Bert L. Vallee's research while affiliated with Harvard Medical School and other places
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Publications (578)
We have previously established that ATP binds to mammalian metallothionein-2 (MT). The interaction between ATP and MT and the associated conformational change of the protein affect the sulfhydryl reactivity and zinc transfer potential of MT [Jiang, L.-J., Maret, W., and Vallee, B. L. (1998) The ATP-metallothionein complex. Proc. Natl. Acad. Sci. U....
The isolation of thionein (T) from tissues has not been reported heretofore. T contains 20 cysteinyl residues that react with 7-fluorobenz-2-oxa-1,3-diazole-4-sulfonamide to form fluorescent adducts. In metallothionein (MT) the cysteinyl residues, which are bound to zinc, do not react. However, they do react in the presence of a chelating agent suc...
Metallothionein (MT) localizes in the intermembrane space of liver mitochondria as well as in the cytosol and nucleus. Incubation of intact liver mitochondria with physiological, micromolar concentrations of MT leads to the import of MT into the mitochondria where it inhibits respiration. This activity is caused by the N-terminal beta-domain of MT;...
The alpha- and beta-polypeptides of human metallothionein (isoform 2), obtained by chemical synthesis, were converted into their respective zinc/thiolate clusters, and each domain was investigated separately. Proton titration data for the N-terminal beta-domain fit a simple model with three ionizations of the same apparent pK(a) value of 4.9 and a...
Angiogenin, a potent inducer of neovascularization, is the first tumour-derived angiogenic factor to have been isolated based solely on its ability to induce the formation of new blood vessels. It is a member of the pancreatic ribonuclease superfamily and its characteristic ribonucleolytic activity is necessary for its angiogenic activity. ft induc...
Thionein (T) has not been isolated previously from biological material. However, it is generated transiently in situ by removal of zinc from metallothionein under oxidoreductive conditions, particularly in the presence of selenium compounds. T very rapidly activates a group of enzymes in which zinc is bound at an inhibitory site. The reaction is se...
Selenium has been increasingly recognized as an essential element in biology and medicine. Its biochemistry resembles that of sulfur, yet differs from it by virtue of both redox potentials and stabilities of its oxidation states. Selenium can substitute for the more ubiquitous sulfur of cysteine and as such plays an important role in more than a do...
Medium-chain dehydrogenases/reductases of the liver alcohol dehydrogenase type are zinc metalloenzymes (Vallee and Hoch, 1957; IÅeson, 1964; Drum et al., 1969), with two zinc atoms per subunit, one catalytic at the active site and one structural at a site influencing subunit interactions (Sytkowski and Vallee, 1976; Brändén et al., 1975). In horse...
The application of scanning probe methods to the high-resolution imaging of biological structure has been developing rapidly during the past few years. In contrast to diffraction and electron microscopy methods, imaging is direct and can be carried out under fluid. Scanning Tunneling Microscopy (STM) allows a resolution of electronic as well as top...
We have previously shown that glutathione (GSH) and glutathione disulfide interact with metallothionein (MT) and modulate its capacity to donate and transfer zinc. In this paper, we show that ATP also forms a 1:1 complex with MT (Kd = 176 +/- 33 microM, pH 7. 4) that enhances the transfer of zinc to zinc-depleted sorbitol dehydrogenase, increases t...
Selenium compounds oxidize the thiolate ligands in the zinc clusters of metallothionein and release zinc. This chemistry defines new cellular targets for biological forms of selenium and suggests important interactions between zinc and selenium, two biologically essential elements. In the course of delineating the redox chemistry of biological zinc...
Angiogenin (Ang), an inducer of neovascularization, is secreted by several types of human tumor cells and appears critical for their growth. The murine anti-Ang monoclonal antibody (mAb) 26-2F neutralizes the activities of Ang and dramatically prevents the establishment and metastatic dissemination of human tumor cell xenografts in athymic mice. Ho...
Daidzin, a major active principle of an ancient Chinese herbal treatment (Radix puerariae) for alcohol abuse, selectively suppresses ethanol intake in all rodent models tested. It also inhibits mitochondrial aldehyde dehydrogenase (ALDH-2). Studies on ethanol intake suppression and ALDH-2 inhibition by structural analogs of daidzin established a li...
The release and transfer of zinc from metallothionein (MT) to zinc-depleted sorbitol dehydrogenase (EC 1.1.1.14) in vitro has been used to explore the role of MT in cellular zinc distribution. A 1:1 molar ratio of MT to sorbitol dehydrogenase is required for full reactivation, indicating that only one of the seven zinc atoms of MT is transferred in...
We postulate a novel and general mechanism in which the redox-active sulfur donor group of cyst(e)ine confers oxidoreductive characteristics on stable zinc sites in proteins. Thus, the present, an earlier, and accompanying manuscripts [Maret, W., Larsen, K. S. & Vallee, B. L. (1997) Proc. Natl. Acad. Sci. USA 94, 2233-2237; Jiang, L.-J., Maret, W....
Metallothionein (MT), despite its high metal binding constant (KZn = 3.2 x 10(13) M-1 at pH 7.4), can transfer zinc to the apoforms of zinc enzymes that have inherently lower stability constants. To gain insight into this paradox, we have studied zinc transfer between zinc enzymes and MT. Zinc can be transferred in both directions-i.e., from the en...
Kudzu (Pueraria lobata) is one of the earliest medicinal plants used in traditional Chinese medicine. It has many profound pharmacological actions including antidipsotropic (antialcohol abuse) activity. Although both the roots and flowers of kudzu, Radix and Flos puerariae, respectively, have been used to treat alcohol abuse safely and effectively...
Mitochondrial (class 2) hamster aldehyde dehydrogenase has been purified and characterized. Its primary structure has been determined and correlated with the tertiary structure recently established for this class from another species. The protein is found to represent a constant class within a complex family of multiple forms. Variable segments tha...
Our search for an angiogenesis-inducing factor in culture medium conditioned by human colon adenocarcinoma cells (HT-29) was inspired by the 'organizer' hypothesis originally postulated by Spemann. It led us to the isolation of angiogenin, a 14 kD protein homologous to pancreatic ribonuclease and one of the most potent stimulators of blood vessel f...
Bovine serum and milk contain a basic angiogenic protein that binds tightly to placental ribonuclease inhibitor. It was purified from both sources by ion-exchange and reversed-phase chromatographies. Its amino acid sequence revealed that it is a member of the ribonuclease superfamily. It contains 123 amino acids in a single polypeptide chain, is cr...
Daidzin is the major active principle in extracts of radix puerariae, a traditional Chinese medication that suppresses the ethanol intake of Syrian golden hamsters. It is the first isoflavone recognized to have this effect. Daidzin is also a potent and selective inhibitor of human mitochondrial aldehyde dehydrogenase (ALDH-2). To establish a link b...
![FIG. 1. Dose-dependent stimulation of [ 3 H]thymidine incorporation and...](profile/Guo-Fu-Hu/publication/14096498/figure/fig1/AS:601578297884720@1520438851986/Dose-dependent-stimulation-of-3-Hthymidine-incorporation-and-proliferation-of_Q320.jpg)
![Table 3 . Effect of FBS on angiogenin-stimulated [ 3 H]thymidine...](profile/Guo-Fu-Hu/publication/14096498/figure/tbl2/AS:601578302083073@1520438852091/Effect-of-FBS-on-angiogenin-stimulated-3-Hthymidine-incorporation-in-HME-cells_Q320.jpg)
Angiogenin stimulates both [3H]thymidine incorporation and proliferation of human endothelial cells in sparse cultures. Under these conditions, a 170-kDa cell surface protein can be detected that binds angiogenin specifically. Angiogenin-stimulated cell growth is concentration-dependent and is completely inhibited by an anti-angiogenin monoclonal a...
The almost universal appreciation for the importance of zinc in metabolism has been offset by the considerable uncertainty regarding the proteins that store and distribute cellular zinc. We propose that some zinc proteins with so-called zinc cluster motifs have a central role in zinc distribution, since they exhibit the rather exquisite properties...
As a first step toward the development of stable, selective, and potent inhibitors of those members of the pancreatic RNase superfamily that induce biological responses, we have focussed on low molecular weight compounds and studied their interactions with the active-site of bovine pancreatic ribonuclease A (RNase A). A new inhibitor is described,...
In a previous study, daidzin, a constituent of an ancient Chinese herbal treatment for alcoholism, decreased home-cage ethanol consumption in laboratory Syrian golden hamsters. The present study tested the generality of daidzin's antidipsotropic effects. Rats served as subjects in a two-lever choice procedure. At one lever, responses earned 10% eth...
The ribonucleolytic activity of angiogenin (Ang) is essential to Ang's capacity to induce blood vessel formation. Previous x-ray diffraction and mutagenesis results have shown that the active site of the human protein is obstructed by Gln-117 and imply that the C-terminal region of Ang must undergo a conformational rearrangement to allow substrate...
The dose effect of pure daidzin on the suppression of ethanol intake in Syrian golden hamsters was compared with that of crude daidzin contained in a methanol extract of Radix puerariae (RP). EC50 values estimated from the graded dose-response curves for pure daidzin and RP extract daidzin are 23 and 2.3 mg per hamster per day, respectively. Appare...
Angiogenin-related protein (Angrp), the putative product of a recently discovered mouse gene, shares 78% sequence identity with mouse angiogenin (Ang). In the present study, the relationship of Angrp to Ang has been investigated by producing both proteins in bacteria and comparing their functional properties. We find that mouse Ang is potently angi...
The x-ray absorption fine structure (XAFS) zinc K-edge steps for intact stages I,II and V,VI Xenopus laevis oocytes demonstrate that the zinc concentration is about 3 and 1 mM, respectively. However, the chi(k) function for the early stage oocytes differs markedly from that for the late one. Analysis of the XAFS data for stage I,II oocytes indicate...
Kinetic analysis and molecular modeling have been used to map the ribonucleolytic center of angiogenin (Ang). Pyrimidine nucleotides were found to interact very weakly with Ang, consistent with the inaccessible B1 pyrimidine binding site revealed by x-ray crystallography. Ang also lacks an effective phosphate binding site on the 5' side of B1. Alth...
Xenopus laevis vitellogenin contains 2 g-atoms (g-at) of Zn and 3 g-at of Ca/dimer, transports zinc in plasma, and plays a role in its distribution within the oocyte [Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413; Montorzi et al. (1995) Biochemistry 34, 10851-10858]. We here report the dynamics and time course of Zn65-labeled...
Daidzin is a potent, selective, and reversible inhibitor of human mitochondrial aldehyde dehydrogenase (ALDH) that suppresses free-choice ethanol intake by Syrian golden hamsters. Other ALDH inhibitors, such as disulfiram (Antabuse) and calcium citrate carbimide (Temposil), have also been shown to suppress ethanol intake of laboratory animals and a...
Xenopus laevis vitellogenin is a plasma protein that contains a total of 5 mol of metal/440 kDa dimer, 2 mol of zinc, and 3 mol of calcium (Montorzi et al. (1994) Biochem. Biophys. Res. Commun. 200, 1407-1413]. There are no other group IIB or transition metals in the molecule. The zinc atoms are removed instantaneously by 1,10-phenanthroline (OP) (...
Since its discovery in 1957 metallothionein (MT) has remained a protein in search of a function. After 40 years of frustrating efforts, three areas of research point to its zinc cluster structure as the basis of its functional potential: (1) the regulation of MT gene expression by zinc-dependent transcription factors, (2) neuronal growth inhibition...
The determination of the crystal structure of the ribonuclease inhibitor-ribonuclease A complex provides exciting new insight on how the leucine-rich repeat allows a single molecule to get around the problem of inhibiting an entire family of enzymes.
The capacity of angiogenin (Ang) to induce blood vessel growth is critically dependent on its ribonucleolytic activity. Crystallography and mutagenesis of human Ang have previously shown that its pyrimidine binding site is obstructed by Gln-117, implying that a conformational change is a key part of the mechanism of Ang action. The 1.5-A-resolution...
Elevated levels of metallothionein (MT) found in rapidly growing tissues such as neonatal liver and various types of human tumors have suggested a role for MT in cell proliferation. To further explore this possibility we investigated the concentration of MT in human colonic cancer (HT-29) cells at different stages of proliferation by means of immun...
The chemically stable but stereochemically flexible, non-toxic nature of zinc combined with its amphoteric properties has permitted it to orchestrate a number of zinc-binding motifs critical to life processes. For zinc enzymes, catalytic, cocatalytic, and structural zinc sites exist. DNA-binding proteins have zinc fingers, twists, and clusters exis...
A noncytotoxic neutralizing monoclonal antibody (mAb), 26-2F, to human angiogenin (Ang), a potent inducer of neovascularization, has been reported to prevent or delay the establishment of HT-29 human tumor xenografts in athymic mice. In the present study the tumor model was modified to increase sensitivity to Ang antagonists to facilitate further i...
Angiogenin, a potent inducer of neovascularization in the chicken chorioallantoic membrane and rabbit cornea, promotes endothelial cell invasion of Matrigel basement membrane. A transformed bovine aortic endothelial cell line, GM 7373, is 5 times more invasive when cultured in the presence of 1 microgram of bovine angiogenin per ml than in its abse...
The three-dimensional structure of human angiogenin has been determined by X-ray crystallography and is compared here with an earlier model which predicted its structure, based on the homology of angiogenin with bovine pancreatic ribonuclease A. Comparison of the predicted model and crystal structure shows that the active-site histidine residues an...
Human angiogenin, a potent inducer of neovascularization, is secreted by HT-29 colon adenocarcinoma cells. microgram doses of a monoclonal antibody that neutralizes the in vitro and in vivo activities of angiogenin prevent or delay the appearance of s.c. HT-29 tumors in athymic mice in a statistically significant, dose-dependent manner. The antibod...
Frog oocytes and embryos have long served as traditional subjects of embryological research providing structural and functional information for the interpretation of the biological processes underlying development. A large number of various chemical agents induce typical teratological changes in frog embryos. However, the effects of metal deficienc...
Vitellogenin induced by estrogen administration has been purified from the serum of Xenopus laevis. Six days after hormone injection, serum was collected and treated with 35% sat. (NH4)2SO4 to remove globulins. A single vitellogenin containing fraction was isolated by chromatography on a Mono-Q column. The protein was identified on the basis of its...
The crystal structure of human angiogenin (reported in the preceding paper in this issue) reveals that the site that corresponds to the pyrimidine binding site of RNase A is obstructed by Gln-117. Mutation of this residue to Ala and Gly is here found to increase activity 11- to 18-fold and 21- to 30-fold, respectively, toward dinucleotide, polynucl...
A gel retardation method for studying the interaction between proteins and carbohydrates employing fluorophore-assisted carbohydrate electrophoresis (FACE) has been developed. A carbohydrate (or carbohydrate mixture) is labeled fluorescently at its reducing end with 8-aminonaphthalene-1,3,6-trisulfonic acid, incubated with its binding protein(s), s...
Angiogenin, a potent inducer of neovascularization, is the only angiogenic molecule known to exhibit ribonucleolytic activity. Its overall structure, as determined at 2.4 A, is similar to that of pancreatic ribonuclease A, but it differs markedly in several distinct areas, particularly the ribonucleolytic active center and the putative receptor bin...
An extract of Radix Puerariae (RP), an herb long used in traditional Chinese medicine for alcohol addiction and intoxication, was shown to suppress the free-choice ethanol intake of ethanol-preferring Syrian golden hamsters. Two isoflavones, diadzein (4',7-dihydroxyisoflavone) and daidzin (7-glucoside of daidzein), isolated from the extract were sh...
The role of ethanol in the history of human development is here summarized under seven topics: I. Alcohol: the substitute for water as the major human beverage; II. Alcohol as a component of the diet and source of calories; III. Alcohol, concentration by distillation; IV. The Reformation, Temperance and Prohibition; V. Potable nonalcoholic beverage...
The 39 chapters in this volume consider subjects ranging from genetics, markers, and molecular biology of alcoholism, to clinical observations and treatment. The aim is to integrate pertinent information from the fields of molecular and cell biology with view to establishing a molecular basis of alcohol use and abuse. An initial preview summarizes...
Syrian Golden hamsters prefer and consume large and remarkably constant amounts of ethanol in a simple two-bottle free-choice regimen. Ethanol intake is significantly suppressed by zimelidine, bromocriptine, buspirone, and lithium carbonate, pharmacological agents that have been shown to be beneficial in controlling ethanol intake in alcohol-depend...
Zinc is essential for vertebrate development; its deficiency results in multiple congenital malformations. Knowledge of the zinc biochemistry that underlies embryologic development is very limited. This has led us to investigate the zinc, iron, and copper contents of Xenopus laevis oocytes and embryos. Stage 1-6 oocytes, isolated from ovaries, and...
Substitution of Asn, Ala or His for Asp-116 in angiogenin increases its ribonucleolytic activity towards tRNA and, at least in the case of His, its ability to induce blood-vessel formation (Harper, J.W. and Vallee, B.L. (1988) Proc. Natl. Acad. Sci. USA 85, 7139-7143). Six additional Asp-116 mutants have been examined to further probe the basis for...
The enzymatic and structural properties of alligator liver alcohol dehydrogenase have been determined. Aliphatic and alicyclic alcohols serve as substrates for this first reptilian form of the enzyme characterized, with Km values decreasing rapidly from methanol to hexanol, as for the human class I enzymes, and a Km of 1.2 mM for ethanol at pH 9.9....
The enzymatic and structural properties of alligator liver alcohol dehydrogenase have been determined. Aliphatic and alicyclic alcohols serve as substrates for this first reptilian form of the enzyme characterized, with K m values decreasing rapidly from methanol to hexanol, as for the human class I enzymes, and a K m of 1.2 mM for ethanol at pH 9....
Modification of class III alcohol dehydrogenase (chi chi-ADH) with phenylglyoxal eliminates fatty acid activation by pentanoate and octanoate and concomitantly increases specific activity toward ethanol and 3-methylcrotyl alcohol 2-3-fold. In contrast, chemical modification decreases activity toward S-(hydroxymethyl)glutathione (FDH activity) and 1...
An angiogenin binding protein isolated previously from endothelial cells has been shown to be a member of the actin family. Calf pulmonary artery endothelial (CPAE) cells were investigated for the presence of surface actin by immunoblotting of isolated surface proteins and by immunofluorescence. CPAE cell surface proteins were isolated by selective...
Cocatalytic zinc binding sites are characteristic of enzyme molecules which contain two or more zinc and/or other metal atoms. In each site an aspartate, glutamate, or histidine residue simultaneously binds to two zinc atoms or a zinc and a different metal atom. In the resultant amino acid bridge, two of the cocatalytic metal atoms bind to the same...
Rabbit, pig and mouse angiogenins have been purified from blood serum and characterized, and the rabbit and pig proteins have been sequenced fully. A partial sequence of the mouse protein is consistent with the sequence deduced from the genomic DNA (Bond, M.D. and Vallee, B.L. (1990) Biochem. Biophys. Res. Commun. 171, 988–995). All three angiogeni...
The origin of the fatty acid activation and formaldehyde dehydrogenase activity that distinguishes human class III alcohol dehydrogenase (alcohol:NAD^+ oxidoreductase, EC 1.1.1.1) from all other alcohol dehydrogenases has been examined by site-directed mutagenesis of its Arg-115 residue. The Ala- and Asp-115 mutant proteins were expressed in Escher...
A 26-kDa endonuclease has been purified to homogeneity from zinc-sufficient Euglena gracilis. The protein binds to single-stranded DNA with a higher affinity than to double-stranded DNA, but it exhibits nucleolytic activity toward both. Thus, it converts supercoiled plasmid pBR322 DNA into the linear form, a property characteristic of endonucleases...
Human mitochondrial aldehyde dehydrogenase (ALDH-I) is potently, reversibly, and selectively inhibited by an isoflavone isolated from Radix puerariae and identified as daidzin, the 7-glucoside of 4',7-dihydroxyisoflavone. Kinetic analysis with formaldehyde as substrate reveals that daidzin inhibits ALDH-I competitively with respect to formaldehyde...
The 42-kDa angiogenin binding protein isolated previously has been purified to electrophoretic homogeneity. It has been identified as a member of the actin family by peptide mapping and partial amino acid sequencing. The interaction of bovine muscle actin with angiogenin is similar to that of the angiogenin binding protein. Angiogenin induces the p...
This chapter discusses the recent studies of the mammalian ribonuclease inhibitor, and in particular, human placental ribonuclease inhibitor (PRI). Far less is known about this protein inhibitor of ribonucleases than about protein inhibitors of proteinases, for which a vast literature exists. Nevertheless, recent studies have revealed distinctive p...
Chemical modifications of human angiogenin had suggested that arginines are essential for its ribonucleolytic activity [Shapiro, R., Weremowicz, S., Riordan, J. F., & Vallee, B. L. (1987) Proc. Natl. Acad. Sci. U.S.A. 84, 8783-8787]. Each of the six arginines within or near angiogenin's catalytic or cell-binding sites--i.e., those at positions 5, 3...
Crystals of recombinant human angiogenin have been grown from solutions containing sodium potassium tartrate and polyethylene glycol as precipitants. They belong to the space group C222(1) (a = 83.36 A, b = 120.64 A, c = 37.72 A) and contain a single molecule in the asymmetric unit. The crystals diffract to at least 2.3 A resolution and are suitabl...
X-ray crystallographic analysis and sequence comparisons of zinc enzymes have identified onsensus sequences for catalytic and structural zinc binding sites (1). Such reference structures have served to predict zinc binding ligands among members of related zinc enzyme families and also, more noticeable, in apparently unrelated proteins. Thus, compar...
Metal activation of metallothionein gene transcription is mediated by specific promoter sequences, termed metal regulatory elements (MREs). Nuclear extracts prepared from various human cell lines were assayed for their capacity to bind to a synthetic human MREa (hMREa) oligomer. Electrophoretic mobility-shift assays with extracts from control cells...
The metal-binding motif in the sequence of leukotriene A4 (LTA4) (EC 3.3.2.6), a bifunctional zinc metalloenzyme, contains a glutamic acid that is conserved in several zinc hydrolases. To study its role for the two catalytic activities, Glu-296 in mouse leukotriene A4 hydrolase was replaced by a glutamine or alanine residue by site-directed mutagen...
The metal-binding motif in the sequence of leukotriene A_4 (LTA_4) (EC 3.3.2.6), a bifunctional zinc metalloenzyme, contains a glutamic acid that is conserved in several zinc hydrolases. To study its role for the two catalytic activities, Glu-296 in mouse leukotriene A_4 hydrolase was replaced by a glutamine or alanine residue by site-directed muta...
Two distinct regions of angiogenin are critical for angiogenic activity: a catalytic site capable of cleaving RNA and a noncatalytic site, encompassing residues 60-68, which may bind to an endothelial cell-surface receptor [Hallahan, T. W., Shapiro, R., & Vallee, B. L. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 2222-2226]. We have now shown that Asn-...
A 23-residue peptide was synthesized that incorporates the loop which binds the structural zinc atom of mammalian alcohol dehydrogenases and contributes, in part, to subunit interactions in the native enzyme. Neither the amino acid composition nor the sequence of the peptide resemble those of zinc fingers. The reduced peptide stoichiometrically bin...
A comparative study on the metal environment of Zn(II)-carboxypeptidase A (ZnCPD) and Co(II)-carboxypeptidase A (CoCPD) in their solution and crystalline forms using the X-ray absorption fine structure (XAFS) technique has been conducted. The first coordination sphere of Zn for ZnCPD in its solution state is found to consist of two distributions of...
Both class I and class II alcohol dehydrogenase (ADH) activities are present in human serum. The contribution of each class can be measured using two class-specific, fluorogenic substrates, 4-methoxy-1-naphthaldehyde and 6-methoxy-2-naphthaldehyde. The former is highly selective for class I isozymes, especially those containing alpha or gamma subun...
The glutathione-dependent formaldehyde dehydrogenase from Escherichia coli has been purified to homogeneity and characterized. It is a 83,000-kDa homodimer containing 4 g-atom of zinc per dimer with a specific activity of 60 units/mg toward S-(hydroxymethyl)glutathione and NAD+ as substrates. Its isoelectric point, 4.4, is consistent with both its...
Zinc is now known to be an integral component of a large number and variety of enzymes and proteins involved in virtually all aspects of metabolism, thus accounting for the fact that this element is essential for growth and development. The chemistry of zinc, superficially bland, in reality has turned out to be ideally appropriate and versatile for...
The participation of zinc in widely diversified biological reactions focuses attention on its chemistry. A number of its properties relate to its biological utilization and versatility. Its amphoteric properties allow the zinc-coordinated water to exist as a "hydronium" or hydroxide ion even at neutrality. Its coordination sphere is flexible and ad...
The rapid induction of thionein (apometallothionein) by many endogenous stimuli such as steroid hormones, cytokines, and second messengers suggests that this cysteine-rich, metal binding protein participates in an as yet undefined role in cellular regulatory processes. This study demonstrates with DNA and RNA binding assays and in vitro transcripti...
Recombinant mouse leukotriene A4 hydrolase was expressed in Escherichia coli as a fusion protein with ten additional amino acids at the amino terminus and was purified to apparent homogeneity by means of precipitation, anion exchange, hydrophobic interaction and chromatofocusing chromatographies. By atomic absorption spectrometry, the enzyme was sh...
Three mutants of recombinant mouse leukotriene A4 (LTA4) hydrolase (3.3.2.6) were produced by site-directed mutagenesis on cDNA. The codons corresponding to His-295, His-299, or Glu-318 were replaced by codons encoding tyrosine, tyrosine, and glutamine, respectively. The mutated cDNAs were expressed in Escherichia coli, and the three mutated protei...
Human liver class III alcohol dehydrogenase (chi chi-ADH) and glutathione dependent formaldehyde dehydrogenase are the same enzyme. The enzyme, chi chi-ADH, exhibits a kcat of 200 min-1 and a km of 4 microM for the oxidation of formaldehyde, but only in the presence of GSH. In the absence of GSH the enzyme is essentially inactive toward formaldehyd...
Angiogenin, a potent blood vessel inducing protein, was implanted into experimentally injured menisci of 75 New Zealand white rabbits. Localised neovascularisation occurred in 52% of the angiogenin-treated animals, and in 9% of the controls. Neovascularisation induced by angiogenin may enhance healing of injuries within the poorly vascularised meni...
Class III alcohol dehydrogenase (chi chi-ADH) from human liver binds both ethanol and acetaldehyde so poorly that their Km values cannot be determined, even at ethanol concentrations up to 3 M. However, long-chain carboxylates, e.g., pentanoate, octanoate, deoxycholate, and other anions, substantially enhance the binding of ethanol and other substr...
The residues that are indispensable for the ribonucleolytic activity of angiogenin are also known to be essential for its angiogenic activity. We now demonstrate that residues in another region of the protein, devoid of catalytic residues, are additionally required for angiogenesis. Endoproteinase Lys-C or a baby hamster kidney cell protease cleave...
A 42-kDa bovine protein that binds bovine angiogenin [angiogenin binding protein (AngBP)] has been identified as a dissociable cell-surface component of calf pulmonary artery endothelial cells and a transformed bovine endothelial cell line, GM7373. Binding of 125I-labeled bovine angiogenin (125I-Ang) to AngBP occurs with an apparent Kd approximatel...
4-Hydroxyalkenals, natural cytotoxic products of lipid peroxidation, are substrates for human alcohol dehydrogenases (ADH). Class I and II ADHs reduce aliphatic 4-hydroxyalkenals with chain lengths of from 5 to 15 carbons at pH 7 with kcat and Km values comparable to simple aliphatic aldehydes of the same chain length. Class II is particularly effe...
The class III human liver alcohol dehydrogenase, identical to glutathione-dependent formaldehyde dehydrogenase, separates electrophoretically into a major anodic form (chi 1) of known structure, and at least one minor, also anodic but a slightly faster migrating form (chi 2). The primary structure of the minor form isolated by ion-exchange chromato...
We now recognize three distinct motifs of DNA-binding zinc proteins: (i) zinc fingers, (ii) zinc clusters, and (iii) zinc twists. Until very recently, x-ray crystallographic or NMR three-dimensional structure analyses of DNA-binding zinc proteins have not been available to serve as standards of reference for the zinc binding sites of these families...
The interactions of human placental ribonuclease inhibitor (PRI) with bovine pancreatic ribonuclease (RNase) A and human angiogenin, a plasma protein that induces blood vessel formation, have been characterized in detail in earlier studies. However, studies on the interaction of PRI with the RNase(s) indigenous to placenta have not been performed p...
Human gamma 1 gamma 1 alcohol dehydrogenase is quite insensitive to inactivation by iodoacetate, its equine counterpart EE highly sensitive, and the human beta 1 beta 1 form intermediately sensitive. Imidazole hardly influences the iodoacetate inactivation of gamma 1 gamma 1, enhances that of EE and decreases that of beta 1 beta 1. In all isozymes,...