Eugene P. Kennedy

Symmetrical distribution of phospholipids in the membrane of Bacillus megaterium

The transbilayer distribution of phosphatidylethanolamine in the cytoplasmic membrane of Bacillus megaterium has been determined. Phosphatidylethanolamine, the principal lipid, is arranged in a markedly asymmetric manner, with the cytoplasmic monolayer containing twice as much as the outer monolayer. This conclusion is based on the availability of the amino group of phosphatidylethanolamine to two reagents, trinitrobenzenesulfonate and isethionyl acetimidate. At 3°C, neither reagent can cross the membrane; at this temperature, 31(±3)% and 35(±3)% of the phosphatidylethanolamine can be trinitrophenylated or amidinated, respectively. In inverted, subcellular membrane vesicles, whose external surface apparently corresponds topologically to the cytoplasmic surface of the membrane in the intact cell, 68(±2)% of the phosphatidylethanolamine is accessible to trinitrobenzenesulfonate at 3°C. This finding is consistent with the conclusion that the phosphatidylethanolamine which is not available to external reagents in whole cells resides in the cytoplasmic side of the membrane. At 15°C, trinitrobenzenesulfonate slowly enters the cell, causing the phosphatidylethanolamine to react in a biphasic manner. The outer monolayer lipid reacts with the kinetics of an homogeneous component with a half-time of two minutes while the cytoplasmic monolayer reacts more slowly, with a half-time of about 40 minutes, limited by the rate at which the reagent crosses the membrane. The external phosphatidylethanolamine pool (reacting rapidly at 15°C) contains 35(±3)% of the total. From the preponderance of phosphatidylethanolamine in the inner monolayer, it can be inferred that the outer monolayer is constituted primarily of the other major bacterial lipid, phosphatidylglycerol. The existence of asymmetry in this bacterial membrane supports the view that lipid asymmetry is a general characteristic of biological membranes.

Partial purification and properties of diglyceride kinase from Escherichia coli

Diglyceride kinase (diacylglycerol kinase, E.C. 2.7.1.-), an enzyme localized in the inner membrane of Escherichia coli, has been purified about 600-fold. The purified enzyme exhibits an absolute requirement for magnesium ion; its activity toward both lipid and nucleotide substrates is stimulated by diphosphatidylglycerol or other phospholipids. Adenine nucleotides are much better substrates for the enzyme than are other purine or pyrimidine nucleotides. The purified enzyme preparation catalyzes the phosphorylation of a number of lipids, including ceramide and several ceramide and diacylglycerol-like analogs. The broad lipid substrate specificity of diglyceride kinase suggests that this enzyme may function in vivo for the phosphorylation of an acceptor other than diacylglycerol.

Cyclic glucans produced by Agrobacterium tumefaciens are substituted with sn-1-phosphoglycerol residues

In a previous study (Miller, K.J., Kennedy, E.P. and Reinhold, V.N. (1986) Science 231, 48-51) it was reported that the biosynthesis of periplasmic cyclic beta-1,2-glucans by Agrobacterium tumefaciens is strictly osmoregulated in a pattern closely similar to that found for the membrane-derived oligosaccharides of Escherichia coli (Kennedy, E.P. (1982) Proc. Natl. Acad. Sci. USA 79, 1092-1095). In addition to the well-characterized neutral cyclic glucan, the periplasmic glucans were found to contain an anionic component not previously reported. Biosynthesis of the anionic component is osmotically regulated in a manner indistinguishable from that of the neutral cyclic beta-1,2-glucan. We now find that the anionic component consists of cyclic beta-1,2-glucans substituted with one or more sn-1-phosphoglycerol residues. The presence of sn-1-phosphoglycerol residues represents an additional, striking similarity to the membrane-derived oligosaccharides of E. coli.

Hitler's Gift and the Era of Biosynthesis

Before the Second World War biochemistry in the United States had a strong flavor of clinical chemistry. It was much occupied with problems of analysis of blood and tissues and the determination of the structures of body constituents. This was important and indeed essential work, but American students had to go abroad to Germany or to England for training in what came to be called dynamic aspects of biochemistry. After the war, the flow of students was largely reversed. This transformation was in considerable part the result of new insights and new approaches brought to America by immigrant scientists. It is a remarkable fact that as late as 1945 when I began graduate studies in biochemistry at the University of Chicago almost nothing was known about the linked reactions leading to the biosynthesis of any of the major types of cell constituents, carbohydrates, lipids, proteins, or nucleic acids. However, this picture was about to change with dramatic rapidity. The latter half of the 20th century became the era of biosynthesis. Now, in 2001, we know in great detail the patterns of reactions leading to the formation of each of these classes of cellular materials, although to be sure much remains to be learned about the regulation and integration of biosynthetic processes in living organisms. The achievements of three biochemists, Fritz Lipmann, Rudolf Schoenheimer, and Konrad Bloch, greatly stimulated this flowering of biosynthetic studies in the United States at the mid-20th century. Each had been driven out of Germany by the brutal anti-Semitism of the Nazi regime. Each was an important part of what has been called Hitler’s gift (1) to American and British science. In helping to bring about the transition to the era of biosynthesis, Fritz Lipmann made clear the crucial role of “energy-rich” phosphates in driving biosynthetic reactions and showed how this principle operated in the formation of the much sought and highly elusive “active acetate” involved in so many pathways. Rudolf Schoenheimer helped put into the hands of biochemists their most subtle and versatile approach, that of the isotope tracer technique, and with its aid revealed the dynamic state of body constituents. Konrad Bloch’s work on the formation of cholesterol illustrated how the insights of Lipmann and Schoenheimer could be combined in a masterpiece of biochemistry to solve a problem of great medical as well as biological significance.

Conditional Lethal Phosphatidylserine Decarboxylase Mutants of Escherichia coli

SummaryThe final step in the biosynthesis of phosphatidylethanolamine, the major membrane lipid of Escherichia coli, is catalyzed by the membrane-bound enzyme, phosphatidylserine decarboxylase. A variation of a procedure for localized mutagenesis (Hong and Ames, 1971) was employed to generate conditional lethal mutants in phosphatidylserine decarboxylase. In our modification, an episome carrying the psd gene closely linked to purA+ was heavily mutagenized in vivo in a strain also lysogenic for phage P1 CMclr100. After induction of a phage lytic cycle, the purA+ marker was transduced to a purA- recipient. A majority of the Pur+ transductants thus contained a psd gene originating from the heavily mutagenized episomal strain. Three mutants were isolated in which temperature-sensitive growth is caused by thermosensitive phosphatidylserine decarboxylase activity that is defective in vivo at the non-permissive temperature. All 3 mutations were mapped at the same location as psd1, being cotransduced with melA, purA, and ampA. The gene order in this region, as determined by a phage P1-mediated, three-factor cross is ampA-psd-purA. psd+ is dominant to the psd mutant alleles.

A sensitive radiochemical assay for choline acetylase

Abstract A sensitive radiochemical assay for choline acetylase has been devised based on the formation of acetylcholine from tritium-labeled acetate. The acetylcholine is separated from other labeled components by chromatography on a short column of Amberlite CG-50 ion exchanger. The procedure is sufficiently specific and sensitive so that it can be used for the measurement of the acetylase in crude, unfractionated homogenates of brain.

A micro radiochemical assay for thiogalactoside transacetylase

Abstract This assay for thiogalactoside transacetylase is based on the galactoside-specific transfer of 3H-acetate from acetyl CoA to a Dowex 1 non-adsorbable form. It provides a rapid micro determination of activity for both crude extracts and the purified enzyme. A simple, economical procedure for synthesis of the radioactive substrate is described.

Periplasmic Membrane-Derived Oligosaccharides and Osmoregulation in Escherichia Coli

Membrane-derived oligosaccharides (MDO), found in the periplasmic space of Escherichia coli and other Gram-negative bacteria, are composed of 8–10 glucose united in a branched structure, joined by β1→2 and β1→6 linkages. They are a family of compounds, variously substituted with sn-1-phosphoglycerol residues, derived from the head-groups of phosphatidylglycerol, and with succinate in 0-ester linkage, as well as smaller amounts of phosphoethanolamine. A study of the turnover of membrane lipids led to the discovery of this novel class of cell constituents. The synthesis of MDO is strictly regulated by the osmolarity of the medium in which the cells are growing. A study of the enzymes catalyzing the biosynthesis of MDO may therefore shed light on the fundamental mechanisms by which cells sense the osmolarity of their surroundings and adapt to it. Progress in the elucidation of the biosynthetic pattern is reviewed.