Sasha Englard

[22] Precipitation techniques

Publisher Summary This chapter describes methods for the precipitation of proteins for preparative purposes. Proteins can be precipitated by causing perturbations in the solvent with respect to pH, ionic strength, and temperature. The art and science of protein fractionation by differential solubility and precipitability has reached a pinnacle, and a wide variety of precipitation methods have been discovered empirically. Although some adsorbents, such as alumina and calcium phosphate gels were used, most of the methods employed several steps of ammonium sulfate precipitation. Perturbations that can cause various conformational transitions include (1) a rise in temperature that can weaken the strength of dipolar interactions, such as hydrogen bonds and can favor formation of hydrophobic interactions and (2) a decrease in temperature that can cause the reverse. Quantitative aspects of the resultant transitions in structure depend on the total numbers of specific kinds of interactions and variation in energies among individual interactions of the same kind. The inclusion of water-miscible solvents in the medium in which the protein is dissolved represents a considerable perturbation.

Analysis for protein modifications and nonprotein cofactors.

Publisher Summary This chapter discusses protein modifications and non-protein cofactors. Numerous peptide hormones, hormone-releasing factors, and neurotransmitters are amidated at their carboxyl-terminal residues. Peptides in this group are generally synthesized with a glycine residue on the carboxyl terminus, as confirmed by the sequence of the specifying DNA or terminal codon of the mRNA. An indication that a given peptide undergoes α-amidation can be gleaned from comparison of the amino acid sequence deduced from coding nucleotide sequences with the amino acid sequence determined from analysis of the protein. The anchor is attached covalently to the carboxyl-terminal residue of the protein through an amide linkage with a phosphoethanolamine residue; it is noncovalently inserted into the membrane by means of the inositol phospholipid portion of the anchor. In contrast to the enzymes involved in chemotaxis that methylate L-glutamate residues, another class of enzymes known as “D-aspartate,” L-isoaspartate-methyltransferases catalyze the formation of methyl esters of D-aspartate and L-isoaspartate residues. The residual protein contains residues of diaminobutyric acid in place of methyl esters of glutamic acid and diaminopropionic acid in place of residues of aspartic acid.

Beef-heart malic dehydrogenases III. Comparative studies of some properties of M-malic dehydrogenase and S-malic dehydrogenase

Abstract The two malic dehydrogenases from beef heart, identified as being supernatant and mitochondrial in origin, have been compared further with regard to kinetic behavior and additional striking differences have been observed. With respect to sulfhydryl groups of mitochondrial malic dehydrogenase and supernantant malic dehydrogenase the following have been established. First, mitochondrial malic dehydrogenase has twice the number of half-cystine residues contained in supernatant malic dehydrogenase (12 as opposed to 6). Secondly, all sulfhydryl groups of undenatured mitochondrial malic dehydrogenase can be titrated with p -mercuribenzoate, although reaction occurs slowly, while only half of the sulfhydryl groups of supernantant malic dehydrogenase can be titrated even in presence of excess reagent. Finally, the titration of mitochondrial malic dehydrogenase with p -mercuribenzoate results in loss of enzymic activity after addition of only three equivalents of reagent, whereas no loss of activity of supernatant-malic dehydrogenase occurs after half its sulfhydryl groups have been titrated. Significant differences in amino acid composition exist between mitochondrial malic dehydrogenase and supernatant malic dehydrogenase. The latter enzyme contains more lysine, arginine, tyrosine, methionine, aspartic acid and tryptophan than does the former. On the other hand, mitochondrial malic dehydrogenase contains more phenylalanine, glycine, proline and threonine.

[18] Mitochondrial l-malate dehydrogenase of beef heart: [EC 1.1.1.37 l-Malate: NAD oxidoreductase]

Publisher Summary This chapter discusses the Mitochondrial L-malate dehydrogenase of beef heart activity measured spectrophotometrically by the increase in absorption at 340 mμ because of the NAD+ reduction. Various methods and modifications of existing procedures are reported for the purification of mammalian heart muscle mitochondrial malate dehydrogenases. The procedure described is for the purification of beef heart mitochondrial malate dehydrogenase that includes a number of steps for the preparation of pig heart malate dehydrogenase. The precautions for the steps requiring calcium chloride and ethanol are incorporated. All operations are performed at 0-5° unless otherwise specified. The enzyme appears to be homogeneous as determined by ultracentrifugation studies and electrophoretic analysis over a wide pH range. Although the mitochondrial malate dehydrogenases from several species are reported to contain no tryptophan, the corresponding enzyme from beef heart muscle contains one residue of tryptophan per mole.

Transcriptional control of glucoamylase synthesis in vegetatively growing and sporulating Saccharomyces species

Three unlinked, homologous genes, STA1, STA2, and STA3, encode the extracellular glycosylated glucoamylase isozymes I, II, and III, respectively, in Saccharomyces species. S. cerevisiae, which is sta0 (absence of functional STA genes in haploids), does carry a glucoamylase gene, delta sta, expressed only during sporulation (W. J. Colonna and P. T. Magee, J. Bacteriol. 134:844-853, 1978; I. Yamashita and S. Fukui, Mol. Cell. Biol. 5:3069-3073, 1985). In this study we examined some of the physiological and genetic factors that affect glucoamylase expression. It was found that STA2 strains grown in synthetic medium produce glucoamylase only in the presence of either Maltrin M365 (a mixture of maltooligosaccharides) or starch. Maximal levels of glucoamylase activity were found in cells grown in rich medium supplemented with glycerol plus ethanol, starch, or Maltrin. When various sugars served as carbon sources they all supported glucoamylase synthesis, although at reduced levels. In any given growth medium glucoamylase isozyme II synthesis was modulated by functionality of the mitochondria. Synthesis of glucoamylase is continuous throughout the growth phases, with maximal secretion taking place in the early stationary phase. In the various regimens, the differences in enzyme accumulation are accounted for by differences in the levels of glucoamylase mRNA. Both glucoamylase mRNA and enzyme activity were drastically and coordinately inhibited in MATa/MAT alpha diploids and by the presence of the regulatory gene STA10. Both effects were partially overcome when the STA2 gene was present on a multicopy plasmid. The STA2 mRNA and glucoamylase were coinduced in sporulating STA2/STA2 diploids. A smaller, coinduced RNA species was also detected by Northern blotting with a STA2 probe. The same mRNA species was detected in sporulating sta0 diploids and is likely to encode the sporulation-specific glucoamylase.

Evaluation of the proportion of the keto form of D-fructose and related 2-ketohexoses present in aqueous solution

Abstract The acyclic forms of D -fructose and some related compounds have been studied by absorption spectroscopy and circular dichroism. These compounds exhibit absorption and ellipticity bands near 275 nm attributed to the keto group of the acyclic form. At mutarotational equilibrium in aqueous solution, D -fructose exists to the extent of 2% in the acyclic form, whereas solutions of D -fructose 1-phosphate, D -fructose 6-phosphate, and D -fructose 1,6-diphosphate contain up to 10 times this proportion of the acyclic form. On the basis of circular dichroism spectra, these phosphoric esters, as well as certain disaccharides of D -fructose, could be differentiated with respect to the position of substitution. The differences in position and magnitude of the dichroic bands were related to specific, conformational features of these molecules.

γ-Butyrobetaine hydroxylation to carnitine in mammalian kidney

Abstract Activity of γ-butyrobetaine hydroxylase (γ-butyrobetaine, 2-oxoglutarate dioxygenase; EC 1.14.11.1) in liver and kidney of several mammalian species was assayed by measurement of tritium release from γ-[2,3- 3 H]butyrobetaine. Crude extracts from cat, hamster, rabbit, and Rhesus monkey kidneys effectively converted γ-butyrobetaine to carnitine. In these species, the levels of hydroxylating activity in kidney exceeded or nearly equaled the level of γ-butyrobetaine hydroxylase activity in the corresponding liver. In contrast, dog, guinea pig, mouse, and rat kidney exhibited no or insignificant capacity to hydroxylate γ-butyrobetaine. The notion that the liver is the exclusive or primary site of carnitine synthesis must be reconsidered at least for some mammalian species.

Hydroxylation of γ‐butyrobetaine to carnitine in human and monkey tissues

Carnitine serves as a carrier of acyl groups into and out of mitochondria and hence is considered to have a central function in the metabolic utilization and synthesis of fatty acids [l-4]. In the rat, the species most extensively studied, adipose tissue, heart, kidney and skeletal muscle catalyze the entire sequence of reactions from e-N-trimethyl-L-lysie to r-butyrobetaine; the same tissues, however, are reported not to hydroxylate the latter compound to carnitine [5-81. Indeed, several studies suggested that the liver is the primary site of carnitine biosynthesis, and that the testis is a secondary site with much less capacity for such synthesis [6-91. In fact it has been considered that extrahepatically synthesized y-butyrobetaine is converted to carnitine exclusively in the liver, and that the carnitine so formed is rapidly transported to other organs [7,10]. That species differences may exist in tissue localization of carnitine synthesis was evidenced by the finding that, in the sheep, kidney and muscle tissues have significant capacity to hydroxylate y-butyrobetaine, albeit relatively low as compared with liver (11). More recently, crude extracts from kidneys of cat, hamster, rabbit, and a single Rhesus monkey were shown to convert y-butyrobetaine to camitine [ 12,131. In those species, the levels of y-butyrobetainehydroxylating activity in kidney nearly equaled or exceeded that measured in the corresponding liver. In contrast, dog, guinea pig, mouse and rat kidney exhibited little or no capacity to hydroxylate y-butyrobetaine. In this study, in which additional primate species

Biochemical and immunological characterization of the STA2-encoded extracellular glucoamylase from saccharomyces diastaticus☆

In Saccharomyces diastaticus each one of three unlinked genes (STA1, STA2, STA3) encodes a glucoamylase (alpha-1,4 glucanglucohydrolase, EC 3.2.1.3) that allows yeast to grow on starch. The enzyme encoded by the STA2 gene (glucoamylase II) has been purified from culture medium to near homogeneity by ethanol precipitation, Trisacryl M DEAE chromatography, and HPLC gel filtration. Glucoamylase II consists of two identical subunits whose average size is 300 kDa. Under denaturing conditions, the native dimeric enzyme readily dissociates to a monomer. Enzymatic deglycosylation of denatured enzyme gives rise to intermediate, partially glycosylated forms and to a 56-kDa completely deglycosylated protein. Glucoamylase releases glucose units by cleaving alpha-1,4 bonds from the nonreducing end of different oligosaccharides, but has only a barely detectable alpha-1,6 hydrolyzing activity. The pH optimum for the purified enzyme was found to be 5.1. The enzyme has a greater affinity for maltohexaose (Km = 0.98 mM, V/Km = 2.39) than for maltotriose (Km = 2.38, V/Km = 0.68) or maltose (Km = 3.20, V/Km = 0.39). Both polyclonal and monoclonal antibodies have been raised against glucoamylase II. The polyclonal antibodies specifically inhibit yeast glucoamylase II activity in a dose-dependent manner, but are found to immunoblot other yeast glycoproteins as well. This oligosaccharide-specific reaction can be competed out by adding excess mannan without affecting glucoamylase reactivity. The cross-reactivity of the polyclonal antibodies with other amylolytic enzymes correlates well with evolutionary distance. Evidence is presented that monoclonal antibodies specific for either carbohydrate or protein epitopes have been obtained.

Purification and properties of calf liver γ-butyrobetaine hydroxylase

Abstract Calf liver γ-butyrobetaine hydroxylase has been purified some 400-fold by DEAE, gel permeation, and hydroxylapatite chromatography. The homogeneous enzyme is a dimer of 46,000-dalton subunits. The K m values for substrates and cofactors and the apparent activation constants for ascorbate and catalase have been determined. Inhibition of the enzyme by a number of divalent metals supports the function of sulfhydryl groups in metal binding. An antibody to the enzyme has been obtained; this does not cross-react with homogeneous γ-butyrobetaine hydroxylase from a Pseudomonas strain. The antibody, coupled to Sepharose 4B, has been used to purify the calf liver hydroxylase 350-fold in one step.