Irving Zabin

Activation and oxidation of long chain fatty acids by rat brain.

I t is generally considered that the oxidation of fatty acids by brain tissue occurs to only a very limited extent or not a t all (QUASTEL and WHEATLEY, 3933; WEINHOUSE, MILLINGTON and VOLK, 1950). It has been shown, however, that fatty acid oxidation can take place in brain slices and minces (GEYER, MAITHEWS and STARE, 1949; VOLK et al., 1952). We have been interested in the metabolism of lipids by brain, and have studied the presence and distribution of some of the enzymes involved in the metabolism of long chain fatty acids. The extent of oxidation of long chain fatty acids by brain mitochondria has been followed with radioactive patmitic acid and palmityl coenzyme A. The results obtained are presented below.

Synthesis and properties of palmityl adenylate, palmityl coenzyme A, and palmityl glutathione☆

Abstract Procedures for the preparation of palmityl adenylate, palmityl coenzyme A, and palmityl glutathione have been described. The stability of these compounds under different pH conditions has been investigated. Hydrolytic activities for each of the palmityl derivatives were demonstrated in several tissue extracts. Palmityl transferase activities from palmityl adenylate to coenzyme A and to glutathione were shown, and ATP formation by guinea-pig liver was detected from palmityl adenylate and pyrophosphate.

Co-linearity of β-Galactosidase with Its Gene by Immunological Detection of Incomplete Polypeptide Chains

Many nonsense mutants that map in the β-galactosidase structural gene produce material that forms precipitin lines when tested by double diffusion on agar against antiserum prepared from native β-galactosidase. Relative sizes of the cross-reacting materia!s measured by sucrose density gradient centrifugation are the same as sizes calculated from genetic mapping of nonsense mutants. Orientation of the protein to its gene is also indicated.

High resolution electron microscopy on highly purified β-galactosidase from Escherichia coli

β-Galactosidase, a lactose-splitting enzyme of high molecular weight, was isolated from Escherichia coli ML 308. The crystalline enzyme was investigated by electron microscopy using the negative staining technique. Two structural patterns were commonly observed and interpreted to represent different orientations of the molecule. The size and shape of unfixed molecules and molecules fixed in formaldehyde solution were compared. A subdivision into four subunits was indicated for the molecule in the amorphous state. The molecular weight calculated from the observed dimensions of the molecule was estimated to be about 500,000. A substructure of the subunit was indicated. The molecule has been found to be asymmetric with regard to its quaternary structure.

β-Galactosidase: Immunological studies of nonsense, missense and deletion mutants

Abstract A series of nonsense mutants which map throughout the β-galactosidase structural gene, and several missense and deletion mutants, were analysed for antigenic activity with purified antiserum produced from native β-galactosidase. Quantitative analyses by the Ouchterlony test, by inhibition studies, and by microcomplement fixation are in general agreement. These show, relative to the position of the nonsense mutation, no immunological activity from mutants of the early (operator) part of the gene, a peak at the center, next a much lower level, and finally a relatively high level in mutants mapping in the terminal part of the gene. No antigenic activity was found in a deletion mutant which produces both the alpha and omega fragments of β-galactosidase. Examination of precipitin lines on Ouchterlony plates of nonsense, missense and deletion mutants, as well as other data, suggests the presence of three classes of antigenic sites, those of relatively long chains, those of relatively short polypeptide chains and, tentatively, those due to the polymer. Heat treatment or addition of sodium dodecyl sulfate destroys immunological activity of nonsense mutant protein. It is suggested that variations in level of antigenic activity of incomplete β-galactosidase chains are due not only to the presence or absence of antigenic sites, but in addition to differences in conformation of these chains.

β-Galactosidase α-complementation

SummaryStudies on β-galactosidase α-complementation are reviewed. The isolation and structure of two β-galactosidase fragments that form an enzymically active complex are described. One of these is a cyanogen bromide peptide from whole β-galactosidase; the other is a dimeric-protein from a lacZ deletion mutant of Escherichia coli. The mechanism most likely involves an initial binding of two cyanogen bromide peptides to the dimer, followed by formation of a tetramer, and finally a slow conformational change of the complex to a native-like enzyme. The overall reaction is essentially irreversible. A region of the polypeptide chain involved in dimer-dimer contact must be supplied by the cyanogen bromide peptide. α-Complemented enzyme contains overlapping sequences. Proteolytic experiments were carried out to determine the origin of the funtionally important segment. The effect on a-complementation of amino acid substitutions at four positions in the polypeptide chain was investigated. The implications of these results for β-galactosidase structure and for proteins in general are discussed.

β -Galactosidase, the Lactose Permease Protein, and Thiogalactoside Transacetylase

INTRODUCTION The three structural genes of the lac operon, Z , Y , and A , code for β -galactosidase, the lactose permease protein, and thiogalactoside transacetylase, respectively. Since the discussion of these gene products in The Lactose Operon (Zabin and Fowler 1970; Kennedy 1970), a considerable amount of work has been carried out, particularly on β -galactosidase. In 1970 the subunit structure of this enzyme was known with reasonable certainty, but some controversy remained. Now the primary structure has been completed. Much new information is available on β -galactosidase from strains with mutations in the lacZ gene, on complementation, and on immunological properties. Interesting fusion proteins have been described. Considerably less information has been accumulated on the lacY gene product, and in fact, the lactose permease protein has not yet been obtained in chemically pure form. The function of thiogalactoside transacetylase has been a mystery for a long time. Recently evidence has been presented for a possible physiological role in the cell. A detailed study of its enzymatic characteristics has been carried out, and a beginning has been made toward determining the amino acid sequence of this protein. Such information on the three proteins is presented here and is discussed primarily from a biochemical point of view. β -GALACTOSIDASE Production and Isolation β -Galactosidase ( β -D-galactoside galactohydrolase E. C. 3.2.1.23) accounts for up to 5% of the total protein in haploid strains of Escherichia coli. Considerably higher levels can be obtained from certain partial diploids (Fowler 1972a). When the strain A324–5 was grown on minimal medium containing...

β-Galactosidase: α-complementation of a deletion mutant with cyanogen bromide peptides☆

Abstract Peptides produced by cleavage of aminoethyl β-galactosidase with cyanogen bromide complement extracts of M15, a mutant with a deletion in the α region of the z gene. Determinations by gel filtration and acrylamide gel electrophoresis in sodium dodecyl sulfate show that the active fraction is in the molecular weight range of 8–11,000. Peptides produced by cyanogen bromide cleavage of crude extracts of strains with intact a regions also complement with M15. In contrast, cyanogen bromide peptides from M15 and from an uninduced wild-type strain do not complement.

Gene fusion techniques cloning vectors for manipulating lacZ gene fusions

Abstract A simple vector system for cloning gene fusions of lacZ is described. We apply one of these new vectors to the cloning and transcriptional analysis of the promoter region of the ompF gene of Escherichia coli .

Immunological studies on β-galactosidase and thiogalactoside transacetylase: Proteins of the lactose system in Escherichia coli*

Tests with anti-thiogalactoside transacetylase have shown that the only serological reactant in a constitutive and in a wild-type Escherichia coli induced for the lactose system was enzymically active protein. This confirms the previous estimate based on enzyme isolation: the rate of synthesis of β -galactosidase is at least ten times higher than that of thiogalactoside transacetylase. The trans-acetylase-anti-transacetylase precipitate was enzymically inactive. No detectable cross-reaction was observed between β -galactosidase and thiogalactoside transacetylase when the pure enzymes and antisera to each were tested by double diffusion in agar gel.