Carl J. Lauter

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.

Cerebral Cortical Metabolism After Chronic Exposure to Ozone

This study represents a biochemical survey of cerebral cortex in dogs, in which an increased latency of evoked electroencephalogram EEG potentials of visual cortex had been observed after prolonged exposure to ozone. Portions of occipital and parietal cortex were analyzed from 44 dogs which had been exposed to 1 to 3 ppm O3 for 18 months. Contents of norepinephrine and epinephrine, lipid hydroperoxides, K+, Na+, and Cl- were measured. In addition, the activity of the following enzymes was determined: monoamine oxidase, catechol-O-methyltransferase, cholinesterase, 5’-nu-cleotidase, and Na+, K+, and Mg++ adenosine triphosphatases (ATP’ases). Catechol-O-methyltransferase activity showed a steady decline as the daily exposure to 1 ppm O3 was increased from 8 to 24 hours. Concurrently, the catecholamine content of the tissues declined.

Composition of plasma lipoproteins of the spiny dogfish Squalus acanthias

Abstract 1. 1. The protein, free fatty acid, triglyceride, cholesterol, phospholipid and total lipid content of the plasma lipoproteins from the spiny dogfish Squalus acanthias have been determined. 2. 2. The chylomicron and the very low density lipoprotein fractions carry the major portion of the lipids. 3. 3. The free fatty acid content in the lipoprotein fractions is considerably higher than in the comparable mammalian fractions with the exception that the amount carried in the d >1·21 fraction is lower. 4. 4. A different role for lipoproteins as a lipid-carrier mechanism in the elasmobranch and the mammal is suggested.

Lipoprotein synthesis. I. rat plasma lipoprotein composition and synthesis from radioactive precursors

The in vivo synthesis of rat plasma lipoproteins was studied by the use of isotopic protein and lipid precursors. Labelled amino acids, palmitic acid and tripalmitin were administered by stomach tube and the radioactivity in the plasma lipoproteins was determined following preparative ultracentrifugal isolation at densities of 1.006, 1.019, 1.063 and 1.21 g/ml.In response to triglyceride feeding, amino acid composition of the high density lipoprotein changed little, but in the low density lipoproteins proportionality in the amino acid pattern was changed as reflected by increases and decreases in certain amino acids.Isotopic amino acids were not incorporated in proportion to the relative abundance with which they occurred in the lipoproteins. Triglyceride feeding markedly stimulated isotope utilization, especially in the low density fractions. Methionine, though only present in small amounts, was extensively utilized and it is suggested that this amino acid may play a significant role in the synthesis of lipoproteins, other than the role of a methyl donor for phosphatidylcholine.

Plasma membranes from isolated liver cells

Abstract Plasma membranes have been prepared from isolated rat or guinea pig liver cells by a method established for whole liver. However, plasma membranes of liver cells isolated by disruption of the tissue with collagenase and hyaluronidase are in some detail dissimilar to those obtained from whole liver. This was indicated by lower cholesterol content and low specific activities of some marker enzymes.

Mn2+-stimulated ATPase in rat brain.

Divalent cation ATPases were prepared from rat brain synaptic vesicles, synaptosomal plasma membranes, and plasma membranes from the brain stem and sciatic nerve and tested for optimal stimulation by Mn2+, Mg2+, or Ca2+. ATPase in the synaptic vesicle subfraction was optimally stimulated by Mn2+. All plasma membrane preparations were optimally stimulated by Mg2+. Separate Mn2+ and Mg2+ ATPases could not be distinguished by either chemical inactivation or substrate preference criteria. Mn2+ stimulated ATPase in the micromolar range and it is suggested that Mn2+ interaction with ATPase may be of physiological and/or toxicological importance by being related to the cellular metabolism of this element.

Synaptic effects of the synthetic pyrethroid resmethrin in rat brain in vitro

Resmethrin (30 microM) induced release of transmitters was not affected by manipulation of the Na+ current with either choline or tetrodotoxin agents which readily reversed the effects of veratridine, deltamethrin and cypermethrin. Resmethrin (I50: 2.2 microM) inhibited the ATP dependent uptake of Ca2+ but deltamethrin and cypermethrin were much less effective. Resmethrin also displaced Ca2+ from crude synaptosomal membranes. The release promoting effects of resmethrin in rat brain in vitro are better explained by its effects on Ca2+ rather than through a specific effect on the Na+ channel. In contrast, the effects of deltamethrin and cypermethrin promote transmitter release by a Na+ dependent process.

A comparative study of brain Ca2+-ATPases

1. Particulate brain ATPases from various vertebrates were optimally activated by Ca2+, Mg2+ or Mn2+. 2. Specific enzyme activity with AT32P as substrate was low in lower vertebrates and increased on the evolutionary scale. 3. The properties of the brain ATPases suggested that most of the activity was associated with plasma membrane ecto-ATPase.