Cationic activation of galactosyltransferase from rat mammary Golgi membranes by polyamines and by basic peptides and proteins.

Galactosyltransferase (EC 2.4.1.22) requires bivalent metal ions for its activity. However, preparations of this enzyme solubilized from Golgi membranes of lactating rat mammary gland were shown to be activated not only by Mn2+, Ca2+ and Mg2+, but also by spermine, spermidine, lysyl-lysine, ethylenediamine and other diaminoalkanes, and by a range of basic proteins and peptides, including clupeine, histone, polylysine, ribonuclease, pancreatic trypsin inhibitor, cytochrome c, melittin, avidin and myelin basic protein. Both N-acetyl-lactosamine synthetase and lactose synthetase activities were enhanced. A basic protein fraction was isolated from bovine milk and shown to activate galactosyltransferase at low concentrations. The polyanions ATP, casein, chondroitin sulphate and heparin reversed the activation of galactosyltransferase by several of the above substances. Galactosyltransferase, assayed as a lactose synthetase, showed a 10-fold greater affinity for glucose when Mn2+ ions were replaced by clupeine or by ribonuclease as cationic activator. Evidence was obtained for the presence of an endogenous cationic activator in solubilized Golgi membrane preparations which evoked a similar low apparent Km,glucose. The findings are discussed in the light of cationic activations of glycosyltransferases generally, of the porous nature of the Golgi membrane, and of the unlikelihood of bivalent metal ions being the physiological activators of galactosyltransferase. It is suggested that the natural cationic activator of lactose synthetase may be a secretory protein acting in a manner analogous to the enzyme's activation by alpha-lactalbumin. A scheme is proposed for the two-stage synthesis of lactose and phosphorylation of casein within the cell, to accommodate the apparent incompatibility of these two processes.     

Quantifying the ion selectivity of the Ca2+ site in photosystem II: evidence for direct involvement of Ca2+ in O2 formation.

Calcium is an essential cofactor in the oxygen-evolving complex (OEC) of photosystem II (PSII). The removal of Ca2+ or its substitution by any metal ion except Sr2+ inhibits oxygen evolution. We used steady-state enzyme kinetics to measure the rate of O2 evolution in PSII samples treated with an extensive series of mono-, di-, and trivalent metal ions in order to determine the basis for the affinity of metal ions for the Ca2+-binding site. Our results show that the Ca2+-binding site in PSII behaves very similarly to the Ca2+-binding sites in other proteins, and we discuss the implications this has for the structure of the site in PSII. Activity measurements as a function of time show that the binding site achieves equilibrium in 4 h for all of the PSII samples investigated. The binding affinities of the metal ions are modulated by the 17 and 23 kDa extrinsic polypeptides; their removal decreases the free energy of binding of the metal ions by 2.5 kcal/mol, but does not significantly change the time required to reach equilibrium. Monovalent ions are effectively excluded from the Ca2+-binding site, exhibiting no inhibition of O2 evolution. Di- and trivalent metal ions with ionic radii similar to that of Ca2+ (0.99 A) bind competitively with Ca2+ and have the highest binding affinity, while smaller metal ions bind more weakly and much larger ones do not bind competitively. This is consistent with a size-selective Ca2+-binding site that has a rigid array of coordinating ligands. Despite the large number of metal ions that competitively replace Ca2+ in the OEC, only Sr2+ is capable of partially restoring activity. Comparing the physical characteristics of the metal ions studied, we identify the pK(a) of the aqua ion as the factor that determines the functional competence of the metal ion. This suggests that Ca2+ is directly involved in the chemistry of water oxidation and is not only a structural cofactor in the OEC. We propose that the role of Ca2+ is to act as a Lewis acid, binding a substrate water molecule and tuning its reactivity.     

Purification and characterization of an erythroid cell-specific factor that binds the murine alpha- and beta-globin genes.

An erythroid cell-specific nuclear factor that binds tightly to a sequence motif (5'-GATAAGGA-3') shared by many erythroid cell-specific promoters was purified to homogeneity by DNA sequence affinity chromatography. Visualization of the purified factor, which we term EF-1, showed a simple pattern comprising a polypeptide doublet with Mrs of 18,000 and 19,000. We confirmed that these species account for EF-1-binding activity by eluting the polypeptides from sodium dodecyl sulfate-polyacrylamide gels and renaturing the appropriate binding activity. Using the purified polypeptides, we mapped seven factor-binding sites that are dispersed across the murine alpha- and beta-globin genes. The murine alpha-globin gene is flanked by at least two EF-1-binding sites. One site is centered at nucleotide (nt) -180 (with respect to the alpha-globin cap site). A fivefold-weaker site is located downstream of the alpha-globin poly(A) addition site, at nt +1049. We mapped five EF-1-binding sites near the murine beta-globin gene. The strongest site was centered at nt -210. Four additional sites were centered at nt -266 (adjacent to the binding site of a factor present in both murine erythroleukemia and Raji cells), -75 (overlapping the beta-globin CCAAT box), +543 (within the second intervening sequence), and -111.     

Investigation of the forms of pig duodenal Ca2+-binding protein produced by limited tryptic hydrolysis.

Vitamin D-dependent Ca2+-binding protein from pig duodenum was hydrolysed with trypsin in the presence of Ca2+ and two products were obtained: T1, which differed from the native protein by loss of Ac-Ser-Ala-Gln-Lys from the N-terminus and Ile-Ser-Gln-OH from the C-terminus, and T2, which differed from T1 by loss of a C-terminal lysine. The hydrolysis inactivated one of the two high-affinity Ca2+-binding sites on the native protein, and the remaining site was stable in T1 but labile in T2 when the proteins were Ca2+-free. Binding studies showed that T1 had Kd values of 2.8 +/- 0.1 nM, 57 +/- 13 microM and 0.8 +/- 0.3 microM for Ca2+, Mg2+ and Mn2+ respectively, and T2 had Kd 2.2 +/- 0.3 nM for Ca2+. The affinity for Mn2+, together with the other Kd values, identified the site on T1 as the site on the native protein previously found to have Kd 0.6 microM for Mn2+, rather than one with Kd 50 microM for Mn2+. In contrast with both the native protein and another form of the protein with a single Ca2+-binding site, the intrinsic fluorescence of T1 and T2 was little affected by the addition of Ca2+. It was concluded that the active binding site in T1 and T2, and also the site in the native protein with the higher affinity for Mn2+, was probably in the C-terminal half of the molecule.     

Characterization of calcium binding to brush-border membranes from rat duodenum.

The Ca2+-binding properties of isolated brush-border membranes at physiological ionic strength and pH were examined by rapid Millipore filtration. A comprehensive analysis of the binding data suggested the presence of two types of Ca2+-binding sites. The high-affinity sites, Ka = (6.3 +/- 3.3) X 10(5) M-1 (mean +/- S.E.M.), bound 0.8 +/- 0.1 nmol of Ca2+/mg of protein and the low-affinity sites, Ka = (2.8 +/- 0.3) X 10(2) M-1, bound 33 +/- 3.5 nmol of Ca2+/mg of protein. The high-affinity site exhibited a selectivity for Ca2+, since high concentrations of competing bivalent cations were required to inhibit Ca2+ binding. The relative effectiveness of the competing cations (1 and 10 mM) for the high-affinity site was Mn2+ approximately equal to Sr2+ greater than Ba2+ greater than Mg2+. Data from the pH studies, treatment of the membranes with carbodi-imide and extraction of phospholipids with aqueous acetone and NH3 provided evidence that the low-affinity sites were primarily phospholipids and the high-affinity sites were either phosphoprotein or protein with associated phospholipid. Two possible roles for the high-affinity binding sites are suggested. Either high-affinity Ca2+ binding is involved with specific enzyme activities or Ca2+ transport across the luminal membrane occurs via a Ca2+ channel which contains a high-affinity Ca2+-specific binding site that may regulate the intracellular Ca2+ concentration and gating of the channel.     

Thallium binding to native and radiation-inactivated Na+/K+-ATPase

The number of high-affinity K+-binding sites on purified Na+/K+-ATPase from pig kidney outer medulla has been assessed by measurement of equilibrium binding of thallous thallium, Tl+, under conditions (low ionic strength, absence of Na+ and Tris+) where the enzyme is in the E2-form. Na+/K+-ATPase has two identical Tl+ sites per ADP site, and the dissociation constant varies between 2 and 9 microM. These values are identical to those for Tl+ occlusion found previously by us, indicating that all high-affinity binding leads to occlusion. The specific binding was obtained after subtraction of a separately characterized unspecific adsorption of Tl+ to the enzyme preparations. Radiation inactivation leads to formation of modified peptides having two Tl+-binding sites with positive cooperativity, the second site-dissociation constant approximating that for the native sites. The radiation inactivation size (RIS) for total, specific Tl+ binding is 71 kDa, and the RIS for Tl+ binding with original affinity is approx. 190 kDa, equal to that of Na+/K+-ATPase activity and to that for Tl+ occlusion with native affinity. This latter RIS value confirms our recent theory that in situ the two catalytic peptides of Na+/K+-ATPase are closely associated. The 71 kDa value obtained for total Tl+ sites is equal to that for total binding of ATP and ADP and it is clearly smaller than the molecular mass of one catalytic subunit (112 kDa). The Tl+-binding experiments reported thus supports the notion that radiation inactivation of Na+/K+-ATPase is a stepwise rather than an all or none process.     

Binding of interleukin 2 to its 75-kDa intermediate affinity receptor is sufficient to activate Na+/H+ exchange

High affinity interleukin 2 (IL-2) binding sites are composed of two IL-2-binding molecules: one of 55 kDa, commonly called TaC, and another of 75 kDa. In the absence of the other IL-2-binding molecule, the 55-kDa molecule binds IL-2 with a relatively low affinity and the 75 kDa molecule binds IL-2 with an intermediate affinity. One of the earliest events following interaction of IL-2 with its receptor on the surface of cells is an increase in intracellular pH due to activation of the Na+/H+ antiport. In contrast to IL-2-induced proliferation of human IL-2-sensitive T cells, interaction of IL-2 with a low affinity binding site was sufficient to activate the Na+/H+ antiport. By determining the effect of IL-2 on cytosolic pH in cells that express one of the two IL-2-binding molecules in the absence of the other IL-2-binding molecule, we have demonstrated that interaction of IL-2 with the 75 kDa IL-2-binding molecule is sufficient to activate the Na+/H+ antiport and thus induce cytosolic alkalinization. This indicates that the 75-kDa IL-2-binding molecule, in the absence of the 55-kDa IL-2-binding molecule, forms a functional receptor that can transduce an activation signal across the cell membrane.     

Characterization of the cation-binding properties of porcine neurofilaments

In the presence of physiological levels of Na+ (10 mM), K+ (150 mM), and Mg2+ (2 mM), dephosphorylated neurofilaments contained two Ca2+ specific binding sites with Kd = 11 microM per unit consisting of eight low, three middle, and three high molecular subunits, as well as 46 sites with Kd = 620 microM. Only one class of 126 sites with Kd = 740 microM was detected per unit of untreated neurofilaments. A chymotryptic fraction enriched in the alpha-helical domains of neurofilament subunits contained one high-affinity Ca2+-binding site (Kd = 3.6 microM) per domain fragment of approximately 32 kDa. This site may correspond to a region in coil 2b of the alpha-helical domain, which resembles the I-II Ca2+-binding site in intestinal Ca2+-binding protein. Homopolymeric filaments composed of the low or middle molecular weight subunits contained low-affinity Ca2+-binding sites with Kd = 37 microM and 24 microM, respectively, while the Kd values for the low-affinity sites in heteropolymeric filaments were 8-10-fold higher. Competitive binding studies, using the chymotryptic fraction to assay the high-affinity Ca2+-binding sites and 22Na+ to monitor binding to the phosphate-containing low-affinity sites, yielded Kd values for Al3+ of 0.01 microM and 4 microM, respectively. This suggests that the accumulation of Al3+ in neurons may be due in part to its binding to neurofilaments.     

Determinants for binding of a 40 kDa protein to the leaders of yeast mitochondrial mRNAs.

An abundant yeast mitochondrial 40 kDa protein (p40) binds with high specificity to the 5'-untranslated region of cytochrome c oxidase subunit II (COX2) mRNA. Using mobility shift and competition assays, we show here that purified p40 complexes with the leaders of all eight mitochondrial mRNAs of Saccharomyces cerevisiae. The location of the protein binding site on the different leaders is not conserved with respect to the AUG start codon. In vitro RNA footprint and deletion experiments have been used to define the p40-binding site on the leaders of COX1 and ATP9 mRNAs. Nucleotides at, and near, a single stranded region are protected or exposed for DEPC modification by binding of p40 to these leaders. Removal of this region from the COX1 messenger shows that it is essential for the protein-RNA interaction. While no obvious sequence similarity can be detected between the single stranded regions in different leaders, a nearby helical segment is conserved. A consensus model for p40-RNA interactions is presented and the possible biological function of p40 is discussed.     

Activation of three types of membrane currents by various divalent cations in identified molluscan pacemaker neurons

We investigated membrane currents activated by intracellular divalent cations in two types of molluscan pacemaker neurons. A fast and quantitative pressure injection technique was used to apply Ca2+ and other divalent cations. Ca2+ was most effective in activating a nonspecific cation current and two types of K+ currents found in these cells. One type of outward current was quickly activated following injections with increasing effectiveness for divalent cations of ionic radii that were closer to the radius of Ca2+ (Ca2+ greater than Cd2+ greater than Hg2+ greater than Mn2+ greater than Zn2+ greater than Co2+ greater than Ni2+ greater than Pb2+ greater than Sr2+ greater than Mg2+ greater than Ba2+). The other type of outward current was activated with a delay by Ca2+ greater than Sr2+ greater than Hg2+ greater than Pb2+. Mg2+, Ba2+, Zn2+, Cd2+, Mn2+, Co2+, and Ni2+ were ineffective in concentrations up to 5 mM. Comparison with properties of Ca2(+)-sensitive proteins related to the binding of divalent cations suggests that a Ca2(+)-binding protein of the calmodulin/troponin C type is involved in Ca2(+)-dependent activation of the fast-activated type of K+ current. Th sequence obtained for the slowly activated type is compatible with the effectiveness of different divalent cations in activating protein kinase C. The nonspecific cation current was activated by Ca2+ greater than Hg2+ greater than Ba2+ greater than Pb2+ greater than Sr2+, a sequence unlike sequences for known Ca2(+)-binding proteins.     

The binding of Mn2+ to bovine plasma protein C, des(1-41)-light chain protein C, and activated des(1-41)-light chain activated protein C

The binding isotherms of Mn2+ to bovine plasma protein C (PC), des(1-41)-light chain protein C (GDPC), and activated GDPC (GDAPC) have been measured. PC contains 14-16 total Mn2+ binding sites, a value that is reduced to approximately 7-8 in the presence of NaCl. The average Kd of the latter sites is 230 +/- 30 microM. Upon removal of a 41-residue peptide from the amino terminus of the light chain of PC, and, concomitantly, all of the gamma-carboxyglutamic acid residues, the resulting protein, GDPC, possesses a single Mn2+ site of Kd = 120 +/- 20 microM. Activation of GDPC to GDAPC results in a slight lowering of the Kd for the single Mn2+ binding site to 53 +/- 8 microM, a value that is essentially unchanged in the presence of monovalent cations, a competitive inhibitor of the enzyme, or an active site directed affinity label. The Mn2+ on GDAPC is displaced by Ca2+, suggesting that the protein binding site for these two divalent cations is the same. These studies establish that Mn2+ is a suitable spectroscopic probe for the Ca2+ binding site of GDAPC, and that the divalent cation site is separate from the monovalent cation site(s) and the active site of the enzyme.     

67 kDa calcimedin, a new Ca2+-binding protein.

A set of four proteins, termed calcimedins, are isolatable from smooth, cardiac and skeletal muscle by using a fluphenazine-Sepharose affinity column. The calcimedins show apparent Mr values of 67,000, 35,000, 33,000 and 30,000 by SDS/polyacrylamide-gel electrophoresis. The 67,000-Mr calcimedin (67 kDa calcimedin) has now been purified to homogeneity by using DEAE-cellulose chromatography followed by Ca2+-dependent binding to phenyl-Sepharose. The amino acid analysis of the 67 kDa calcimedin shows this protein does not contain trimethyl-lysine but does contain 2 mol of tryptophan/mol of protein. The 67 kDa calcimedin shows positive ellipticity in the near-u.v. range with c.d. Ca2+-binding studies indicate one high-affinity Ca2+-binding site with Kd 0.4 microM. The data show that the 67 kDa calcimedin is distinct from other Ca2+-binding proteins described to date.     

Metal binding to DNA polymerase I, its large fragment, and two 3',5'-exonuclease mutants of the large fragment

DNA polymerase I (Pol I) is an enzyme of DNA replication and repair containing three active sites, each requiring divalent metal ions such as Mg2+ or Mn2+ for activity. As determined by EPR and by 1/T1 measurements of water protons, whole Pol I binds Mn2+ at one tight site (KD = 2.5 microM) and approximately 20 weak sites (KD = 600 microM). All bound metal ions retain one or more water ligands as reflected in enhanced paramagnetic effects of Mn2+ on 1/T1 of water protons. The cloned large fragment of Pol I, which lacks the 5',3'-exonuclease domain, retains the tight metal binding site with little or no change in its affinity for Mn2+, but has lost approximately 12 weak sites (n = 8, KD = 1000 microM). The presence of stoichiometric TMP creates a second tight Mn2+ binding site or tightens a weak site 100-fold. dGTP together with TMP creates a third tight Mn2+ binding site or tightens a weak site 166-fold. The D424A (the Asp424 to Ala) 3',5'-exonuclease deficient mutant of the large fragment retains a weakened tight site (KD = 68 microM) and has lost one weak site (n = 7, KD = 3500 microM) in comparison with the wild-type large fragment, and no effect of TMP on metal binding is detected. The D355A, E357A (the Asp355 to Ala, Glu357 to Ala double mutant of the large fragment of Pol I) 3',5'-exonuclease-deficient double mutant has lost the tight metal binding site and four weak metal binding sites. The binding of dGTP to the polymerase active site of the D355A,E357A double mutant creates one tight Mn2+ binding site with a dissociation constant (KD = 3.6 microM), comparable with that found on the wild-type enzyme, which retains one fast exchanging water ligand. Mg2+ competes at this site with a KD of 100 microM. It is concluded that the single tightly bound Mn2+ on Pol I and a weakly bound Mn2+ which is tightened 100-fold by TMP are at the 3',5'-exonuclease active site and are essential for 3',5'-exonuclease activity, but not for polymerase activity. Additional weak Mn2+ binding sites are detected on the 3',5'-exonuclease domain, which may be activating, and on the polymerase domain, which may be inhibitory. The essential divalent metal activator of the polymerase reaction requires the presence of the dNTP substrate for tight metal binding indicating that the bound substrate coordinates the metal.(ABSTRACT TRUNCATED AT 400 WORDS)     

Purification and properties of a novel nucleotide-hydrolysing enzyme (5''-nucleotidase) from Boophilus microplus.

Conformational changes in the beta-subunit of the bovine brain Ca2+-binding protein S100b (S100-beta) accompanying Ca2+ binding were investigated by analysis of the spectroscopic properties of the single tyrosine residue (Tyr17 beta) and flow-dialysis binding experiments. S100-beta binds Ca2+ sequentially at two sites to change the conformation of the protein. The first Ca2+ ion binds to site II beta, a typical Ca2+-binding site in the C-terminal region, and it does not significantly perturb the proximal environment of Tyr17 beta. After the first site is occupied, another Ca2+ ion binds to the N-terminal Ca2+-binding site, I beta, and strengthens a hydrogen bond between Tyr17 beta and a neighbouring carboxylate acceptor group, which results in a large increase in the Tyr17 beta fluorescence spectrum half-width and a positive absorption and c.d. signal between 290 and 275 nm. Ca2+ binding to the S100b.Zn2+6 complex, studied by flow-dialysis and fluorescence measurements showed that, although Zn2+ ions increase the affinity of S100b protein for Ca2+, the Ca2+-binding sequence was not changed. Tb3+ (terbium ion) binding studies on the S100b.Zn2+6 complex proved that Tb3+ antagonizes only Ca2+ binding site II beta and confirmed the sequential occupation of Ca2+-binding sites on the S100b.Zn2+6 complex.     

Neomycin Is an Agonist at a Polyamine Site on the N-Methyl-D-Aspartate Receptor

Neomycin appears as a full agonist and spermidine as a partial agonist at the site where polyamines enhance 1-[1-(2-thienyl)cyclohexyl][3H]piperidine ([3H]TCP) binding on the N-methyl-D-aspartate (NMDA) receptor. Other aminoglycosides also enhance [3H]TCP binding with efficacies roughly proportional to the number of primary amine groups. The polyamine antagonists ifenprodil and arcaine inhibit enhancement of [3H]TCP binding by spermidine or neomycin. The inhibition of [3H]TCP binding by arcaine is apparently competitively reduced by neomycin and spermidine, supporting a common site. Diethylenetriamine (previously described as a polyamine antagonist) may be a partial agonist. Enhancement by neomycin or spermidine is not additive to that of Mg2+, consistent with competition of Mg2+ and spermidine or neomycin at the site where these compounds enhance [3H]TCP binding. Polyamines also enhance the binding of the competitive antagonist 2-(2-carboxypiperazin-4-yl)[3H]propyl-1-phosphonic acid ([3H]CPP). Neomycin, which does not enhance [3H]CPP binding, inhibits the enhancement by spermidine. That this site is distinct from the site where spermidine and neomycin increase [3H]TCP binding is supported by different pharmacology. Arcaine and diethylenetriamine do not inhibit spermidine enhancement of [3H]CPP binding. Mg2+ also does not compete with the spermidine enhancement of [3H]CPP binding. Ifenprodil inhibits the spermidine enhancement of [3H]CPP binding. The data suggest two or more polyamine sites, with arcaine selective for the site that enhances [3H]TCP binding. Neomycin is an agonist at one polyamine site and antagonist to the second.     

Studies on the metal binding sites in the catalytic domain of beta1,4-galactosyltransferase

The catalytic domain of bovine beta1,4-galactosyltransferase (beta4Gal-T1) has been shown to have two metal binding sites, each with a distinct binding affinity. Site I binds Mn(2+) with high affinity and does not bind Ca(2+), whereas site II binds a variety of metal ions, including Ca(2+). The catalytic region of beta4Gal-T1 has DXD motifs, associated with metal binding in glycosyltransferases, in two separate sequences: D(242)YDYNCFVFSDVD(254) (region I) and W(312)GWGGEDDD(320) (region II). Recently, the crystal structure of beta4Gal-T1 bound with UDP, Mn(2+), and alpha-lactalbumin was determined in our laboratory. It shows that in the primary metal binding site of beta4Gal-T1, the Mn(2+) ion, is coordinated to five ligands, two supplied by the phosphates of the sugar nucleotide and the other three by Asp254, His347, and Met344. The residue Asp254 in the D(252)VD(254) sequence in region I is the only residue that is coordinated to the Mn(2+) ion. Region II forms a loop structure and contains the E(317)DDD(320) sequence in which residues Asp318 and Asp319 are directly involved in GlcNAc binding. This study, using site-directed mutagenesis, kinetic, and binding affinity analysis, shows that Asp254 and His347 are strong metal ligands, whereas Met344, which coordinates less strongly, can be substituted by alanine or glutamine. Specifically, substitution of Met344 to Gln has a less severe effect on the catalysis driven by Co(2+). Glu317 and Asp320 mutants, when partially activated by Mn(2+) binding to the primary site, can be further activated by Co(2+) or inhibited by Ca(2+), an effect that is the opposite of what is observed with the wild-type enzyme.     

Magnetic resonance and kinetic studies of the partial complex and Component I subunit forms of Salmonella typhimurium anthranilate synthase

Metal ion interactions of the monofunctional partial complex of Salmonella typhimurium anthranilate synthase were investigated using kinetic, NMR, and EPR methods. Mn2+ activates AS-partial complex in place of Mg2+, with a Km of 0.08 microM for Mn2+ and of 3.5 microM for Mg2+ in glutamine-dependent anthranilate synthase activity. The kinetics indicated that the metal interacts at the active site with chorismate, not glutamine. EPR and NMR water proton relaxation rate (PRR) studies supported this conclusion. EPR binding analysis showed that chorismate dramatically tightens Mn2+ binding by the partial complex. PRR experiments indicated that stoichiometric amounts of chorismate cause a substantial decrease in the enhancement of water relaxation by Mn2+, while millimolar amounts of glutamine have no effect. Analysis of the frequency dependence of water proton relaxation rates yielded dipolar correlation times of 2.5 x 10(-9) s and 4.1 x 10(-9) s for the Mn2+-partial complex and Mn2+-partial complex-chorismate complexes, respectively. These studies also indicated that chorismate binding reduces the number of fast-exchanging water molecules on enzyme-bound Mn2+ from 1 to 0.25. PRR experiments with the native bifunctional anthranilate synthase-phosphoribosyltransferase enzyme indicated the existence of additional Mn2+-binding sites which presumably function to activate the phosphoribosyltransferase activity of the Component II subunit.     

Sodium-23 and lanthanum-139 nuclear magnetic resonance studies of cation binding to aqualysin I,a thermostable serine protease

Aqualysin I has at least two Ca2+-binding sites that have different affinities for Ca2+. The binding of various metal ions to aqualysin I was studied using 23Na- and 139La-NMR spectrometry. Evidence is presented that Ca2+, La3+, and Na+ bind to the low-affinity Ca2+-binding site of aqualysin I, but Mg2+ does not. Our results confirm that binding of metals at the low-affinity Ca2+-binding site is essential for thermostabilization, since the addition of Mg2+ did not result in thermostabilization. La3+ was found to bind to both the low-affinity Ca2+-binding site and an additional metal ion-binding site that can also be involved in the thermostabilization of aqualysin I.     

Structure-function studies of the functional and binding epitope of the human 37 kDa laminin receptor precursor protein.

Expression of the 37 kDa laminin receptor precursor protein (37LRP) correlates directly with increased invasiveness and the metastatic potential of tumors. The 37LRP matures to a 67 kDa protein which facilitates the binding of cancer cells to basement membranes. The palindrome peptide sequence LMWWML, corresponding to the 173-178-residue stretch of the human 37LRP sequence, has been identified as the laminin-1-binding site. Peptides from 37LRP of species that contain this palindrome-bind laminin-1 with high affinity. Nuclear magnetic resonance (NMR) conformational studies have been undertaken on a synthetic 15-residue peptide (KGAHSVGLMWWMLAR) containing the palindrome to establish the structural basis of this activity. To further correlate the structural data with laminin-1-binding function, analogous structural studies were conducted for a similar peptide (RGKHSIGLIWYLLAR) lacking the palindrome, originating from 37LRP sequence of Saccharomyces cerevisiae and exhibiting low laminin-1-binding affinity. Finally, in vitro cell invasion assays were performed to investigate the possibility that the laminin-1-binding affinity of the peptides influences their inhibitory activity.