 implants was (0.17±0.05) mm, including (0.17±0.06) mm for mandibular and (0.16±0.05) mm for maxillary, showing no statistical difference (P>0.05). Conclusions: In the limited scope of this clinical study, it has been proved that early loading of superhydrophilic implant is a safe and feasible treatment scheme, and the bone resorption at the implant edge after long-term early loading restoration needs further follow-up study.

Parametrically Tunable Soliton-Induced Resonant Radiation by Three-Wave Mixing.

We show that a temporal soliton can induce resonant radiation by three-wave mixing nonlinearities. This constitutes a new class of resonant radiation whose spectral positions are parametrically tunable. The experimental verification is done in a periodically poled lithium niobate crystal, where a femtosecond near-IR soliton is excited and resonant radiation waves are observed exactly at the calculated soliton phase-matching wavelengths via the sum- and difference-frequency generation nonlinearities. This extends the supercontinuum bandwidth well into the mid IR to span 550-5000 nm, and the mid-IR edge is parametrically tunable over 1000 nm by changing the three-wave mixing phase-matching condition. The results are important for the bright and broadband supercontinuum generation and for the frequency comb generation in quadratic nonlinear microresonators.

Inhibitory effects of different substituted transition metal-based krebs-type sandwich structures on human hepatocellular carcinoma cells.

Two new transition metal-substituted Krebs-type sandwich structures of (H2imi)2[(BiW9O33)2{W0.5Ni0.5(H2O)}2{Na4 (H2O)14}{Ni(H2O)3}2]·17.5H2O (3) and (H2imi)2[(BiW9O33)2(WO(OH))2Na4(H2O)13(Zn(H2O)3)2]·12H2O (4) (imi = iminazole) were synthesized using the aqueous solution method, and their structures were identified using elemental analysis, infrared, and ultraviolet spectoscopy, thermogravimetry, and single crystal X-ray diffraction. Two polyoxometalates (POMs) were isomorphic with manganese (Mn2+) and cobalt (Co2+) compounds, (Himi)2[(BiW9O33)2(W(OH)2)2(Mn(H2O)3)2(Na3(H2O)14)]·16H2O (1) and (H2imi)2[(BiW9O33)2(W(OH)2)2(Co(H2O)3)2Na4 (H2O)14]·17H2O (2), which were reported in a previous study. This study continued with the determination of the inhibitory effects of four POMs on human hepatic carcinoma Hep G2 cells. Four compounds were used to compare their effects on the measurement of the sub-G1 (sub-G1 hypodiploid cell population) DNA content, the analysis of nuclear morphology, and the protein expression. The results showed that compound 3 (IC50 = 25.6 ± 1.07 µmol L-1) had a much higher anti-tumor activity than compounds 1 and 4 in Hep G2 cells. Compound 2 had the lowest anti-tumor activities in Hep G2 cells. However, compound 3 showed much higher cytotoxicity than compounds 1, 2 and 4 in QSG-7701 cells. Compound 4 had the lowest cytotoxicity of all of these compounds. These findings indicate the effective relationship between the metal components and structures, and their antitumor activities in Hep G2 cells.

Design and performance of an ultra-high vacuum scanning tunneling microscope operating at dilution refrigerator temperatures and high magnetic fields.

We describe the construction and performance of a scanning tunneling microscope capable of taking maps of the tunneling density of states with sub-atomic spatial resolution at dilution refrigerator temperatures and high (14 T) magnetic fields. The fully ultra-high vacuum system features visual access to a two-sample microscope stage at the end of a bottom-loading dilution refrigerator, which facilitates the transfer of in situ prepared tips and samples. The two-sample stage enables location of the best area of the sample under study and extends the experiment lifetime. The successful thermal anchoring of the microscope, described in detail, is confirmed through a base temperature reading of 20 mK, along with a measured electron temperature of 250 mK. Atomically resolved images, along with complementary vibration measurements, are presented to confirm the effectiveness of the vibration isolation scheme in this instrument. Finally, we demonstrate that the microscope is capable of the same level of performance as typical machines with more modest refrigeration by measuring spectroscopic maps at base temperature both at zero field and in an applied magnetic field.

Ultrafast and octave-spanning optical nonlinearities from strongly phase-mismatched quadratic interactions.

Cascaded nonlinearities have attracted much interest, but ultrafast applications have been seriously hampered by the simultaneous requirements of being near phase matching and having ultrafast femtosecond response times. Here we show that in strongly phase-mismatched nonlinear frequency conversion crystals the pump pulse can experience a large and extremely broadband self-defocusing cascaded Kerr-like nonlinearity. The large cascaded nonlinearity is ensured through interaction with the largest quadratic tensor element in the crystal, and the strong phase mismatch ensures an ultrafast nonlinear response with an octave-spanning bandwidth. We verify this experimentally by showing few-cycle soliton compression with noncritical cascaded second-harmonic generation: Energetic 47 fs infrared pulses are compressed in a just 1-mm long bulk lithium niobate crystal to 17 fs (under 4 optical cycles) with 80% efficiency, and upon further propagation an octave-spanning supercontinuum is observed. Such ultrafast cascading is expected to occur for a broad range of pump wavelengths spanning the near- and mid-IR using standard nonlinear crystals.

Optical Cherenkov radiation by cascaded nonlinear interaction: an efficient source of few-cycle energetic near- to mid-IR pulses.

When ultrafast noncritical cascaded second-harmonic generation of energetic femtosecond pulses occur in a bulk lithium niobate crystal optical Cherenkov waves are formed in the near- to mid-IR. Numerical simulations show that the few-cycle solitons radiate Cherenkov (dispersive) waves in the λ = 2.2 - 4.5 µm range when pumping at λ1 = 1.2 - 1.8 µm. The exact phase-matching point depends on the soliton wavelength, and we show that a simple longpass filter can separate the Cherenkov waves from the solitons. The Cherenkov waves are born few-cycle with an excellent Gaussian pulse shape, and the conversion efficiency is up to 25%. Thus, optical Cherenkov waves formed with cascaded nonlinearities could become an efficient source of energetic near- to mid-IR few-cycle pulses.

DomNet: protein domain boundary prediction using enhanced general regression network and new profiles.

The accurate and stable prediction of protein domain boundaries is an important avenue for the prediction of protein structure, function, evolution, and design. Recent research on protein domain boundary prediction has been mainly based on widely known machine learning techniques. In this paper, we propose a new machine learning based domain predictor namely, DomNet that can show a more accurate and stable predictive performance than the existing state-of-the-art models. The DomNet is trained using a novel compact domain profile, secondary structure, solvent accessibility information, and interdomain linker index to detect possible domain boundaries for a target sequence. The performance of the proposed model was compared to nine different machine learning models on the Benchmark_2 dataset in terms of accuracy, sensitivity, specificity, and correlation coefficient. The DomNet achieved the best performance with 71% accuracy for domain boundary identification in multidomains proteins. With the CASP7 benchmark dataset, it again demonstrated superior performance to contemporary domain boundary predictors such as DOMpro, DomPred, DomSSEA, DomCut, and DomainDiscovery.

Inhibition of the G2 DNA damage checkpoint and of protein kinases Chk1 and Chk2 by the marine sponge alkaloid debromohymenialdisine.

Cells can respond to DNA damage by activating checkpoints that delay cell cycle progression and allow time for DNA repair. Chemical inhibitors of the G(2) phase DNA damage checkpoint may be used as tools to understand better how the checkpoint is regulated and may be used to sensitize cancer cells to DNA-damaging therapies. However, few inhibitors are known. We used a cell-based assay to screen natural extracts for G(2) checkpoint inhibitors and identified debromohymenialdisine (DBH) from a marine sponge. DBH is distinct structurally from previously known G(2) checkpoint inhibitors. It inhibited the G(2) checkpoint with an IC(50) of 8 micrometer and showed moderate cytotoxicity (IC(50) = 25 micrometer) toward MCF-7 cells. DBH inhibited the checkpoint kinases Chk1 (IC(50) = 3 micrometer) and Chk2 (IC(50) = 3.5 micrometer) but not ataxia-telangiectasia mutated (ATM), ATM-Rad3-related protein, or DNA-dependent protein kinase in vitro, indicating that it blocks two major branches of the checkpoint pathway downstream of ATM. It did not cause the activation or inhibition of different signal transduction proteins, as determined by mobility shift analysis in Western blots, suggesting that it inhibits a narrow range of protein kinases in vivo.

Ataxia telangiectasia mutated (ATM) kinase and ATM and Rad3 related kinase mediate phosphorylation of Brca1 at distinct and overlapping sites. In vivo assessment using phospho-specific antibodies.

Recent studies have provided evidence that breast cancer susceptibility gene products (Brca1 and Brca2) suppress cancer, at least in part, by participating in DNA damage signaling and DNA repair. Brca1 is hyperphosphorylated in response to DNA damage and co-localizes with Rad51, a protein involved in homologous-recombination, and Nbs1.Mre11.Rad50, a complex required for both homologous-recombination and nonhomologous end joining repair of damaged DNA. Here, we report that there is a qualitative difference in the phosphorylation states of Brca1 between ionizing radiation (IR) and UV radiation. Brca1 is phosphorylated at Ser-1423 and Ser-1524 after IR and UV; however, Ser-1387 is specifically phosphorylated after IR, and Ser-1457 is predominantly phosphorylated after UV. These results suggest that different types of DNA-damaging agents might signal to Brca1 in different ways. We also provide evidence that the rapid phosphorylation of Brca1 at Ser-1423 and Ser-1524 after IR (but not after UV) is largely ataxia telangiectasia mutated (ATM) kinase-dependent. The overexpression of catalytically inactive ATM and Rad3 related (ATR) kinase inhibited the UV-induced phosphorylation of Brca1 at these sites, indicating that ATR controls Brca1 phosphorylation in vivo after the exposure of cells to UV light. Moreover, ATR associates with Brca1; ATR and Brca1 foci co-localize both in cells synchronized in S phase and after exposure of cells to DNA-damaging agents. ATR can itself phosphorylate the region of Brca1 phosphorylated by ATM (Ser-Gln cluster in the C terminus of Brca1, amino acids 1241-1530). However, there are additional uncharacterized ATR phosphorylation site(s) between residues 521 and 757 of Brca1. Taken together, our results support a model in which ATM and ATR act in parallel but somewhat overlapping pathways of DNA damage signaling but respond primarily to different types of DNA lesion.

The plant isoflavenoid genistein activates p53 and Chk2 in an ATM-dependent manner.

Genistein is an isoflavenoid that is abundant in soy beans. Genistein has been reported to have a wide range of biological activities and to play a role in the diminished incidence of breast cancer in populations that consume a soy-rich diet. Genistein was originally identified as an inhibitor of tyrosine kinases; however, it also inhibits topoisomerase II by stabilizing the covalent DNA cleavage complex, an event predicted to cause DNA damage. The topoisomerase II inhibitor etoposide acts in a similar manner. Here we show that genistein induces the up-regulation of p53 protein, phosphorylation of p53 at serine 15, activation of the sequence-specific DNA binding properties of p53, and phosphorylation of the hCds1/Chk2 protein kinase at threonine 68. Phosphorylation and activation of p53 and phosphorylation of Chk2 were not observed in ATM-deficient cells. In contrast, the topoisomerase II inhibitor etoposide induced phosphorylation of p53 and Chk2 in ATM-positive and ATM-deficient cells. In addition, genistein-treated ATM-deficient cells were significantly more susceptible to genistein-induced killing than were ATM-positive cells. Together our data suggest that ATM is required for activation of a DNA damage-induced pathway that activates p53 and Chk2 in response to genistein.

The DNA damage response: putting checkpoints in perspective.

The inability to repair DNA damage properly in mammals leads to various disorders and enhanced rates of tumour development. Organisms respond to chromosomal insults by activating a complex damage response pathway. This pathway regulates known responses such as cell-cycle arrest and apoptosis (programmed cell death), and has recently been shown to control additional processes including direct activation of DNA repair networks.

Caffeine abolishes the mammalian G(2)/M DNA damage checkpoint by inhibiting ataxia-telangiectasia-mutated kinase activity.

Recent evidence indicates that arrest of mammalian cells at the G(2)/M checkpoint involves inactivation and translocation of Cdc25C, which is mediated by phosphorylation of Cdc25C on serine 216. Data obtained with a phospho-specific antibody against serine 216 suggest that activation of the DNA damage checkpoint is accompanied by an increase in serine 216 phosphorylated Cdc25C in the nucleus after exposure of cells to gamma-radiation. Prior treatment of cells with 2 mM caffeine inhibits such a change and markedly reduces radiation-induced ataxia-telangiectasia-mutated (ATM)-dependent Chk2/Cds1 activation and phosphorylation. Chk2/Cds1 is known to localize in the nucleus and to phosphorylate Cdc25C at serine 216 in vitro. Caffeine does not inhibit Chk2/Cds1 activity directly, but rather, blocks the activation of Chk2/Cds1 by inhibiting ATM kinase activity. In vitro, ATM phosphorylates Chk2/Cds1 at threonine 68 close to the N terminus, and caffeine inhibits this phosphorylation with an IC(50) of approximately 200 microM. Using a phospho-specific antibody against threonine 68, we demonstrate that radiation-induced, ATM-dependent phosphorylation of Chk2/Cds1 at this site is caffeine-sensitive. From these results, we propose a model wherein caffeine abrogates the G(2)/M checkpoint by targeting the ATM-Chk2/Cds1 pathway; by inhibiting ATM, it prevents the serine 216 phosphorylation of Cdc25C in the nucleus. Inhibition of ATM provides a molecular explanation for the increased radiosensitivity of caffeine-treated cells.

Mammalian Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway.

In response to DNA damage and replication blocks, cells activate pathways that arrest the cell cycle and induce the transcription of genes that facilitate repair. In mammals, ATM (ataxia telangiectasia mutated) kinase together with other checkpoint kinases are important components in this response. We have cloned the rat and human homologs of Saccharomyces cerevisiae Rad 53 and Schizosaccharomyces pombe Cds1, called checkpoint kinase 2 (chk2). Complementation studies suggest that Chk2 can partially replace the function of the defective checkpoint kinase in the Cds1 deficient yeast strain. Chk2 was phosphorylated and activated in response to DNA damage in an ATM dependent manner. Its activation in response to replication blocks by hydroxyurea (HU) treatment, however, was independent of ATM. Using mass spectrometry, we found that, similar to Chk1, Chk2 can phosphorylate serine 216 in Cdc25C, a site known to be involved in negative regulation of Cdc25C. These results suggest that Chk2 is a downstream effector of the ATM-dependent DNA damage checkpoint pathway. Activation of Chk2 might not only delay mitotic entry, but also increase the capacity of cultured cells to survive after treatment with gamma-radiation or with the topoisomerase-I inhibitor topotecan.

Quantitative measurement of the catastrophe rate of dynamic microtubules.

Previous work has shown that catastrophe frequency is the predominant dynamic parameter of microtubules that changes dramatically during the cell cycle. As an alternative to videomicroscopy assays, we have developed a biochemical assay to measure directly the average catastrophe rate of a population of microtubules. In this assay, the growing plus end of the microtubules, polymerized off seeds, are labeled with a brief pulse of alpha-32P-GTP, followed by a cold GTP chase. The rate of loss of 32P label in microtubules measured by this method is equal to the catastrophe frequency at microtubule plus ends measured by videomicroscopy of individual microtubules. Addition of mitotic extract from Xenopus eggs increases the catastrophe rate of purified tubulin by almost 100-fold, while interphase extract alters the catastrophe rate by about 20-fold as compared to pure tubulin. Most of the catastrophe-promoting activities in both mitotic and interphase extracts is found in particulate fractions. High-speed centrifugation of extracts appears to eliminate the components required for increasing microtubule catastrophe, but does not eliminate the cell cycle difference in microtubule dynamics. This assay provides a new approach to quantitate microtubule catastrophe rates. It will be of particular interest to search for catastrophe factors associated with intracellular membranes or other insoluble components.

Caspase-dependent activation of cyclin-dependent kinases during Fas-induced apoptosis in Jurkat cells.

The activation of cyclin-dependent kinases (cdks) has been implicated in apoptosis induced by various stimuli. We find that the Fas-induced activation of cdc2 and cdk2 in Jurkat cells is not dependent on protein synthesis, which is shut down very early during apoptosis before caspase-3 activation. Instead, activation of these kinases seems to result from both a rapid cleavage of Wee1 (an inhibitory kinase of cdc2 and cdk2) and inactivation of anaphase-promoting complex (the specific system for cyclin degradation), in which CDC27 homolog is cleaved during apoptosis. Both Wee1 and CDC27 are shown to be substrates of the caspase-3-like protease. Although cdk activities are elevated during Fas-induced apoptosis in Jurkat cells, general activation of the mitotic processes does not occur. Our results do not support the idea that apoptosis is simply an aberrant mitosis but, instead, suggest that a subset of mitotic mechanisms plays an important role in apoptosis through elevated cdk activities.

Association of the catalytic subunit of aspartate transcarbamoylase with a zinc-containing polypeptide fragment of the regulatory chain leads to increases in thermal stability.

The regulatory enzyme aspartate transcarbamoylase (ATCase), comprising 2 catalytic (C) trimers and 3 regulatory (R) dimers, owes its stability to the manifold interchain interactions among the 12 polypeptide chains. With the availability of a recombinant 70-amino acid zinc-containing polypeptide fragment of the regulatory chain of ATCase, it has become possible to analyze directly the interaction between catalytic and regulatory chains in a complex of simpler structure independent of other interactions such as those between the 2 C trimers, which also contribute to the stability of the holoenzyme. Also, the effect of the interaction between the polypeptide, termed the zinc domain, and the C trimer on the thermal stability and other properties can be measured directly. Differential scanning microcalorimetry experiments demonstrated that the binding of the zinc domain to the C trimer leads to a complex of markedly increased thermal stability. This was shown with a series of mutant forms of the C trimer, which themselves varied greatly in their temperature of denaturation due to single amino acid replacements. With some C trimers, for which tm varied over a range of 30 degrees C due to diverse amino acid substitutions, the elevation of tm resulting from the interaction with the zinc domain was as large as 18 degrees C. The values of tm for a variety of complexes of mutant C trimers and the wild-type zinc domain were similar to those observed when the holoenzymes containing the mutant C trimers were subjected to heat denaturation.(ABSTRACT TRUNCATED AT 250 WORDS)

A 70-amino acid zinc-binding polypeptide fragment from the regulatory chain of aspartate transcarbamoylase causes marked changes in the kinetic mechanism of the catalytic trimer.

Interaction between a 70-amino acid and zinc-binding polypeptide from the regulatory chain and the catalytic (C) trimer of aspartate transcarbamoylase (ATCase) leads to dramatic changes in enzyme activity and affinity for active site ligands. The hypothesis that the complex between a C trimer and 3 polypeptide fragments (zinc domain) is an analog of R state ATCase has been examined by steady-state kinetics, heavy-atom isotope effects, and isotope trapping experiments. Inhibition by the bisubstrate ligand, N-(phosphonacetyl)-L-aspartate (PALA), or the substrate analog, succinate, at varying concentrations of substrates, aspartate, or carbamoyl phosphate indicated a compulsory ordered kinetic mechanism with carbamoyl phosphate binding prior to aspartate. In contrast, inhibition studies on C trimer were consistent with a preferred order mechanism. Similarly, 13C kinetic isotope effects in carbamoyl phosphate at infinite aspartate indicated a partially random kinetic mechanism for C trimer, whereas results for the complex of C trimer and zinc domain were consistent with a compulsory ordered mechanism of substrate binding. The dependence of isotope effect on aspartate concentration observed for the Zn domain-C trimer complex was similar to that obtained earlier for intact ATCase. Isotope trapping experiments showed that the compulsory ordered mechanism for the complex was attributable to increased "stickiness" of carbamoyl phosphate to the Zn domain-C trimer complex as compared to C trimer alone. The rate of dissociation of carbamoyl phosphate from the Zn domain-C trimer complex was about 10(-2) that from C trimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Peptide-protein interaction markedly alters the functional properties of the catalytic subunit of aspartate transcarbamoylase.

Interaction of a 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase (ATCase) with the catalytic (C) subunit leads to dramatic changes in enzyme activity and affinity for ligand binding at the active sites. The complex between the polypeptide (zinc domain) and wild-type C trimer exhibits hyperbolic kinetics in contrast to the sigmoidal kinetics observed with the intact holoenzyme. Moreover, the Scatchard plot for binding N-(phosphonacetyl)-L-aspartate (PALA) to the complex is linear with a Kd corresponding to that evaluated for the holoenzyme converted to the relaxed (R) state. Additional evidence that the binding of the zinc domain to the C trimer converts it to the R state was attained with a mutant form of ATCase in which Lys 164 in the catalytic chain is replaced by Glu. As shown previously (Newell, J.O. & Schachman, H.K., 1990, Biophys. Chem. 37, 183-196), this mutant holoenzyme, which exists in the R conformation even in the absence of active site ligands, has a 50-fold greater affinity for PALA than the free C subunit. Adding the zinc domain to the C trimer containing the Lys 164-->Glu substitution leads to a 50-fold enhancement in the affinity for the bisubstrate analog yielding a value of Kd equal to that for the holoenzyme. A different mutant ATCase containing the Gln 231 to Ile replacement was shown (Peterson, C.B., Burman, D.L., & Schachman, H.K., 1992, Biochemistry 31, 8508-8515) to be much less active as a holoenzyme than as the free C trimer. For this mutant holoenzyme, the addition of substrates does not cause its conversion to the R state. However, the addition of the zinc domain to the Gln 231-->Ile C trimer leads to a marked increase in enzyme activity, and PALA binding data indicate that the complex resembles the R state of the holoenzyme. This interaction leading to a more active conformation serves as a model of intergenic complementation in which peptide binding to a protein causes a conformational correction at a site remote from the interacting surfaces resulting in activation of the protein. This linkage was also demonstrated by difference spectroscopy using a chromophore covalently bound at the active site, which served as a spectral probe for a local conformational change. The binding of ligands at the active sites was shown also to lead to a strengthening of the interaction between the zinc domain and the C trimer.

A 70-amino acid zinc-binding polypeptide from the regulatory chain of aspartate transcarbamoylase forms a stable complex with the catalytic subunit leading to markedly altered enzyme activity.

In an effort to clarify effects of specific protein-protein interactions on the properties of the dodecameric enzyme aspartate transcarbamoylase (carbamoyl-phosphate:L-aspartate carbamoyltransferase, EC 2.1.3.2), we initiated studies of a simpler complex containing an intact catalytic trimer and three copies of a fragment from the regulatory chain. The partial regulatory chain was expressed as a soluble 9-kDa zinc-binding polypeptide comprising 11 amino acids encoded by the polylinker of pUC18 fused to the amino terminus of residues 84-153 of the regulatory chain; this polypeptide includes the zinc domain detected in crystallographic studies of the holoenzyme. In contrast to intact regulatory chains, the zinc-binding polypeptide is monomeric in solution because it lacks the second domain responsible for dimer formation and assembly of the dodecameric holoenzyme. The isolated 9-kDa protein forms a tight, zinc-dependent complex with catalytic trimer, as shown by the large shift in electrophoretic mobility of the trimer in nondenaturing polyacrylamide gels. Enzyme assays of the complex showed a hyperbolic dependence of initial velocity on aspartate concentration with Vmax and Km for aspartate approximately 50% lower than the values for free catalytic subunit. A mutant catalytic subunit containing the Lys-164----Glu substitution exhibited a striking increase in enzyme activity at low aspartate concentrations upon interaction with the zinc domain because of a large reduction in Km upon complex formation. These changes in functional properties indicate that the complex of the zinc domain and catalytic trimer is an analog of the high-affinity R ("relaxed") state of aspartate transcarbamoylase, as proposed previously for a transiently formed assembly intermediate composed of one catalytic and three regulatory subunits. Conformational changes at the active sites, resulting from binding the zinc-containing polypeptide chains, were detected by difference spectroscopy with trinitrophenylated catalytic trimers. Isolation of the zinc domain of aspartate transcarbamoylase provides a model protein for study of oligomer assembly, communication between dissimilar polypeptides, and metal-binding motifs in proteins.

Mechanism of adenylate kinase. Critical evaluation of the X-ray model and assignment of the AMP site.

The substrate binding sites of adenylate kinase (AK) proposed by X-ray crystallographic studies [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45, and subsequent revisions] were evaluated by site-specific mutagenesis in conjunction with structural analysis by NMR. The residues examined in this report include two near an adenosine site (threonine-39 and arginine-44) and two in the phosphate binding region (arginine-128 and arginine-149). The results and conclusions are summarized as follows: (a) Although Thr-39 is very close to an adenine site [Egner, U., Tomasselli, A. G., & Schulz, G. E. (1987) J. Mol. Biol. 195, 649-658], it is nonessential either structurally or functionally. (b) The R44M mutant enzyme showed significant increases in the Michaelis and dissociation constants of adenosine 5'-monophosphate (AMP) (36- and 22-fold, respectively) while all other kinetic parameters were relatively unperturbed. The proton NMR property of this mutant was unchanged in the free enzyme and only slightly perturbed in the binary complexes with AMP and with MgATP (adenosine 5'-triphosphate), and in the ternary complex with MgAP5A [P1,P5-bis(5'-adenosyl) pentaphosphate]. These results indicate that Arg-44 interacts specifically with AMP starting at the binary complex, and suggest that the MgATP site proposed by Pai et al. (1977) is likely to be the AMP site. (c) The kinetic parameters of R149M were dramatically perturbed: kcat decreased by a factor of 1540, Km increased to 130-fold, and kcat/Km decreased by a factor of 2 X 10(5).(ABSTRACT TRUNCATED AT 250 WORDS)