Formation Of Inactive Complex Research Articles

Protocellular Heme and Iron-Sulfur Clusters.

ConspectusCentral to the quest of understanding the emergence of life is to uncover the role of metals, particularly iron, in shaping prebiotic chemistry. Iron, as the most abundant of the accessible transition metals on the prebiotic Earth, played a pivotal role in early biochemical processes and continues to be indispensable to modern biology. Here, we discuss our recent contributions to probing the plausibility of prebiotic complexes with iron, including heme and iron-sulfur clusters, in mediating chemistry beneficial to a protocell. Laboratory experiments and spectroscopic findings suggest plausible pathways, often facilitated by UV light, for the synthesis of heme and iron-sulfur clusters. Once formed, heme displays catalytic, peroxidase-like activity when complexed with amphiphiles. This activity could have been beneficial in two ways. First, heme could have catalytically removed a molecule (H2O2) that could have had degradative effects on a protocell. Second, heme could have helped in the synthesis of the building blocks of life by coupling the reduction of H2O2 with the oxidation of organic substrates. The necessity of amphiphiles to avoid the formation of inactive complexes of heme is telling, as the modern-day electron transport chain possesses heme embedded within a lipid membrane. Conversely, prebiotic iron-sulfur peptides have yet to be reported to partition into lipid membranes, nor have simple iron-sulfur peptides been found to be capable of participating in the synthesis of organic molecules. Instead, iron-sulfur peptides span a wide range of reduction potentials complementary to the reduction potentials of hemes. The reduction potential of iron-sulfur peptides can be tuned by the type of iron-sulfur cluster formed, e.g., [2Fe-2S] versus [4Fe-4S], or by the substitution of ligands to the metal center. Since iron-sulfur clusters easily form upon stochastic encounters between iron ions, hydrosulfide, and small organic molecules possessing a thiolate, including peptides, the likelihood of soluble iron-sulfur clusters seems to be high. What remains challenging to determine is if iron-sulfur peptides participated in early prebiotic chemistry or were recruited later when protocellular membranes evolved that were compatible with the exploitation of electron transfer for the storage of energy as a proton gradient. This problem mirrors in some ways the difficulty in deciphering the origins of metabolism as a whole. Chemistry that resembles some facets of extant metabolism must have transpired on the prebiotic Earth, but there are few clues as to how and when such chemistry was harnessed to support a (proto)cell. Ultimately, unraveling the roles of hemes and iron-sulfur clusters in prebiotic chemistry promises to deepen our understanding of the origins of life on Earth and aids the search for life elsewhere in the universe.

Mechanism of Protease Resistance of D-Amino Acid Residue Containing Cationic Antimicrobial Heptapeptides.

Antimicrobial peptides (AMPs) have been an alternate promising class of therapeutics in combating global antibiotic resistance threat. However, the short half-life of AMPs, owing to protease degradability, is one of the major bottlenecks in its commercial success. In this study, we have developed all-D-amino acid containing small cationic peptides P4C and P5C, which are completely protease-resistant, noncytotoxic, nonhemolytic, and potent against the ESKAPE pathogens in comparison to their L analogues. MD simulations suggested marginal improvement in the peptide-binding affinity to the membrane-mimetic SDS micelle (∼ 1 kcal/mol) in response to L → D conversion, corroborating the marginal improvement in the antimicrobial activity. However, L → D chirality conversion severely compromised the peptide:protease (trypsin) binding affinity (≥10 kcal/mol). The relative distance between the scissile peptide carbonyl and the catalytic triad of the protease (H57, D102, and S195) was found to be significantly altered in the D-peptide:protease complex (inactive conformation) relative to the active L-peptide:protease complex. Thus, the poor binding affinity between D-peptides and the protease, resulting in the inactive complex formation, explained their experimentally observed proteolytic stability. This mechanistic insight might be extended to the proteolytic stability of the D-peptides in general and stimulate the rational design of protease-resistant AMPs.

Minimal RNA self-reproduction discovered from a random pool of oligomers.

The emergence of RNA self-reproduction from prebiotic components would have been crucial in developing a genetic system during the origins of life. However, all known self-reproducing RNA molecules are complex ribozymes, and how they could have arisen from abiotic materials remains unclear. Therefore, it has been proposed that the first self-reproducing RNA may have been short oligomers that assemble their components as templates. Here, we sought such minimal RNA self-reproduction in prebiotically accessible short random RNA pools that undergo spontaneous ligation and recombination. By examining enriched RNA families with common motifs, we identified a 20-nucleotide (nt) RNA variant that self-reproduces via template-directed ligation of two 10 nt oligonucleotides. The RNA oligomer contains a 2'-5' phosphodiester bond, which typically forms during prebiotically plausible RNA synthesis. This non-canonical linkage helps prevent the formation of inactive complexes between self-complementary oligomers while decreasing the ligation efficiency. The system appears to possess an autocatalytic property consistent with exponential self-reproduction despite the limitation of forming a ternary complex of the template and two substrates, similar to the behavior of a much larger ligase ribozyme. Such a minimal, ribozyme-independent RNA self-reproduction may represent the first step in the emergence of an RNA-based genetic system from primordial components. Simultaneously, our examination of random RNA pools highlights the likelihood that complex species interactions were necessary to initiate RNA reproduction.

Immune control of Mycobacterium tuberculosis is dependent on both soluble TNFRp55 and soluble TNFRp75.

Tuberculosis presents a global health challenge, and tumour necrosis factor (TNF) signalling is required for host immunity against Mycobacterium tuberculosis (Mtb). TNF receptor shedding, however, compromises effective immunity by reducing bioactive TNF through the formation of inactive complexes. In this study, we first compared the effect of total soluble TNF receptors using a transgenic p55ΔNS /p75-/- murine strain on host protection during a low-dose aerosol Mtb H37Rv challenge. We report that the presence of membrane-bound TNFRp55 alone in the absence of TNFRp75 results in superior control of a primary Mtb infection where p55ΔNS /p75-/- hyperactive dendritic cells displayed an increased capacity to induce a hyperactive Mtb-specific CD4+ T-cell response. p55ΔNS /p75-/- dendritic cells expressed a higher frequency of MHCII and increased MFIs for both CD86 and MHCII, while CD4+ T cells had higher expression of CD44 and IFN-γ. Next, the relative contributions of soluble TNFRp55 and soluble TNFRp75 to host protection against either primary Mtb infection or during reactivation of latent tuberculosis were delineated by comparing the experimental outcomes of control C57BL/6 mice to transgenic p55ΔNS /p75-/- , p55ΔNS and p75-/- mouse strains. We found that soluble TNFRp55 is redundant for immune regulation during the chronic stages of a primary Mtb infection. However, TNFRp55 together with soluble TNFRp75 has a crucial role in immune regulation of reactivation of latent tuberculosis.

POLYMERIZATION IN SOLUTION AS A WAY TO OPTIMIZE THE SYNTHESIS OF BETA-DIKETONATE MACROINITIATORS BASED ON POLYMETHYLMETHACRYLATE

The polymerization of methyl methacrylate initiated by Co (II) 5-methyl-5-hexene-2,4-dionate (MHD-Co) solution in dimethylformamide, methyl ethyl ketone and toluene has been studied. It is shown that polymerization does not take place in the solution of dimethylformamide due to the formation of inactive complexes «β-diketonate-solvent». Maximal polymerization rate has been observed in methyl ethyl ketone. The polymerization rate increases with decreasing of initiator concentration due to the MHD-Co inhibitory effect at high concentrations of chelate. The optimal concentration range of MHD-Co for polymerization initiation of methyl methacrylate in methyl ethyl ketone is 1,0-2,5×10-3 mol/l. The differences are noted in the behavior of the MHD-Co which chelate ring is conjugated with the double bond of the ligand, in comparison to cobalt 3-allylpentane-2,4-dionate where such conjugation is absent. The synthesized products are soluble in the monomers, they have a large number of metal chelate groups and can be used as macroinitiators in the preparation of branched and graft polymers. The total molecular weight of the grafted chains is much longer than the main chain.

Acute hypoxia affects P-TEFb through HDAC3 and HEXIM1-dependent mechanism to promote gene-specific transcriptional repression.

Hypoxia is associated with a variety of physiological and pathological conditions and elicits specific transcriptional responses. The elongation competence of RNA Polymerase II is regulated by the positive transcription elongation factor b (P-TEFb)-dependent phosphorylation of Ser2 residues on its C-terminal domain. Here, we report that hypoxia inhibits transcription at the level of elongation. The mechanism involves enhanced formation of inactive complex of P-TEFb with its inhibitor HEXIM1 in an HDAC3-dependent manner. Microarray transcriptome profiling of hypoxia primary response genes identified ∼79% of these genes being HEXIM1-dependent. Hypoxic repression of P-TEFb was associated with reduced acetylation of its Cdk9 and Cyclin T1 subunits. Hypoxia caused nuclear translocation and co-localization of the Cdk9 and HDAC3/N-CoR repressor complex. We demonstrated that the described mechanism is involved in hypoxic repression of the monocyte chemoattractant protein-1 (MCP-1) gene. Thus, HEXIM1 and HDAC-dependent deacetylation of Cdk9 and Cyclin T1 in response to hypoxia signalling alters the P-TEFb functional equilibrium, resulting in repression of transcription.

Deactivation of phosphorus in silicon due to implanted nitrogen

AbstractDeactivation of phosphorus due to nitrogen incorporation was quantitatively investigated. Coimplantation was performed to produce nitrogen profiles overlapping phosphorus profiles in preamorphized silicon. Nitrogen was found to retard phosphorus activation after recrystallization of the amorphous layer. When phosphorus activation was completed, a gap of dopant activation level was observed between samples implanted with and without nitrogen. The gap of the activation level increased linearly with the doses of nitrogen and phosphorus. This implies formation of inactive complexes by pairing of phosphorus and nitrogen atoms. However, the gap disappeared when the peak profile of nitrogen shifted toward the surface during additional annealing at 750 °C for long times. This indicates that the complexes were not stable and therefore dissolved with nitrogen diffusion. (© 2014 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Ability of phytoremediation for absorption of strontium and cesium from soils using Cannabis sativa

Aims: This study is aimed at assessing the effectiveness of Cannabis sativa in the absorption of cesium and strontium elements from the soil. Materials and Methods: This study was conducted in 2011, in Tehran, Iran. We employed the phytoremediation technology to refine the contamination of soil with radioactive material such as cesium and strontium. Cannabis sativa was selected because of its capability for potential radioactive absorption. It was planted in various soils with different concentrations of cesium and strontium (20 ppm, 40 ppm, 60 ppm, and 80 ppm), and after sufficient growth for about six months, it was separated into root, stem, and leaves for measuring the absorption of these elements in the main parts of the plant. The samples were measured by using the Inductively Coupled Plasma (ICP) method. Results: Strontium absorption and the main parts of the plant showed a significant relationship. The percentage of strontium absorption was 45% in the root, 40% in the stem, and the minimum absorption was found in the leaves (15%), but the corresponding figure was not significant for the cesium element. A strontium concentration of 60 ppm was possibly the maximum absorption concentration by Cannabis. Conclusion: Our findings suggest that strontium can be absorbed by Cannabis sativa, with the highest absorption by the roots, stems, and leaves. However, cesium does not reach the plant because of its single capacity and inactive complex formation.

Amisyn Regulates Exocytosis and Fusion Pore Stability by Both Syntaxin-dependent and Syntaxin-independent Mechanisms

Amisyn and tomosyn are related by the possession of a C-terminal vesicle-associated membrane protein-like domain that allows them to bind to syntaxin 1 and assemble into SNARE complexes. The formation of inactive complexes may sequester syntaxin and allow tomosyn and amisyn to act as inhibitors of exocytosis. We aimed to use adrenal chromaffin and PC12 cells to probe this possible mode of action of amisyn and tomosyn in dense core granule exocytosis. Although tomosyn is expressed by adrenal chromaffin and PC12 cells, amisyn expression could not be detected allowing examination of the effect of introduction of amisyn expression onto a neuronal-like background. Overexpression of m-tomosyn1 and expression of amisyn both inhibited Ca2+-induced exocytosis in transfected PC12 cells. Surprisingly, this inhibition was not removed when amisyn and tomosyn constructs were used in which key residues required for efficient binding to syntaxin1 were mutated. The effect of amisyn was further characterized using carbon fiber amperometry in chromaffin cells. Expression of amisyn had no effect on the basic characteristics of the amperometric spikes but reduced the number of spikes elicited. This inhibitory action on the extent of exocytosis was also seen with the amisyn mutant deficient in syntaxin1 binding. In addition, expression of amisyn resulted in an increase in the lifetime of the prespike foot, and this effect was abolished by the mutations. These results show that tomosyn and amisyn can negatively regulate exocytosis independently of syntaxin and also that amisyn can regulate the stability of the fusion pore.

The different behavior of nitrogen and phosphorus as n-type dopants in SiC

In this work, the experimentally observed differences in the behavior of nitrogen and phosphorus as n-type dopants in silicon carbide have been investigated within the framework of density functional theory. A key to the understanding of complex formation is the investigation of the dynamics of these dopant atoms. We have found essential differences in the recombination behavior of nitrogen and phosphorus atoms with vacancies. In contrast to nitrogen, which prefers to be built in on the carbon site exclusively (NC), our dynamical calculations show phosphorus to be built in on both substitutional lattice sites (PSi and PC). By this, PC-containing complexes should be considered as possible candidates for P-related electron paramagnetic resonance spectra in 6H–SiC which up to now have not been explained consistently. Furthermore, we explain why the formation of inactive complexes is likely in case of nitrogen but not in case of phosphorus.

Cerebrospinal fluid plasminogen, plasmin and protease inhibitors in multiple sclerosis

Objective: to study plasma and cerebrospinal fluid (CSF) concentrations of plasminogen, plasmin, plasmin- α 2-antiplasmin (PAP) complex and the protease inhibitory capacity in multiple sclerosis (MS) patients. Design: patients diagnosed as having MS were involved in this study. The reference subjects had lumbar punctures for clinical reasons but were exclusive of having either MS or other types of neurological disease. The identities of MS and reference samples were unknown to the researcher until laboratory results were ready. Setting: department of Neurology, Helsinki University Central Hospital, Finland. Subjects and methods: plasminogen, plasmin, α 2-antiplasmin, PAP complex, α 2-macroglobulin and α 1-antitrypsin levels were studied by microplate chromogenic assays, ELISA and zymography in 34 patients with MS and 24 reference subjects. Results: significantly lower mean levels of CSF plasminogen concentrations were observed in MS in comparison with reference subjects (MS: 0.40±0.003%, REF: 0.67±0.12%; P<0.01). Higher concentrations of PAP complex were detected in CSF of MS patients when compared with reference subjects ( P<0.05). Zymography was employed for the qualitative screening of samples for plasmin activity. Plasmin activity could not be detected in either plasma or CSF samples in MS or reference samples. There was no significant differences between CSF α 2-antiplasmin, α 2-macroglobulin and α 1-antitrypsin in MS when compared to reference subjects. There were no significant correlation amongst the CSF anti-proteolytic analytes either in MS or reference subjects. Finally, plasma plasminogen, PAP complex and inhibitor concentrations were within reference ranges in MS patients. Conclusions: Lower levels of CSF plasminogen and quantifiable amounts of PAP complex in MS CSF provide further evidence of plasminogen activation in the studied patients. We previously found highly raised tissue-type plasminogen activator activity in MS CSF. 4,5 Inability to detect plasmin activity in CSF with the highly sensitive zymography, suggests formation of inactive complexes with α 2-antiplasmin. Furthermore, MS CSF has normal serine protease inhibitory capacity when compared to reference subjects.

Expression and kinetics of fibrinolytic components in plasma and peritoneum during abdominal surgery

Summary Objective: A reduced peritoneal tissue fibrinolytic capacity is thought to be pathogenetic in intra-abdominal adhesion formation. Therefore, the aim of this study was to characterize the expression of fibrinolytic components in peritoneal tissue and peripheral blood at the beginning and end of abdominal surgery. Design: Peripheral blood and peritoneal tissue were sampled simultaneously from consecutive patients operated for non-septic causes (Group 1, n = 27) or for intra-abdominal infections (Group 2, n = 10). Setting: Surgical department, University hospital. Subjects: Thirty-seven surgical patients. Interventions: Abdominal surgery. Main outcome measures: Tissue-type plasminogen activator (t-PA), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor type-1 (PAI-1) and the inactive complex formation between t-PA and PAI-1 were assayed in plasma and peritoneal tissue extracts sampled at the beginning and end of surgery. Results: The expression of fibrinolytic components in plasma was similar in the two groups when surgery commenced. However, the intraoperative response was partly different. In Group 1, significant increases in t-PA antigen, PAI-1 antigen and t-PA/PAI complex were observed during surgery as opposed to Group 2. When surgery started, Group 2 displayed a reduced fibrinolytic capacity in peritoneal tissue compared with Group 1. In the latter, there was a rapid decline in t-PA activity, followed by an increase in uPA, PAI-1 and t-PA-PAI complex. The changes in Group 2 were similar, but attenuated. There was no correlation in concentrations of fibrinolytic parameters between peritoneal and blood samples. Conclusion: These observations provide novel insights into the early systemic and tissue response to surgery and may lead to new therapeutic strategies.

Chemoprotective properties of chlorophyllin against vinyl carbamate, p-nitrophenyl vinyl ether and their electrophilic epoxides.

Chlorophyllin (CHL), a water-soluble sodium and copper derivative of chlorophyll, has been shown to be a strong antimutagen in several test systems, but its mechanism of antimutagenic action is largely unknown. In the present study, we have found the protective properties of CHL against vinyl carbamate, p-nitrophenyl vinyl ether and their electrophilic epoxides. CHL exhibited dose-related inhibition of his+ reversion in Salmonella typhimurium TA 1535 induced by these mutagens. Formation of DNA adducts from vinyl carbamate epoxide (VCO) and 2'-(4-nitrophenoxy)oxirane (NPO) was also markedly attenuated in the presence of CHL. Oral administration of CHL prior to the topical application of each of the above carcinogens resulted in significant reduction in both incidence and multiplicity of skin tumors in mice. The effective protection by CHL against VCO and NPO suggest that its formation of inactive complexes with these carcinogens is mediated by mechanisms other than pi-pi interactions.

MDM2 Overexpression Does Not Account for Stabilization of Wild-Type p53 Protein in Non-Hodgkin's Lymphomas

p53 protein overexpression is a frequent finding in non-Hodgkin's lymphomas (NHL), being detected in over 25% of the cases. Moreover, some high-grade lymphomas and a large fraction of low-grade tumors show a pattern of scattered p53 accumulation in a limited percentage of neoplastic cells. In contrast, NHLs show a low frequency of p53 gene mutations. To investigate the molecular bases of p53 protein overexpression, a large series of NHLs was analyzed for p53 gene status. The analysis of the entire coding region of the gene (exons 2-11) and corresponding donor and acceptor splicing sites indicated that a significant proportion of p53-positive tumors overexpresses a wild-type form of p53 protein (wt-p53). To assess whether wt-p53 accumulation was related to the formation of inactive complexes with endogenous proteins, MDM2 oncogene expression and amplification were analyzed. MDM2 overexpression was detected only in one third of the wt-p53-positive cases, thus excluding that MDM2 accounts tout court for the accumulation of a normal p53 protein. However, the fact that MDM2 overexpression was detected in only the p53-positive cases and the observation that MDM2-positive cells were a subpopulation of p53-positive cells suggest a link between the two phenomena. In particular, our results indicate that the accumulation of a wt form of p53 protein could promote the overexpression of the MDM2 gene product. In addition, the prevalence of MDM2 positivity in intermediate/high-grade tumors together with the concordant expression of wt-p53 and MDM2 only in the high-grade component of a 'composite' lymphoma suggests that perturbation in the MDM2/p53 critical ratio could play a role in lymphoma progression.

Functional antagonism between the estrogen receptor and Fos in the regulation of c-fos protooncogene transcription.

Estrogen hormones induce transient transcriptional activation of c-fos during the early phases of mitogenic stimulation of target cells. This is mediated by a functional estrogen response element (ERE) that in the human c-fos gene is localized 1kb up-stream of the transcription start site. This is the first known example of transient transcriptional activation induced by a steroid hormone acting via its nuclear receptor. Starting with the hypothesis that the product of c-fos (Fos) interferes with estrogen receptor (ER) activity on this gene promoter, generating in this way a feedback inhibition mechanism responsible for the rapid transcriptional down-regulation detected in vivo, we tested the effects of Fos overexpression on ER-mediated activation of the c-fos promoter in transfected HeLa cells. Transient transfection of an ER expression vector is followed by hormone-dependent trans-activation of reporter genes comprising the c-fos ERE linked to its own promoter. Coexpression of Fos in the cell induces a significant reduction in the activity of ER on the reporter genes. Fos antagonism is effective on both transcription activation functions of the receptor molecule and is independent of the nature of the target promoter. Furthermore, under the same experimental conditions, the estrogen-receptor complex antagonizes activation of an AP-1-responsive test gene by Fos. ER mutants deprived of the DNA-binding domain are efficient inhibitors of Fos activity, indicating that reciprocal antagonism is likely to be mediated by the formation of inactive complexes between the two factors. These results reveal the existence of a functional interference between the ER and Fos for regulation of c-fos protooncogene transcription. It is the first case in which the product of an estrogen-induced growth-related gene is shown to exert a negative feedback control on ER regulation of its own promoter.

Adenine nucleotide binding at a noncatalytic site of mitochondrial F1-ATPase accelerates a Mg(2+)- and ADP-dependent inactivation during ATP hydrolysis.

The evidence is presented that the ADP- and Mg(2+)-dependent inactivation of MF1-ATPase during MgATP hydrolysis requires binding of ATP at two binding sites: one is catalytic and the second is noncatalytic. Binding of the noncatalytic ATP increases the rate of the inactive complex formation in the course of ATP hydrolysis. The rate of the enzyme inactivation during ATP hydrolysis depends on the medium Mg2+ concentration. High Mg2+ inhibits the steady-state activity of MF1-ATPase by increasing the rate of formation of inactive enzyme-ADP-Mg2+ complex, thereby shifting the equilibrium between active and inactive enzyme forms. The Mg2+ needed for MF1-ATPase inactivation binds from the medium independent from the MgATP binding at either catalytic or noncatalytic sites. The inhibitory ADP molecule arises at the MF1-ATPase catalytic site as a result of MgATP hydrolysis. Exposure of the native MF1-ATPase with bound ADP at a catalytic site to 1 mM Mg2+ prior to assay inactivates the enzymes with kinact 24 min-1. The maximal inactivation rate during ATP hydrolysis at saturating MgATP and Mg2+ does not exceed 10 min-1. The results show that the rate-limiting step of the MF1-ATPase inactivation during ATP hydrolysis with excess Mg2+ precedes binding of Mg2+ and likely is the rate of formation of enzyme with ADP bound at the catalytic site without bound P(i). This complex binds Mg2+ resulting in inactive MF1-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)

Characterization of the intermediates in the hydroformylation reaction catalyzed by platinum diphenylphosphinous acid complexes

This report describes the reactivity of compounds of the type (Ph 2 POHOPPh 2 )PtHL (1), in particular the reactions related to hydroformylation. Ligands L carrying alkenyl and alkynyl groups (Ph 2 PCH 2 CH=CH 2 , Ph 2 PCH=CH 2 , P(CH 2 CH=CH 2 ) 3 , Ph 2 POC 2 H 4 CH=CH 2 , Ph 2 POC 2 H 4 C≡CH) undergo intramolecular insertion into the adjacent platinum-hydride bond. Diphenylvinylphosphine initially gives a phosphaplatinacyclopropane, which rearranges to (Ph 2 POHOPPh 2 )PtPh 2 PCH 2 CH 2 (10), a phosphaplatinacyclobutane complex for which the crystal structure has been determined. Prolonged hydroformylation with 1 gives rise to the formation of inactive complexes. The crystal structure of one of these dimers has been determined, {(Ph 2 POHOPPh 2 )Pt} 2 (μ-PPh 2 )(μ-H)

Cathepsin D inactivates cysteine proteinase inhibitors cystatins

The formation of inactive complexes in excess molar amounts of human cathepsins H and L with their protein inhibitors human stefin A, human stefin B and chicken cystatin at pH 5.6 has been shown by measurement of enzyme activity coupled with reversephase HPLC not to involve covalent cleavage of the inhibitors. Inhibition must be the direct result of binding. On the contrary the interaction of cystatins with aspartic proteinase cathepsin D at pH 3.5 for 60 min followed by HPLC resulted in their inactivation accompanied by peptide bond cleavage at several sites, preferentially those involving hydrophobic amino acid residues. The released peptides do not inhibit papain and cathepsin L. These results explain reported elevated levels of cysteine proteinases and lead to the proposal that cathepsin D exerts an important function, through inactivation of cystatins, in the increased activities of cysteine proteinases in human diseases including muscular distrophy.

Mg 2+ is bound to glutamine synthetase extracted from bovine or ovine brain in the presence of l-Methionine- S-sulfoximine phosphate

Purified glutamine synthetase from bovine or ovine brain had no tightly bound Mn 2+. By extraction of bovine or ovine brain glutamine synthetase in the presence of l-Met- S-sulfoximine phosphate and ADP in metal ion-free water and 0.1 m KCl, only endogenously bound divalent cations were trapped on the enzyme. Enzyme complexes isolated by immunoprecipitation contained <0.05 Mn 2+ and 1.5 ± 0.2 Mg 2+ per subunit. Without inactive complex formation, the enzyme immunoprecipitated from extracts contained undetectable Mn 2+ (<0.01 eq per subunit) and 0.1–2.0 eq of Mg 2+ per subunit. Direct binding measurements showed that the purified bovine brain enzyme contained two divalent cations bound at the active site of each subunit. Thus, although either Mg 2+ or Mn 2+ supports enzyme activity in vitro, Mg 2+ rather than Mn 2+ appears to be bound to brain glutamine synthetase in vivo.

Heparin: Do We Understand Its Antithrombotic Actions?

Heparin: Do We Understand Its Antithrombotic Actions?