K. Bruce Jacobson

The interaction of cadmium and certain other metal ions with proteins and nucleic acids

The toxic effects of cadmium and other selected divalent cations are presumed to be related to specific chemical and physical characteristics of the ion. The chemistry of cadmium and metal ions in general is reviewed from the viewpoint of such relevant properties as ion polarizability, electronic structure, and the hard-soft characteristics. The softness of metal ions is seen as a useful single parameter to correlate with the affinity for nucleic acids and proteins and with toxic effects. The effects of cadmium on nucleic acids and proteins are examined for a number of specific cases to illustrate the variety of interactions that are well recognized and to demonstrate the utility of soft metal ions as reagents and probes for examining the relationship of structure and function in these macromolecules.

Comparison of techniques for enzyme immobilization on silicon supports

Abstract Enzyme immobilization onto silicon substrates has been investigated by five different coupling procedures. The methods included covalent coupling either through a metal link reagent or silane reagents containing pendant amino or epoxide linkers, an entrapment technique using a thin layer of gelatin, or an adsorption technique using poly- l -lysine. These immobilization procedures were evaluated using glucose oxidase and a simple spectrophotometric method employing Fenton’s reagent. Retention of enzyme activity and surface loading were assessed. The immobilization techniques were also evaluated by electron microscopy to characterize the evenness of the surface coatings. All of the covalent coupling procedures led to surface loadings, approaching 1 pmol mm −2 ; however, the surfaces appeared irregular on a microscopic scale. The poly- l -lysine adsorption technique provided the smoothest surface. With the exception of the entrapment technique, all immobilization procedures provided immobilized enzyme that retained >75% activity after several weeks of storage.

ALTERATIONS OF GENETIC MATERIAL FOR ANALYSIS OF ALCOHOL DEHYDROGENASE ISOZYMES OF DROSOPHILA MELANOGASTER

The supply of gene mutations, chromosome rearrangements, and techniques in Drosophila genetics is the result of over 50 years of research. This material allows manipulation of genetic factors that is not feasible with other animals. The methods of classical Drosophila genetics should be useful in the study of the control of enzymes. Gel electrophoresis provides a convenient and powerful method which is easily used on Drosophila enzymes in combination with genetic methods. It is with this system that we have investigated alcohol dehydrogenase (ADH) of D . melanogaster. This enzyme catalyzes the NAD-linked oxidation of alcohols to their corresponding aldehydes or ketones and the reverse reaction. Alcohols of from two to five carbons are good substrates for Drosophila ADH. Crude extracts of flies from inbred strains have three isozymes of ADH (Grell et al., 1965; Unprung and Leone, 1965). Another report described two of the isozymes (Johnson and Denniston, 1964). Furthermore, there are two kinds of inbreds with respect to the migration of their trios of ADH in gel electrophoresis. Hybrids from crosses between these two kinds contain hybrid isozymes not present in either parental type.

Mechanism of suppression in Drosophila: A change in tyrosine transfer RNA☆

Abstract The mechanism of suppression of the vermilion locus in Drosophila melanogaster is examined. The suppressor locus, su(s)2, is shown to control directly the amount of a specific tyrosine transfer RNA which occurs in the adult fly. Wild-type flies have three chromatographic forms of tyrosine tRNA but flies that are homozygous for the suppressor gene su(s)2 contain little or none of the second chromatographic form. The isoacceptor patterns of tRNA for leucine, phenylalanine and serine are identical in the suppressor mutant and wild-type fly. Genetic data show that the phenotypic expression of su(s)2 and the altered chromatographic pattern of tyrosine tRNA are recessive and that both map at the same position on the left tip of the X chromosome. Furthermore, another suppressor of vermilion was induced by ethyl methane sulfate, su(s)e1, that is at the same locus as su(s)2 and that produces the same change in tyrosine tRNA as su(s)2. The recessive character of the suppressor mutants is evident both in the phenotypic expression and in the alteration of the tyrosine tRNA. This recessive character indicates that the suppressor locus is not the locus for the primary structure of tyrosine tRNA but that it may control an enzyme that modifies the tyrosine tRNA in some way.

Lactic dehydrogenases: subfractionation of isozymes.

Electrophoresis in polyacrylamide gel of homogenates of various organs from the mouse yields five major lactic dehydrogenase bands. If the gels are treated with β-mercaptoethanol, subsequent electrophoresis produces 15 bands which show lactic dehydrogenase activity. This could be explained if one molecule of nicotinamide adenine dinucleotide (coenzyme) is attached to each of the monomeric subunits of lactic dehydrogenase and if mercaptoethanol can remove the coenzyme only from the muscle type. This is consistent with the hypothesis that intact lactic dehydrogenase is a tetramer.

Applications of mass spectrometry to DNA sequencing

The ability of the mass spectrometer to analyze collectively the masses of DNA fragments that are produced in the Sanger procedure for sequencing may allow the gel electrophoresis step to be eliminated. On the other hand, if gel electrophoresis is required, the use of resonance ionization spectroscopy coupled to a mass spectrometer may enable much faster analysis of DNA bands labeled with stable isotopes. Other combinations of labeling of the DNA and its mass spectrometric analysis with or without gel electrophoresis are also considered. Recent advances in these areas of mass spectrometry are reviewed.

Alcohol Dehydrogenase of Drosophila: Interconversion of Isoenzymes

Isoenzymes of alcohol dehydrogenase extracted from Drosophila melanogaster are interconvertible and can be distinguished by electrophoretic mobility. When adsorbed on diethylaminoethyl cellulose, the faster-moving forms are converted to the slowest-moving form; the latter is converted to the former in the presence of 0.05 molar nicotinamide-adenine dinucleotide, and the conversion is accompanied by the binding of 3.5 moles of the dinucleotide per mole of enzyme. A change in heat stability accompanies the conversion of the slowest form of alcohol dehydrogenase to the fastest form; the latter becomes stable at 45�C. The increased heat stability may indicate that a conformational change in the alcohol dehydrogenase occurs along with the binding of nicotinamide-adenine dinucleotide.

Multiple forms of Drosophila alcohol dehydrogenase: III. Conversion of one form to another by nicotinamide adenine dinucleotide or acetone☆

Abstract Alcohol dehydrogenase occurs in five electrophoretic forms in inbred strains of Drosophila melanogaster . It was previously reported that the slowest migrating form of the enzyme (ADH 5 ) could be isolated and converted into the faster forms (ADH 4 , ADH 3 , ADH 2 , and ADH 1 ) by NAD + . This report is concerned with the conditions that affect the rate of interconversion of the isoenzymes, the interaction of NAD + and NADH with ADH 5 , and the role that acetone plays in the conversion process. The rate of conversion of ADH 5 to the faster forms is dependent upon the concentrations of ADH 5 and NAD + and upon the pH of the medium. The purified ADH 5 becomes inactivated upon conversion to ADH 1 in a manner that is reversed by the electrophoresis procedure. When high concentrations of mercaptoethanol, in addition to NAD + , are present, the inactivation of the enzyme is prevented, but abnormal electrophoretic forms of the enzyme may be produced. The conversion of ADH 5 to ADH 1 by NAD + is accompanied by binding of variable amounts of NAD to the enzyme and the production of significant amounts of NADH. The presence of acetone enhances the effect of NAD + in causing conversion, and at 0.1 m concentration, acetone alone can accomplish the conversion of ADH 5 to ADH 1 . The heat stability of the ADH 1 produced from ADH 5 is greater than that of ADH 5 . We suggest that Drosophila alcohol dehydrogenase can exist in different configurations that differ in electrostatic charge and heat stability, that these differences represent changes in the protein conformation, and that the fly accomplishes the interconversion of the various forms in a controlled fashion.

Isolation and characterization of pteridines from heads of Drosophila melanogaster by a modified thin-layer chromatography procedure

An improved thin-layer chromatography technique is described for the separation of fluorescent compounds found in extracts of heads of Drosophila melanogaster. Eighteen to twenty fluorescent spots are resolved, two of which are xanthurenic acid and 3-hydroxykynurenine, and the remaining spots are presumably pteridines. Of these, nine have been identified and quantitated directly on the chromatograms with a fluorometer. One of the spots present on the chromatogram apparently has not been described previous to this work. Characteristics of this substance, termed “quench spot,” are presented, several of which indicate that it may be a pteridine or pteridine derivative.

Electrochemically induced adsorption of radio-labeled DNA on gold and HOPG substrates for STM investigations

In a scanning tunneling microscope (STM) electrochemical cell we have studied the effects of electrode potential on both the surface topography and the adsorption of deoxyribonucleic acid (DNA) to graphite and gold surfaces. Images of the surface of highly oriented pyrolytic graphite (HOPG), of the same area, in response to a positive increase in surface potential show degradation of the step edges with little change in the crystal plane. Images of the same area of a gold surface demonstrate the formation of and the progressive increase in nodular structures on the crystal planes, in response to increased potential, with little effect on the step edges. Using radio-labeled DNA we monitored electrochemical absorption onto HOPG and gold surfaces. Although at no applied potential and at negative surface potentials some DNA was bound, at positive potentials 3 to 5 times more DNA was incorporated onto both surfaces. DNA adsorbed to a surface at a positive potential was not removed by reversing the potential.