A lectin recognizes differential arrangements of O-glycans on mucin repeats

Interaction of Vicia villosa agglutinin-B4 (VVA-B4) to glycopeptides with O-linked GalNAc residues was investigated by surface plasmon resonance. The affinity was shown to be influenced by the arrangement of O-glycosylation sites on a peptide, PTTTPITTTTK, representing the tandem repeat of MUC2. The association rate constant was relatively high with a particular category of GalNAc-peptides in which more than three amino acid residues were placed between GalNAc-Thr residues. PTT^∗T^∗PITT^∗T^∗TK (T^∗ indicates GalNAc-Thr) had the highest association rate constant among the glycopeptides tested. The dissociation rate constant was low in the peptides containing consecutive GalNAc residues and PT^∗TTPIT^∗T^∗T^∗TK was the lowest of the glycopeptides tested. Dissociation constant (K_D), calculated as k_d/k_a was the lowest with PTT^∗T^∗PITT^∗T^∗TK. Therefore, the arrangement but not the quantity of GalNAc residues apparently determines the affinity between VVA-B4 and peptides with attached GalNAc residues.     

Studies on the Behavior of 14C-chlorpyrifos in a Model Rice Ecosystem

Fate of ¹⁴C-chlorpyrifos was studied in a model rice ecosystem. The level of ¹⁴C-residues in floodwater showed initially a rapid decline in first 10 days. These residues were observed till 30 days. The insecticide residues in soil did not show any appreciable build-up, thereby indicating that the residue levels of this insecticide may not be significant. Extractable residues were formed up to 10–13% of the applied ¹⁴C-activity during the period of 136 days, while the bound fraction of ¹⁴C-residues reached a maximum of 2.9% after 92 days. Algae and rice plants showed ¹⁴C-residues to the extent of only 0.01% of the applied ¹⁴C-activity. Rice grains did not show any residues at all. These results indicate that chlorpyrifos undergoes considerable degradation in rice soils and does not leave residues, which may be of environmental concern.     

A new polymer of 8,9-Di-O-Glucosylated 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid from a cell wall of Brevibacterium casei ACM Ac-2114T

A polysaccharide containing the residues of 2-keto-3-deoxy-D-glycero-D-galacto-nonulosonic acid (Kdn) was found in the cell wall of the Brevibacterium casei strain ACM Ac-2114^T. The polymer structure was elucidated by analyzing one-dimensional spectra of ¹H and ¹³C NMR and bidimentional experiments ¹H/¹H-COSY, TOCSY, ¹H/¹³C-gHSQC, and ¹H/¹³C-gHMBC. The polymer is built up of the 2 → 4-linked Kdn residues substituted by β-D-Glcp residues at 8- and 9-hydroxyls; such a polymer with disubstituted Kdn residues was found for the first time. A glycosylated teichoic acid of the 1,3-poly(glycerolphosphate) type was also identified among other anionic polymers of cell wall.     

Structural and Functional Analysis of Transmembrane Segment VI of the NHE1 Isoform of the Na+/H+ Exchanger

The Na⁺/H⁺ exchanger isoform 1 is a ubiquitously expressed integral membrane protein. It resides on the plasma membrane of cells and regulates intracellular pH in mammals by extruding an intracellular H⁺ in exchange for one extracellular Na⁺. We characterized structural and functional aspects of the transmembrane segment (TM) VI (residues 227–249) by using cysteine scanning mutagenesis and high resolution NMR. Each residue of TM VI was mutated to cysteine in the background of the cysteineless NHE1 protein, and the sensitivity to water-soluble sulfhydryl-reactive compounds (2-(trimethylammonium)ethyl)methanethiosulfonate (MTSET) and (2-sulfonatoethyl)methanethiosulfonate (MTSES) was determined for those residues with significant activity remaining. Three residues were essentially inactive when mutated to Cys: Asp²³⁸, Pro²³⁹, and Glu²⁴⁷. Of the remaining residues, proteins with the mutations N227C, I233C, and L243C were strongly inhibited by MTSET, whereas amino acids Phe²³⁰, Gly²³¹, Ala²³⁶, Val²³⁷, Ala²⁴⁴, Val²⁴⁵, and Glu²⁴⁸ were partially inhibited by MTSET. MTSES did not affect the activity of the mutant NHE1 proteins. The structure of a peptide representing TM VI was determined using high resolution NMR spectroscopy in dodecylphosphocholine micelles. TM VI contains two helical regions oriented at an approximate right angle to each other (residues 229–236 and 239–250) surrounding a central unwound region. This structure bears a resemblance to TM IV of the Escherichia coli protein NhaA. The results demonstrate that TM VI of NHE1 is a discontinuous pore-lining helix with residues Asn²²⁷, Ile²³³, and Leu²⁴³ lining the translocation pore.     

A comparison of direct and indirect 15N isotope techniques for estimating crop N uptake from organic residues

Experiments were carried out to compare the direct approach for estimating crop N uptake from ¹⁵N labelled organic inputs, to two indirect approaches, ¹⁵N isotope dilution and A value. In the first experiment soils received 25, 50, 75, or 100 mg N kg soil⁻¹ in the form of Casuarina equisitifolia residues in addition to ammonium sulphate fertiliser, to give a total of 100 mg N kg soil⁻¹ added. This was a cross labelling design, thus two matching sets of treatments, were set up, identical in all but the position of the ¹⁵N label. Maize (Zea mays L.) plants were grown in the soils amended with residues for 11 weeks and N derived from residues (Ndfr) estimated using the A-value or the direct approach. The A-value approach appeared to significantly overestimate %Ndfr compared to the direct method. In the second experiment contrasting residues were added to soil, fababean (Vicia faba L. var. minor), alfalfa (Medicago sativa L.), soyabean fixing, (Glycine max (L.) Merrill), soyabean non-fixing, barley (Hordeum vulgare L.) and maize. This was also cross-labelling design, labelled and unlabelled residues were used. Maize plants were grown in these soils for 11 weeks and %Ndfr in the maize plants estimated using ¹⁵ N isotope dilution and the direct approach. The ¹⁵ N isotope dilution approach also overestimated %Ndfr compared to the direct method in this experiment. Pool substitution appeared to be responsible for the discrepancy between the direct and indirect techniques. It was concluded that ¹⁵N isotope dilution and A-value approaches as used in these experiments (i.e where residues and ¹⁵N label are added simultaneously) were not appropriate techniques for estimating N derived from organic residues in soils. This revised version was published online in June 2006 with corrections to the Cover Date.     

Role of strong anchor residues in the effective binding of 10-mer and 11-mer peptides to HLA-A*2402 molecules

The binding capacity of one-hundred-and-seventy-two 8-mer to 11-mer peptides carrying HLA-A24 anchor residues to HLA-A^*2402 molecules was analyzed by using a HLA class I stabilization assay. Most (76.2%) of these peptides bound to HLA-A^*2402 molecules. These results confirmed previous findings that Tyr and Phe at P2 as well as Phe, Trp, Ile, and Leu at the C-terminus were main anchor residues for HLA-A^*2402. Tyr at P2 was a stronger anchor residue than Phe, while bulky aromatic hydrophobic residues Phe and Trp at the C-terminus are stronger anchors than aliphatic hydrophobic residues Ile and Leu. These results were also supported by an analysis using a panel of mutated 9-mer peptides at P2 and P9. Taken together, these results suggest that HLA-A^*2402 molecules have deep B- and F-pockets because they favor peptides carrying bulky aromatic hydrophobic residues at P2 and the C-terminus. The affinity of 8-mer peptides was significantly lower than that of 9-mer to 11-mer peptides, while there was no difference in affinity between 9-mer, 10-mer, and 11-mer peptides. The affinity of peptides carrying bulky aromatic hydrophobic residues at the C-terminus was higher than that of peptides carrying aliphatic hydrophobic residues in each of the 8-mer to 11-mer peptides, though the greatest difference in affinity was observed in 11-mer peptides. The strong interaction of side chains of these anchor residues with the corresponding pockets may permit the effective binding of 10-mer and 11-mer peptides to HLA-A^*2402 molecules.     

Identification of Important Amino Acid Residues of the Na+-Ca2+ Exchanger Inhibitory Peptide,XIP

The Na⁺-Ca²⁺ exchanger plays an important role in cardiac contractility by moving Ca²⁺ across the plasma membrane during excitation-contraction coupling. A 20 amino acid peptide, XIP, synthesized to mimic a region of the exchanger, inhibits exchange activity. We identify here amino acid residues important for inhibitory function. Effects of modified peptides on Na⁺-Ca²⁺ exchange activity were determined. Exchange activity was assessed as ⁴⁵Ca²⁺ uptake into Na⁺-loaded cardiac sarcolemmal vesicles. We find that the entire length of XIP is important for maximal potency, though the major inhibitory components are between residues 5 and 16. Basic and aromatic residues are most important for the inhibitory function of XIP. Substitutions of arginine 12 and arginine 14 with alanine or glutamine dramatically decrease the potency of XIP, suggesting that these residues play a key role in possible charge-charge interactions. Substitutions of other basic residues with alanines or glutamines had less effect on the potency of XIP. All aromatic residues participate in binding with the exchanger, probably via hydrophobic interactions as indicated by tryptophan fluorescence. A tyrosine is required at position 6 for maximal inhibition and phenylalanine 5 and tyrosine 8 can only be replaced by other aromatic residues. Tyrosine 10 and tyrosine 13 can be replaced with other bulky residues. A specific conformation of XIP, with structural constrains provided by all parts of the molecule, is required for optimal inhibitory function. Received: 19 September 1996/Revised: 20 November 1996     

Heat- and alkali-induced deamination of 5-methylcytosine and cytosine residues in DNA

5-Methylcytosine residues in DNA underwent deamination at high temperatures. Furthemore, their rate of deamination at neutral or alkaline pH was greater than that of cytosine residues in DNA. As sources of [¹⁴C]5-methylcytosine-containing DNA, we used bacteriophage XP-12 DNA, in which 5-methylcytosine residues completely replace C residues, and calf thymus DNA experimentally substituted with [¹⁴C]5-methylcytosine residues. Upon incubation at 95°C in a physiological buffer or at 60°C in 1 M NaOH, the respective rates of deamination of 5-methylcytosine residues were about 3- and 1.5-times those of cytosine residues. Under the same conditions, the free 5-methyldeoxycytidine was converted to thymidine more rapidly than deoxycytidine was converted to deoxyuridine. The reactions at physiological pH and elevated temperature suggest that deamination of 5-methylcytosine residues may yield a significant portion of spontaneous mutations in vivo, especially in view of the lack of thymine-specific mismatch repair systems with specificity and efficiency comparable to that of uracil excision repair systems.     

Functional Differences of Invariant and Highly Conserved Residues in the Extracellular Domain of the Glycoprotein Hormone Receptors

Multiple interactions exist between human follicle-stimulating hormone (FSH) and the N-terminal hormone-binding fragment of the human FSH receptor (FSHR) extracellular domain (ECD). Binding of the other human glycoprotein hormones to their cognate human receptors (luteinizing hormone receptor (LHR) and thyroid-stimulating hormone receptor (TSHR)) was expected to be similar. This study focuses on amino acid residues in β-strands 2 (Lys⁷⁴), 4 (Tyr¹²⁴, Asn¹²⁹, and Thr¹³⁰), and 5 (Asp¹⁵⁰ and Asp¹⁵³) of the FSHR ECD identified in the human FSH·FSHR ECD crystal structure as contact sites with the common glycoprotein hormone α-subunit, and on noncontact residues in β-strands 2 (Ser⁷⁸) and 8 (Asp²²⁴ and Ser²²⁶) as controls. These nine residues are either invariant or highly conserved in LHR and TSHR. Mutagenesis and functional characterization of these residues in all three human receptors allowed an assessment of their contribution to binding and receptor activation. Surprisingly, the six reported α-subunit contact residues of the FSHR ECD could be replaced without significant loss of FSH binding, while cAMP signaling potency was diminished significantly with several replacements. Comparative studies of the homologous residues in LHR and TSHR revealed both similarities and differences. The results for FSH/FSHR were analyzed on the basis of the crystal structure of the FSH·FSHR ECD complex, and comparative modeling was used to generate structures for domains, proteins, and complexes for which no structures were available. Although structural information of hormone-receptor interaction allowed the identification of hormone-receptor contact sites, functional analysis of each contact site was necessary to assess its contribution to hormone binding and receptor activation.     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ^? cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon.     

Effects of the Protonation State of the EEEE Motif of a Bacterial Na-channel on Conduction and Pore Structure

A distinctive feature of prokaryotic Na⁺-channels is the presence of four glutamate residues in their selectivity filter. In this study, how the structure of the selectivity filter, and the free-energy profile of permeating Na⁺ ions are altered by the protonation state of Glu177 are analyzed. It was found that protonation of a single glutamate residue was enough to modify the conformation of the selectivity filter and its conduction properties. Molecular dynamics simulations revealed that Glu177 residues may adopt two conformations, with the side chain directed toward the extracellular entrance of the channel or the intracellular cavity. The likelihood of the inwardly directed arrangement increases when Glu177 residues are protonated. The presence of one glutamate residue with its chain directed toward the intracellular cavity increases the energy barrier for translocation of Na⁺ ions. These higher-energy barriers preclude Na⁺ ions to permeate the selectivity filter of prokaryotic Na⁺-channels when one or more Glu177 residues are protonated.     

Analysis and prediction of calcium-binding pockets from apo-protein structures exhibiting calcium-induced localized conformational changes

Calcium binding in proteins exhibits a wide range of polygonal geometries that relate directly to an equally diverse set of biological functions. The binding process stabilizes protein structures and typically results in local conformational change and/or global restructuring of the backbone. Previously, we established the MUG program, which utilized multiple geometries in the Ca²⁺‐binding pockets of holoproteins to identify such pockets, ignoring possible Ca²⁺‐induced conformational change. In this article, we first report our progress in the analysis of Ca²⁺‐induced conformational changes followed by improved prediction of Ca²⁺‐binding sites in the large group of Ca²⁺‐binding proteins that exhibit only localized conformational changes. The MUG^SR algorithm was devised to incorporate side chain torsional rotation as a predictor. The output from MUG^SR presents groups of residues where each group, typically containing two to five residues, is a potential binding pocket. MUG^SR was applied to both X‐ray apo structures and NMR holo structures, which did not use calcium distance constraints in structure calculations. Predicted pockets were validated by comparison with homologous holo structures. Defining a “correct hit” as a group of residues containing at least two true ligand residues, the sensitivity was at least 90%; whereas for a “correct hit” defined as a group of residues containing at least three true ligand residues, the sensitivity was at least 78%. These data suggest that Ca²⁺‐binding pockets are at least partially prepositioned to chelate the ion in the apo form of the protein.     

Effects of the Protonation State of the EEEE Motif of a Bacterial Na+-channel on Conduction and Pore Structure

A distinctive feature of prokaryotic Na⁺-channels is the presence of four glutamate residues in their selectivity filter. In this study, how the structure of the selectivity filter, and the free-energy profile of permeating Na⁺ ions are altered by the protonation state of Glu177 are analyzed. It was found that protonation of a single glutamate residue was enough to modify the conformation of the selectivity filter and its conduction properties. Molecular dynamics simulations revealed that Glu177 residues may adopt two conformations, with the side chain directed toward the extracellular entrance of the channel or the intracellular cavity. The likelihood of the inwardly directed arrangement increases when Glu177 residues are protonated. The presence of one glutamate residue with its chain directed toward the intracellular cavity increases the energy barrier for translocation of Na⁺ ions. These higher-energy barriers preclude Na⁺ ions to permeate the selectivity filter of prokaryotic Na⁺-channels when one or more Glu177 residues are protonated.     

Ability of growth hormone fragments to compete with 125I-iodinated human growth hormone for specific binding to isolated adipocytes of hypophysectomized rats

Several noncovalent complexes of large fragments of human GH, which are less active than native human GH in stimulating glucose metabolism in adipose tissue of hypophysectomized rats, were tested for their ability to compete with ¹²⁵I-iodinated human GH for specific binding to isolated adipocytes of hypophysectomized rats. The complexes tested were A (residues 1–134 + residues 141–191; S-carbamidomethylated), B (residues 1–134 + residues 135–191; S-carbamidomethylated) and C (residues 1–134 + residues 135–191; S-carboxymethylated). When compared to native human GH, the complexes were less active in competing with ¹²⁵I-iodinated human GH for specific binding to adipocytes, and their order of potency in the binding assay (A > B > C) was similar to that of their respective activities in stimulating glucose metabolism in isolated adipose tissue of hypophysectomized rats.     

Conformational Activation of Antithrombin by Heparin Involves an Altered Exosite Interaction with Protease

Heparin allosterically activates antithrombin as an inhibitor of factors Xa and IXa by enhancing the initial Michaelis complex interaction of inhibitor with protease through exosites. Here, we investigate the mechanism of this enhancement by analyzing the effects of alanine mutations of six putative antithrombin exosite residues and three complementary protease exosite residues on antithrombin reactivity with these proteases in unactivated and heparin-activated states. Mutations of antithrombin Tyr²⁵³ and His³¹⁹ exosite residues produced massive 10–200-fold losses in reactivity with factors Xa and IXa in both unactivated and heparin-activated states, indicating that these residues made critical attractive interactions with protease independent of heparin activation. By contrast, mutations of Asn²³³, Arg²³⁵, Glu²³⁷, and Glu²⁵⁵ exosite residues showed that these residues made both repulsive and attractive interactions with protease that depended on the activation state and whether the critical Tyr²⁵³/His³¹⁹ residues were mutated. Mutation of factor Xa Arg¹⁴³, Lys¹⁴⁸, and Arg¹⁵⁰ residues that interact with the exosite in the x-ray structure of the Michaelis complex confirmed the importance of all residues for heparin-activated antithrombin reactivity and Arg¹⁵⁰ for native serpin reactivity. These results demonstrate that the exosite is a key determinant of antithrombin reactivity with factors Xa and IXa in the native as well as the heparin-activated state and support a new model of allosteric activation we recently proposed in which a balance between attractive and repulsive exosite interactions in the native state is shifted to favor the attractive interactions in the activated state through core conformational changes induced by heparin binding.     

Nitrogen cycling in leucaena (Leucaena leucocephala) alley cropping in semi-arid tropics

In an alley cropping system, prunings from the hedgerow legume are expected to supply nitrogen (N) to the associated cereal. However, this may not be sufficient to achieve maximum crop yield. Three field experiments with alley-cropped maize were conducted in a semi-arid environment in northern Australia to determine: (1) the effect of N fertilizer on maize growth in the presence of fresh leucaena prunings; (2) the effect of incorporation of leucaena and maize residues on maize yield and the fate of plant residue¹⁵N in the alley cropping system; and (3) the¹⁵N recovery by maize from¹⁵N-labelled leucaena, maize residues and ammonium sulphate fertilizer.Leucaena residues increased maize crop yield and N uptake although they did not entirely satisfy the N requirement of the alley crop. Additional N fertilizer further increased the maize yield and N uptake in the presence of leucaena residues. Placement of leucaena residues had little effect on the availability of N to maize plants over a 2 month period. The incorporation of leucaena residues in the soil did not increase the recovery of leucaena¹⁵N by maize compared with placement of the residues on the soil surface. After 2 months, similar proportions of the residue¹⁵N were recovered by maize from mulched leucaena (6.3%), incorporated leucaena (6.1%) and incorporated maize (7.6%). By the end of one cropping season (3 months after application) about 9% of the added¹⁵N was taken up by maize from either¹⁵N-labelled leucaena as mulch or¹⁵N-labelled maize residues applied together with unlabelled fresh leucaena prunings as mulch. The recovery of the added¹⁵N was much higher (42.7%) from the¹⁵N-labelled ammonium sulphate fertilizer at 40 kg N ha^-1 in the presence of unlabelled leucaena prunings. Most of the added¹⁵N recovered in the 200 cm soil profile was distributed in the top 25 cm soil with little leached below that. About 27–41% of the leucaena¹⁵N was apparently lost, largely through denitrification from the soil and plant system, in one cropping season. This compared with 35% of the fertilizer¹⁵N lost when the N fertilizer was applied in the presence of prunings. ei]H Lambers     

GAP45 Phosphorylation Controls Assembly of the Toxoplasma Myosin XIV Complex

Toxoplasma gondii motility is powered by the myosin XIV motor complex, which consists of the myosin XIV heavy chain (MyoA), the myosin light chain (MLC1), GAP45, and GAP50, the membrane anchor of the complex. MyoA, MLC1, and GAP45 are initially assembled into a soluble complex, which then associates with GAP50, an integral membrane protein of the parasite inner membrane complex. While all proteins in the myosin XIV motor complex are essential for parasite survival, the specific role of GAP45 remains unclear. We demonstrate here that final assembly of the motor complex is controlled by phosphorylation of GAP45. This protein is phosphorylated on multiple residues, and by using mass spectroscopy, we have identified two of these, Ser¹⁶³ and Ser¹⁶⁷. The importance of these phosphorylation events was determined by mutation of Ser¹⁶³ and Ser¹⁶⁷ to Glu and Ala residues to mimic phosphorylated and nonphosphorylated residues, respectively. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to either Ala or Glu residues does not affect targeting of GAP45 to the inner membrane complex or its association with MyoA and MLC1. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to Ala residues also does not affect assembly of the mutant GAP45 protein into the myosin motor complex. Mutation of Ser¹⁶³ and Ser¹⁶⁷ to Glu residues, however, prevents association of the MyoA-MLC1-GAP45 complex with GAP50. These observations indicate that phosphorylation of Ser¹⁶³ and Ser¹⁶⁷ in GAP45 controls the final step in assembly of the myosin XIV motor complex.     

Functional dissection of a cell-division inhibitor, SulA, of Escherichia coli and its negative regulation by Lon

SulA is induced in Escherichia coli by the SOS response and inhibits cell division through interaction with FtsZ. To determine which region of SulA is essential for the inhibition of cell division, we constructed a series of N-terminal and C-terminal deletions of SulA and a series of alanine substitution mutants. Arginine at position 62, leucine at 67, tryptophan at 77 and lysine at 87, in the central region of SulA, were all essential for the inhibitory activity. Residues 3–27 and the C-terminal 21 residues were dispensable for the activity. The mutant protein lacking N-terminal residues 3–47 was inactive, as was that lacking the C-terminal 34 residues. C-terminal deletions of 8 and 21 residues increased the growth-inhibiting activity in lon ⁺ cells, but not in lon ⁻ cells. The wild-type and mutant SulA proteins were isolated in a form fused to E. coli maltose-binding protein, and tested in vitro for sensitivity to Lon protease. Lon degraded wild-type SulA and a deletion mutant lacking the N-terminal 93 amino acids, but did not degrade the derivative lacking 21 residues at the C-terminus. Futhermore, the wild-type SulA and the N-terminal deletion mutant formed a stable complex with Lon, while the C-terminal deletion did not. MBP fused to the C-terminal 20 residues of SulA formed a stable complex with, but was not degraded by Lon. When LacZ protein was fused at its C-terminus to 8 or 20 amino acid residues from the C-terminal region of SulA the protein was stable in lon ⁺ cells. These results indicate that the C-terminal 20 residues of SulA permit recognition by, and complex formation with, Lon, and are necessary, but not sufficient, for degradation by Lon. Received: 8 October 1996 / Accepted: 27 November 1996     

Rational redesign of neutral endopeptidase binding to merlin and moesin proteins

Neutral endopeptidase (NEP) is a 90‐ to 110‐kDa cell‐surface peptidase that is normally expressed by numerous tissues but whose expression is lost or reduced in a variety of malignancies. The anti‐tumorigenic function of NEP is mediated not only by its catalytic activity but also through direct protein–protein interactions of its cytosolic region with several binding partners, including Lyn kinase, PTEN, and ezrin/radixin/moesin (ERM) proteins. We have previously shown that mutation of the K¹⁹K²⁰K²¹ basic cluster in NEPs' cytosolic region to residues QNI disrupts binding to the ERM proteins. Here we show that the ERM‐related protein merlin (NF2) does not bind NEP or its cytosolic region. Using experimental data, threading, and sequence analysis, we predicted the involvement of moesin residues E¹⁵⁹Q¹⁶⁰ in binding to the NEP cytosolic domain. Mutation of these residues to NL (to mimic the corresponding N¹⁵⁹L¹⁶⁰ residues in the nonbinder merlin) disrupted moesin binding to NEP. Mutation of residues N¹⁵⁹L¹⁶⁰Y¹⁶¹K¹⁶²M¹⁶³ in merlin to the corresponding moesin residues resulted in NEP binding to merlin. This engineered NEP peptide–merlin interaction was diminished by the QNI mutation in NEP, supporting the role of the NEP basic cluster in binding. We thus identified the region of interaction between NEP and moesin, and engineered merlin into a NEP‐binding protein. These data form the basis for further exploration of the details of NEP‐ERM binding and function.     

Chemical and Enzymatic Properties of Acid-cellulase Produced by Aspergillus niger

Some properties of a purified acid-cellulase produced by Aspergillus niger were investigated. The acid-cellulase was stable at the pH range between 4.0 and 10.0 and exhibited the highest activity toward glycol cellulose at pH 2.5. The optimum temperature of activity was measured to be 50 C, while the enzyme was inactivated above 40‘C by heating for 1 hr. Insoluble cellulose such as filter paper was difficult to be attacked by the enzyme.

Mg²⁺ and Mn²⁺ ions inhibited the activity, while Co²⁺ ion caused a slight activation.

The nitrogen content of the enzyme protein was determined to be 14.37%. The enzyme contained 378 residues of amino acids rich in acidic amino acids, 12 residues of glucosamine and 10 residues of arabinose per molecule. N-terminus was not detected by DNP-method.