Eberhard G. Trams

On the sidedness of plasma membrane enzymes

Abstract 1. 1. The objective of this investigation was to determine if certain marker enzymes were associated with either the exterior or interior aspect of the plasma membrane. 2. 2. Experimentally, the problem was approached by comparing enzymatic activity of intact cells with that of disrupted cells. Because isolated cells from dispersed guinea pig liver were found to be of limited value only, the investigation was pursued in varous cultured cell lines. Permeability of whole cells to substrate was excluded as a possible source of error. 3. 3. Most of the activities of non-specific Mg2+-ATPase and 5′-nucleotidase of several cell lines (i.e. HeLa; KB; human, mouse and guinea pig hepatocyte, mouse neuroblastoma) was the expression of ecto-enzymes. Phosphodiesterase I, p- nitrophenylphosphatase and leucyl-β-naphthylamidase also may occur preferentially as ecto-enzymes, though fewer cell lines were tested. 4. 4. Adenylate cyclase was clearly associated with the interior aspect of the plasma membrane. Nucleotidepyrophosphatase of isolated guinea pig hepatocytes had the characteristics of an ecto-enzyme but was associated with the inside of the plasma membrane in Chang hepatocytes and KB cells.

On the isolation and characterization of gangliosides.

Abstract A new method for the extraction and purification of brain gangliosides is described. Analysis of the gangliosides from various species showed a marked similarity in respect to composition and behavior. Chromatography of the gangliosides indicated the presence of at least four closely related components. Time-course studies on the liberation of neuraminic acid and reducing sugar were suggestive of a branched chain in the polysaccharide part of the molecule. Analytical data indicated that more than one neuraminic acid residue occurs in some units. The probability of more than one type of neuraminic acid linkage to the galactose and hexosamine moiety of the molecule was suggested. From physical studies it was inferred that gangliosides are not high-molecular-weight polymers but form micellar aggregates in aqueous solutions.

Neuron specific protein (NSP) in neuroblastoma cells: relation to differentiation.

The spectrum of enolase enzyme forms has been examined in several lines of neuroblastoma cells and compared to those present in whole brain. The neuron specific enolase (NSP) is greatly decreased in the cultured cells as judged by activity profiles and radioimmunoassay. The synthesis of neuronal enolase appears to be extensively depressed in these cells while the total enolase activity is not affected. The non-neuron form of enolase (NNE) apparently compensates for the lack of the neuronal forms in the cultured cells. The preponderance of NNE in cultured neurons suggests that this enzyme is present in immature neurons, and that neuroblastoma cells are not fully differentiated with respect to the enolase function. Dibutyryl cyclic adenosine monophosphate treatment does increase NSP levels in mouse neuroblastoma cells, but not to the levels expected for fully differentiated neurons. The results indicate that NSP is a molecular correlate of fully differentiated neurons.

A proposal for the role of ecto-enzymes and adenylates in traumatic shock.

Abstract It is proposed that the biochemical mechanisms which lead to the pathology of irreversible traumatic shock are based on the vasodilatory action of adenylates and failure of ecto-enzymes to clear these substances from the vascular bed. Following massive injury, or another assault which produces significant cytolysis, cytoplasmic ATP is released into the circulation. Ecto-phosphoesterhydrolases on erythrocytes normally control plasma adenylate concentrations to be maintained below 10 −7 M. If a critical ATP (or ADP) concentration between 10 −5 and 10 −4 M is reached, erythrocytes become semi-permeable and themselves release additional ATP into the plasma compartment. When ecto-ATPase activity becomes insufficient in metabolizing plasma ATP at rates which are needed to compensate for the release of cytoplasmic ATP, the vasodilatory effects of adenylates will manifest themselves in a sustained manner. It is suggested that ecto-phosphoesterhydrolases on blood formed elements and endothelial cells serve a protective function. Circulatory shock ensues under conditions when these ecto-enzymes are rendered incompetent or when plasma adenylate concentrations exceed their catabolic capacity.

Synthesis of lipids by liver plasma membranes. Incorporation of acyl-coenzyme a derivatives into membrane lipids in vitro

1. 1. Plasma membranes were prepared from rat liver homogenates. The membrane preparation was characterized by its composition and the presence of particular enzymes which have been associated with membrane function. Lipid composition of the membrane was studied in detail and was compared with that reported for other membranes. 2. 2. Palmityl-, stearyl-, oleyl- and linoleyl-CoA esters were prepared enzymatically from radiocarbon labeled acyl precursors. These substrates were incubated with the membranes and incorporation of isotope into various lipids was investigated. 3. 3. Under these conditions, small amounts of neutral glycerides were formed. In this process, unsaturated acyl-CoA derivatives were better utilized precursors than the saturated analogs. Fatty acids were principally incorporated into phosphatidic acid, Phosphatidylethanolamine and phosphatidylcholine, presumably through a reacylation of their lyso derivatives. 4. 4. The major portion of the fatty acids from [14C]acyl-CoA esters was recovered as free fatty acid and in the form of S-acyl pantetheine.

GANGLIOSIDE COMPOSITION AND BIOSYNTHESIS IN CULTURED CELLS DERIVED FROM CNS

Abstract— Cultured mouse neuroblastoma cells (clone N18) contained a homologous series of gangliosides, GM3, GM2, GM1 and GD1a; the total lipid bound sialic acid (LBSA) was 3.3 nmol per mg of protein, of which GD1a comprised two‐thirds. In contrast, neonatal hamster astrocytes (clone NN) and human glioblastoma cells (Cox clone) contained mainly GM3, which represented 95% of the 2 nmol of LBSA per mg protein in these cells. When the cells were grown in the presence of [14C]galactose, label was incorporated into all of the gangliosides isolated from the cells. The labeling pattern corresponded to the ganglioside composition of the cell lines; GD1a was more extensively labeled in N18 cells and GM3 was the major labeled ganglioside extracted from glial cells. In addition to in rivo biosynthesis, in vitro synthesis of gangliosides was also determined. The activities of five glycosyltransferases of the ganglioside biosynthetic pathway were measured in homogenates of the three cell lines. The neuroblastoma cells contained all five enzyme activities whereas the two glial cell lines were deficient in UDP‐N‐acetylgalactosamine: GM3N‐acetylgalactosaminyltransferase activity, which catalyzes the synthesis of GM2 from GM3. The results indicated that cells of neuronal origin contain the more complex gangliosides associated with CNS and the requisite biosynthetic enzymes and that cells of glial origin are missing these complex gangliosides and the key glycosyltransferase required for their synthesis.

Standardized method for the determination of human erythrocyte membrane adenosine triphosphatases

Abstract A standardized assay is described for the simultaneous determination of Mg 2+ -ATPase, Na + , K + -ATPase, and Ca 2+ -ATPase in human erythrocyte (RBC) membrane preparations. Membranes were prepared by lysis of RBCs in hypotonic buffer, and ATPase activity assays were based on the measurement of 32 P-labeled inorganic phosphate release from [γ- 32 P]ATP. The results obtained by this method were compared with those of colorimetric determination of inorganic phosphate and of ATP hydrolysis with high-performance liquid chromatography. The activity of the three enzymes was measured in RBC membranes obtained from 30 normal subjects. Repeated sampling of individuals over a 4-month period showed that interindividual differences were substantial, but that in each individual enzymatic activity was maintained in a narrow range by presumed homeostatic mechanisms. Statistical analysis of the data showed no interdependence of the three enzymes; a correlation of activity with age, sex, or phase of the menstrual cycle was not apparent. The values obtained for the Ca 2+ -ATPase did not follow a normal distribution, and it is suggested that this enzyme has two phenotypic variants. The described method is sufficiently precise and economical to be recommended for adoption as standard procedure in clinical research.

Nucleotide pyrophosphatase activity of rat liver plasma membranes

Abstract 1. 1. Palmityl-CoA is metabolised to S- palmityl pantetheine by plasma membrane preparations. The metabolism of other nucleotide pyrophosphates by the plasma membrane of liver cells was found to be similar. UDPG was converted to glucose i -phosphate and UMP; NAD+ and NADH were hydrolysed at the pyrophosphate bond. 2. 2. The subcellular distribution of the nucleotide pyrophosphatase was studied with NADH as substrate. The similarity in distribution to 5′-nucleotidase, a plasma membrane marker enzyme, indicates that nucleotide pyrophosphatase is an authentic plasma membrane enzyme. 3. 3. NADH breakdown was competitively inhibited by UDPG, CoA and NAD+, and the properties of the enzyme were very similar to those of other mammalian nucleotide pyrophosphatases.

Carotenoid transport in the plasma of the scarlet ibis (Eudocimus ruber)

1. 1. A study was made of the lipoprotein composition of blood plasma from the scarlet ibis (Eudocimus ruber), white ibis and a pink hybrid. Donor animals were obtained either from the delta of the Amazon River or from the National Zoo, Washington, D.C. 2. 2. It was found that the carotenoid composition of the plasma of the captive scarlet ibis differed from that of the wild bird. 3. 3. The carotenoid pigments which are characteristic for scarlet ibis were principally associated with the high-density lipoprotein fractions. Pigment half-life was of the order of 80–100 hr and consistent with the metabolism of high-density lipoproteins (HDL). 4. 4. Further fractionation of the HDL by disc electrophoresis indicated the presence of a component which appears to be a carotenoid carrier protein. Substantial amounts of this protein were present in the scarlet ibis whereas only traces could be detected in the white ibis.

Metabolism of coenzyme A and related nucleotides by liver plasma membranes

Abstract 1. 1. Plasma membranes convert acyl CoA into S -acylpantetheine and several nucleotide residues. The objectives of this investigation were to determine the reaction sequences which are involved in the further metabolism of CoA and of some related nucleotides. 2. 2. The reaction: Acyl-CoA → 4′-phospho- S -acyl pantetheine + 3′,5′-diphosphoadenosine (3′,5′-ADP) was catalyzed by nucleotide pyrophosphatase. 3′,5′-ADP was further metabolized to adenosine and P i with the intermediate formation of 3′-AMP. 3. 3. Liver plasma membrane preparations are characterized by the presence of a variety of phosphoester hydrolases and a labile thioesterase. The properties of these enzymes are described and their possible roles in plasma membranes are discussed.