Bert L. Vallee

The function of metallothionein

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 in brain, and (3) interactions with glutathione and zinc proteins.

Kudzu root: An ancient chinese source of modern antidipsotropic agents

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 in China for more than a millennium, their true efficacy, active constituents, sites and mechanisms of action have never been critically examined. Recently, we have demonstrated that a crude extract of Radix puerariae suppresses the free-choice ethanol intake of ethanol-preferring golden Syrian hamsters and have identified two of its isoflavones, daidzin and daidzein, that account for this effect. Since then, we and other investigators have confirmed these findings in rats that were either trained or genetically bred to prefer and consume large amounts of ethanol. This article summarizes recent progress on the pharmacological and biochemical studies of the antidipsotropic isoflavones isolated from Radix puerariae.

Zinc metallothionein imported into liver mitochondria modulates respiration

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 β-domain of MT; in this system, the isolated C-terminal α-domain is inactive. Free zinc inhibits respiration at concentrations commensurate with the zinc content of either MT or the isolated β-domain, indicating that MT inhibition involves zinc delivery to mitochondria. Respiratory inhibition of uncoupled mitochondria identifies the electron transfer chain as the primary site of inhibition. The apoform of MT, thionein, is an endogenous chelating agent and activates zinc-inhibited respiration with a 1:1 stoichiometry ([zinc binding sites]/[zinc]). Carbamoylation of the lysines of MT significantly attenuates the inhibitory effect, suggesting that these residues are critical for the passage of MT through the outer mitochondrial membrane. Such an import pathway has been proposed for other proteins that also lack a mitochondrial targeting sequence, e.g., apocytochrome c, and possibly Cox17, a mitochondrial copper chaperone that is the only protein known so far to exhibit significant primary sequence homology to MT. The presence and respiratory inhibition of MT in liver, but not heart, mitochondria suggest a hitherto unknown biological modulating activity of MT in cellular respiration and energy metabolism in a tissue-specific manner.

[44] Nitration with tetranitromethane

Publisher Summary Tetranitromethane (TNM) is examined for its suitability to modify amino acid residues in peptides, polymers, and proteins. It is shown that the reagent can be highly specific for the nitration of tyrosyl residues. Selective modification is achieved readily because reaction conditions are very mild. The reaction can be quantitated easily by spectrophotometry because nitrotyrosine absorbs in the visible region of the spectrum, and by amino acid analysis. Further, other products of the reaction, nitroformate and protons, can also serve as a means of quantitation. Under the usual reaction conditions TNM also oxidizes sufhydryl groups. TMN forms a charge-transfer complex with phenoxide ion, in aqueous solution, thus accounting for the pH-dependence of the reaction. An intermediate sulfenyl nitrate is formed which, in the presence of excess thiol, that is, low molar excesses of TNM, gives the disulfide and nitrite ion. TNM can be highly specific for tyrosyl modification when it is employed under the mild conditions described. Nitration with TNM is used successfully to study tyrosyl residues in many enzymes and proteins.

Thermolysin: A zinc metalloenzyme

Metal analyses and inhibitor studies have shown that thermolysin, a neutral protease from B. thermoproteolyticus, is a zinc metalloenzyme. The relevance of this finding to the active site characteristics of other bacterial neutral proteases and to those of alkaline proteases is considered.

The Metallothionein/Thionein System : An Oxidoreductive Metabolic Zinc Link

Metallothioneins (MTs) were discovered more than 50 years ago and identified as low‐molecular weight, sulfhydryl‐rich proteins that were subsequently found to bind zinc predominantly. The binding of seemingly redox inactive zinc ions allows MT to play a central role in oxidoreductive cellular metabolism, cellular zinc distribution and homeostasis. In this interpretive study, we discuss the interaction of MT with physiologically relevant molecules and its effect on zincthiolate bonds. These interactions are linked to recent progress in the functional role of MT in cellular zinc transport, energy production, and protection of the organism against oxidative stress and neurodegenerative diseases.

[36] Reactions with N-ethylmaleimide and p-mercuribenzoate

Publisher Summary This chapter presents the reactions of two reagents, p -mercuribenzoate (PMB) and N-ethylmaleimide (NEM), in particular, are readily quantitated because of an alteration in spectral characteristics and because the products can be measured directly as well. The former has long been thought to be a highly specific, but reversible sulfhydryl reagent and has been utilized successfully as a quantitative reagent for measuring sulfhydryl groups in proteins. NEM has received considerable attention, particularly because its reaction product is stable even to acid hydrolysis and has also been employed as a quantitative reagent. The methods by which these two compounds may be used to modify sulfhydryl groups of enzymes are essentially the same as those established for determining the number of such groups in nonenzymatic proteins, with the exception that prior denaturation is obviously omitted when the establishment of functional significance is the ultimate objective. Quantitation of the reaction is one of the major prerequisites for defining the basis of altered activity consequent to chemical modification of an enzyme, therefore, the methods employed are discussed from that aspect.

Zinc and metalloenzymes.

Publisher Summary The chapter discusses the zinc and metalloenzymes. Two groups of proteins that associate with metals can be differentiated. Members of each group may or may not possess known enzymatic activities.In one group, the metalloproteins, a given metal is combined with the protein in a unique manner, such that the two can be thought of as an “entity” in nature. A second group, in which a protein combines reversibly with one of several different cations, the metal-protein complexes, has been much more resistant to definitive appraisal. Metal-enzyme complexes, a subgroup of metal-protein complexes, exhibit enzymatic activity consequent to readily dissociable combination with a variety of metal ions. The study of metal complexes and particularly metal chelates has provided both new experimental and new theoretical backgrounds for the study of metals in relation to the specificity of enzyme action, metal-enzyme, metal-substrate, and metalloenzyme interaction, as well as nietal-enzyme inhibition. The chapter elaborates on the metalloenzymes several distinct characteristics, which may be well to inspect and analyze. The characteristics of the metal-protein bond serve as the primary parameter for the differentiation of metalloenzymes from metal-enzyme complexes. For the metalloenzymes, an empirical formula based on the molecular weight of the protein, the ratio of moles of metal/moles of protein, and that of moles of nietal/moles of coenzyme when present, expresses the stoichiometric relationships of the components of the active holoenzyme to one another. Four zinc metalloproteins have been characterized in the chapter: the carbonic anhydrase of ox erythrocytes, the zinc-containing protein of human leukocytes, the carboxypeptidase of bovine pancreas, and the alcohol dehydrogenase of yeast.

New human liver alcohol dehydrogenase forms with unique kinetic characteristics

Abstract All adult and infant human liver homogenates studied thus far show two previously unreported forms of alcohol dehydrogenase on starch gel electrophoresis. Under the conditions employed, these forms migrate toward the anode and readily stain for pentanol but virtually not for ethanol oxidizing activity. In contrast, all human ADH isoenzymes identified previously are cathodic and react equally well with either substrate. These new ADH forms have been separated from the other known ones by DEAE-cellulose chromatography and are then purified on Agarose-hexane-AMP. Although the physical characteristics of the new anodic ADH forms are similar to those of the known human ADH isoenzymes, the former are not inhibited by 12 mM 4-methyl pyrazole, oxidize ethanol very poorly and appear to prefer longer chain alcohols as substrates.

The Magnesium-Deficiency Tetany Syndrome in Man

MAGNESIUM is second only to potassium in abundance as an intracellular cation. The whole body of a human adult contains nearly 25 gm.1 The numerous important biochemical reactions dependent upon th...