Sasha Englard

• Albert Einstein College of Medicine

The department of biochemistry is the oldest basic sciencedepartment at the Albert Einstein College of Medicine. Its firstchair, who was also the first faculty member to be appointed tothe medical school itself, was hired in 1952, three years beforethe fledgling institution opened its doors to the first enteringclass. Since those early days, the de...

Forty-one aromatic and aliphatic analogs of alpha-ketoglutarate were studied kinetically for their interaction with the alpha-ketoglutarate binding site of gamma-butyrobetaine hydroxylase obtained from Pseudomonas sp. AK1. Together, the compounds represent structural permutations probing the contribution of: 1) the C5 carboxyl group of alpha-ketogl...

Starting with phosphonates such as (I) or (IX), cyclopropyl-substituted γ-butyrobetaines, e.g. (VIII) or (X), are prepared.

The two C-2 monodeuterated isomers of L-carnitine were synthesized by enzymatic hydration of crotonobetaine in D2O and by enzymatic proton exchange of L-[2-2H2]carnitine in H2O. These reactions, catalyzed by an induced Escherichia coli carnitine hydrolyase proceed stereospecifically. The two isomers of L-[2-2H]carnitine were examined by 1H NMR at 5...

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...

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...

The influence of the bacteriophage phi X174 (phi X) C protein on the replication of bacteriophage phi X174 DNA has been examined. This small viral protein, which is required for the packaging of phi X DNA into proheads, inhibits leading strand DNA synthesis. The inhibitory effect of the phi X C protein requires a DNA template bearing an intact 30-b...

The mono- (2) and bis-phosphate (3) derivatives of D-threo-2,5-hexodiulose (1) (5-keto-D-fructose) were synthesized enzymically and purified by anion-exchange chromatography. The proportions, sizes of ring, and anomeric configurations were determined by F.t. 31P- and 13C-n.m.r. spectroscopy. Compound 2 was found to exist preponderantly (70-78%) in...

The stereochemical course of the aliphatic hydroxylation of gamma-butyrobetaine by calf liver and by Pseudomonas sp AK1 gamma-butyrobetaine hydroxylases has been determined. With [3(RS)-3-3H]-gamma-butyrobetaine or [3(R)-3-3H]-gamma-butyrobetaine as substrate, a rapid and significant loss of tritium to the medium occurred. On the other hand, with [...

The production of carnitine from peptide-bound 6-N-trimethyl-L-lysine (Lys(Me3)) or 4-N-trimethyl-aminobutyrate(gamma-butyrobetaine) perfused through isolated guinea pig livers was investigated. [Methyl-3H] Lys(Me3)-labeled agalacto-orosomucoid (AGOR) and asialofetuin were rapidly taken up and degraded by the perfused liver. Most of the free Lys(Me...

The production of carnitine from peptide-bound 6-N-trimethyl-L-lysine (Lys(Me3)) or 4-N-trimethyl-aminobutyrate(gamma-butyrobetaine) perfused through isolated guinea pig livers was investigated. [Methyl-3H] Lys(Me3)-labeled agalacto-orosomucoid (AGOR) and asialofetuin were rapidly taken up and degraded by the perfused liver. Most of the free Lys(Me...

Primary and secondary tritium kinetic isotope effects have been determined for the reactions catalyzed by purified preparations of gamma-butyrobetaine hydroxylase obtained from Pseudomonas sp AK 1 and from calf liver. With [methyl-14C,(3R)-3-3H]-gamma-butyrobetaine as substrate, the bacterial hydroxylase was found to exhibit a primary T(V/K) of 1.3...

Hydroxylation of 6-N-trimethyl-l-lysine(lys(Me3)) to 3-hydroxy-6-N-trimethyl-l-lysine(3-HO-lys(Me3)) by several rat tissues has been examined and compared. The kidney enzyme, which previously was shown to require molecular oxygen and α-ketoglutarate as cosubstrates, ferrous iron and ascorbate as cofactors, and to be stimulated by catalase, has a br...

Following receptor-mediated endocytosis of trimethyllysine-labeled asialofetuin and agalacto-orosomucoid by liver parenchymal and nonparenchymal cells, respectively, the glycoproteins are degraded and the methylated lysine residues released. The free intracellular trimethyllysine is then converted, in addition to 2-N-acetyl-6-N-trimethyllysine, to...

Uptake of carnitine by cultured human fetal lung flbroblasts (WI-38 and IMR-90) and by smooth muscle cells from calf aorta and from human uterus was found to be temperature dependent and saturable. IMR-90 cells showed an apparent Km of 6–8 μM and a V of 21–28 pmol/h/106 cells for l-carnitine. Transport was abolished by N-ethylmaleimide and was inhi...

5-Keto-D-fructose (5KF) is isolated from cultures of Gluconobacter cerinus growing on D-fructose as the sole carbon source. 5KF is a substrate for hexokinase, fructokinase, and several polyol dehydrogenases. 1H and 13C nuclear magnetic resonance studies show that 5KF exists in different forms in anhydrous dimethyl-d6 sulfoxide and D2O. In dimethyl-...

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 Km values for substrates and cofactors and the apparent activation constants for ascorbate and catalase have been determined. Inhibition of the enzyme by...

6-N-(3-³H)Trimethyl-DL-lysine was synthesized from 6-N-acetyl-L-lysine by the following chemical scheme: 6-N-acetyl-L-lysine ..-->.. 2-keto-6-N-acetylcaproic acid ..-->.. 2-(3-³H)keto-6-N-acetylcaproic acid ..-->.. 2-(3-³H)keto-6-N-acetylcaproic acid oxime ..-->.. 6-N-(3-³H)acetyl-DL-lysine ..-->.. DL-(3-³H)lysine ..-->.. 2-N-(3-³H)formyl-DL-...

A method for the assay of gamma-butyrobetaine hydroxylase activity is described. The procedure is based on the measurement of 3H2O formed from [2,3-3H]gamma-butyrobetaine. The formation of 3H2O was essentially linear with time of incubation and enzyme concentration. Despite a significant isotope effect that causes the extent of hydroxylation to be...

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-[2,3-3H]butyrobetaine. Crude extracts from cat, hamster, rabbit, and Rhesus monkey kidneys effectively converted γ-butyrobetaine to carnitine. In th...

Lysyl hydroxylase from chick embryos was strongly inhibited by heat-denatured collagens from various vertebrate sources, and by separated a chains and β components of rat tail tendon collagen. The kinetics exhibited with this enzyme when heat-denatured calf or rabbit skin collagens was used showed a mixed type of inhibition. On the other hand, a pr...

This chapter describes the assay method, purification procedure, and properties of 5-keto-D-fructose reductase from gluconobacter cerinus. The reduction of 5-keto-D-fructose is followed by measuring the oxidation of NADPH spectrophotometrically at 340 nm. Assays are carried out in cuvettes of 10 mm light path maintained at 30 ° and containing 0.8 m...

This chapter describes the assay method, purification procedure, and properties of 5-keto-D-fructose reductase from yeast. Yeast 5-keto-D-fructose reductase is measured spectrophotometrically by the decrease in absorption at 340 nm due to NADPH oxidation in the presence of 5-keto-D-fructose. An NADPH:5-keto-D-fructose reductase activity was initial...

This chapter describes the assay method, purification procedure, and properties of aldohexose dehydrogenase from gluconobacter cerinus. The assay is based on the spectrophotometric determination of NADPH formed with D-mannose as substrate. The assay system (1.01–1.02 ml), in addition to enzyme, contains: 0.8 ml of Tris HC1, 0.1 ml of D-mannose and...

This chapter focuses on 5-keto-D-fructose and its phosphate esters. 5-ketofructose (5KF) is produced by oxidation of D-fructose through all phases of bacterial growth. It is consumed by the growing cells until the pH of the medium drops to 3.5–3.8. When growth ceases, D-fructose is converted stoichiometrically to 5KF, which then accumulates in the...

cis,cis-Muconate cycloisomerase (cis,cis-muconate lactonizing enzyme, EC 5.5.1.1.) was purified in crystalline form from Pseudomonas putida. Ultracentrifugation studies, as well as gel filtration chromatography and electrophoresis, indicate that the enzyme is an oligomeric protein of molecular weight 252,000 (s20,w 12.20 × 10−13 s), which is built...

A mutant of Gluconobacter cerinus var. ammoniacus, IFO 3267, has been isolated which is deficient with respect to fructose 5-dehydrogenase, the enzyme catalyzing the oxidation of d-fructose to 5-keto-d-fructose (5 KF). Growth of this mutant on fructose as the sole carbon source was impaired unless the culture medium was supplemented with 5 KF. Sign...

Heat-denatured collagens obtained from tissues of several vertebrate species strongly inhibited prolyl hydroxylase activity of both chick embryos and WI-38 fetal human lung fibroblasts. With 3,4-3H-L-proline-labeled “protocollagen” (“deoxycollagen” chains) as substrate and one of several kinds of unlabeled or tritium-labeled collagens as inhibitor,...

The accumulation of 5-keto-d-fructose (5KF) by Gluconobacter cerinus grown on d-fructose in unbuffered medium was shown to be optimal at pH 4.0 after cell growth ceased. During the exponential phase of growth or at neutral pH after the onset of the stationary phase, 5KF production continued but did not accumulate because of its rapid reutilization...

1.1. Skeletal muscle and yeast phosphofructokinase (ATP: D-fructose 6-phosphate 1-phosphotransferase, EC 2.7.1.11) were shown to catalyse the phosphorylation of 5-keto-D-fructose 1-phosphate (D-threo-2,5-hexodiulose 1-phosphate) in the presence of ATP. The product of the reaction was isolated and identified as 5-keto-D-fructose 1,6-bisphosphate.2.2...

Rabbit muscle aldolase was reduced with sodium borohydride (or cyanoborohydride) in the presence of specifically labeled [3H]- and [14C]fructose 1,6-diphosphate. Under steady-state conditions, about 10–15% of the specific ϵ-lysine catalytic sites were bound to fructose 1,6-diphosphate by a covalent ketimine linkage. The remaining substituted lysine...

1.1. Liver fructokinase (ATP: D-fructokinase I-phosphotransferase, EC 2.7.1.3) was shown to catalyze the phosphorylation of 5-keto-D-fructose (D-threo-2,5-hexodiulose) in the presence of ATP. The product was identified as 5-keto-D-fructose I-phosphate.2.2. The monophosphate ester of 5-keto-D-fructose was found to inhibit competitively the action of...

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 2̃% in the acycli...

A specific reduced nicotinamide dinucleotide phosphate-linked 5-keto-d-fructose reductase has been isolated and purified approximately 300-fold from extracts of bakers' yeast. The reaction catalyzed by the enzyme was found to be 5-Keto-d-fructose + NADPH + H+ → l-sorbose + NADP+ Km values for NADPH and 5-keto-d-fructose were 4.6 x 10-5 m and 1 x...

The molecular weight of bovine heart supernatant malic dehydrogenase has been determined by sedimentation equilibrium. The enzyme was shown to be a dimer with a molecular weight of 72,000. Upon reaction with maleic anhydride or in the presence of guanidinium chloride, the enzyme was dissociated into two subunits having identical molecular weights o...

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 pr...

The chapter presents lists of labelled citric acid cycle intermediates and discusses the general procedures for separation, purification, isolation, and analysis of these intermediates. The separation of citric acid cycle intermediates and other organic acids are achieved by partition chromatography on silica gel or similar media and by elution chr...

The chapter describes the method for the preparation of crystalline beef heart muscle extramitochondrial malate dehydrogenase. All operations are carried out at 3-5°. All additions of solid ammonium sulfate are carried out at 0°; the pH of the mixture is maintained between 7.1 and 7.3 by a dropwise addition of approximately 2 N ammonium hydroxide....

The molecular weight of bovine heart supernatant malic dehydrogenase has been determined by sedimentation equilibrium. The enzyme was shown to be a dimer with a molecular weight of 72,000. Upon reaction with maleic anhydride or in the presence of guanidinium chloride, the enzyme was dissociated into two subunits having identical molecular weights o...

The circular dichroism spectra of a number of uronic acids with equatorial hydroxyl groups at C-4 were examined and compared with the spectra of uronic acids with axial hydroxyl groups at C-4. In aqueous solution, the derivatives of glucuronic and mannuronic acids exhibited negative circular dichroism bands near 234 mμ and positive bands with maxim...

The optical rotatory dispersion properties of uronic acids and acetamido sugars have been investigated. For both categories of sugars, two optically active transitions have been implicated as the foremost factors determining the shapes of the rotatory of dispersion curves in the spectral region below 230 nm. By a study of difference curves, the rot...

A soluble NADP-linked aldohexose dehydrogenase has been isolated and purified about 100-fold from extracts of Gluconobacter cerinus cells. The enzyme catalyzes the oxidation of D-glucose, D-mannose, 2-deoxy-D-glucose, and 2-amino-2-deoxy-D-mannose with apparent Km values at pH 8.0 of 5.3 × 10⁻³ M, 1.8 × 10⁻³ M, 3.9 × 10⁻² M, and 2.9 × 10⁻² M, respe...

Yeast hexokinase was shown to catalyze the phosphorylation of 5-keto-D-fructose (D-threo-2,5-hexodiulose) in the presence of ATP. The product was identified as a monophosphate ester. A procedure for the preparation of this ester and some of its properties are described. Reduction of the monophosphate ester by the NADPH-5-keto-D-fructose reductase f...

Only 3 of the 6 sulfhydryl groups of native bovine heart supernatant malate dehydrogenase (S-MDH) react with p-mercuribenzoate (PMB) with no loss of enzymatic activity. In addition, prolonged incubation of S-MDH in 2.6 m urea solutions does not significantly alter the catalytic properties of the enzyme. In 2.6 m urea, however, all 6 sulfhydryl grou...

Only 3 of the 6 sulfhydryl groups of native bovine heart supernatant malate dehydrogenase (S-MDH) react with p-mercuribenzoate (PMB) with no loss of enzymatic activity. In addition, prolonged incubation of S-MDH in 2.6 m urea solutions does not significantly alter the catalytic properties of the enzyme. In 2.6 m urea, however, all 6 sulfhydryl grou...

Ferritin exhibits a plain optical rotatory dispersion (ORD) curve in the spectral region from 600 to 250 mμ. The curve obtained in the far-ultraviolet region with a trough at 233 mμ and peak at 198 mμ is typical of a protein containing an appreciable content of helical segments. The rotatory parameters which were obtained using the Moffitt-Yang and...

Equilibration of maleate in D2O with a partially purified preparation of maleate hydratase from rabbit kidney resulted in the formation of d-malate containing 1 atom of stably bound deuterium per molecule. The enzyme therefore catalyzes a stereospecific addition to the double bond of maleate. The product of the enzymatic hydration in D2O was examin...

The intramolecular interactions of flavinadenine dinucleotide (FAD) have been investigated using optical rotatory dispersion techniques. A Cotton effect with a trough at 395 mμ and a peak at 355 mμ was observed for flavin-adenine dinucleotide in aqueous solution. An equimolar mixture of its constituent mononucleotides, riboflavin 5′-phosphate and a...

Optical rotatory dispersion curves have been obtained for a series of oligosaccharides. The rotatory data for the oligosaccharides, over the spectral region 600−185 mμ, where in general agreement with the sum of the rotations of the constituent monomeric residues. Distinct features of shape of the rotatory dispersion curves, including changes in th...

The direction of optical rotatory dispersion curves in the far-ultraviolet spectral region has been related to configuration and conformation of the ring forms of sugars. Rules have been proposed for the study of ketoses, higher-carbon sugars, and furanoid forms of sugars. Agreement has been established with previous treatments of optical rotatory...

The direction of optical rotatory dispersion curves in the far-ultraviolet spectral region has been related to configuration and conformation of the ring forms of sugars. Rules have been proposed for the study of ketoses, higher-carbon sugars, and furanoid forms of sugars. Agreement has been established with previous treatments of optical rotatory...

Kinetic studies on beef heart supernatant malic dehydrogenase were carried out in 0.1 m triethanolamine-acetate buffer. Analysis of the data by the method of Dalziel revealed that specific relationships between calculated kinetic parameters did not conform to the criteria proposed for mechanisms involving sequential addition for each pair of substr...

Kinetic studies on beef heart supernatant malic dehydrogenase were carried out in 0.1 m triethanolamine-acetate buffer. Analysis of the data by the method of Dalziel revealed that specific relationships between calculated kinetic parameters did not conform to the criteria proposed for mechanisms involving sequential addition for each pair of substr...

The stoichiometry of reduced diphosphopyridine nucleotide binding to supernatant malic dehydrogenase (S-MDH) was determined by fluorometric techniques and a Sephadex gel filtration procedure. Both types of measurement yielded a stoichiometry of 1 mole of DPNH bound per mole of enzyme. This stoichiometry was unaffected by the presence of d-malate, a...

A specific reduced nicotinamide adenine dinucleotide phosphate-linked 5-keto-d-fructose reductase has been isolated and purified approximately 1200-fold from extracts of Gluconobacter cerinus. The reaction catalyzed was found to be 5-Keto-d-fructose + NADPH + H+ ⇌ d-fructose + NADP+ with an equilibrium far towards the right (Keq = 2 x 1010). Km val...

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 b...

Differential extraction of beef-heart muscle revealed the presence of two malic dehydrogenases. One, apparently derived from the supernatant fraction, is easily extractable and exhibits no inhibition of oxidation of DPNH by elevated concentrations of oxaloacetate. The other, presumably mitochondrial in origin, requires more drastic means of extract...

Malic dehydrogenase has been purified from acetone-dried powders of thoroughly washed minces of whole beef heart.

Malic dehydrogenase has been purified from acetone-dried powders of thoroughly washed minces of whole beef heart.The enzyme appears to be homogeneous as determined by ultrcentrifugation studies and electrophoretic analysis over a wide pH range. The enzyme was found to have a sedimentation velocity constant of 4.3·10−13 sec at 20° and a diffusion co...

Some observations on the reactions within the tricarboxylic acid cycle ; involving the reversible formation of carbon--carbon unsaturations are reviewed, ; and the results of a study on an anaerobic exchange of deuterium from the medium ; into succinate, as catalyzed by heart particle succinic oxidase preparations, are ; discussed in terms of the s...