Fernando Zambrano

The molecular organization of nerve membranes: I. Isolation and characterization of plasma membranes from the retinal axons of the squid: an axolemma-rich preparation

Abstract Squid retinal nerves, because of their high ratio of axolemma to Schwann cell membrane, were used to develop a procedure to isolate plasma membranes. NADH oxidoreductase and (Na + K)-ATPase were studied as enzyme of plasma membranes in various fractions separated by differential centrifugation of hypotonic homogenates of retinal axons. The pellet which sedimented at 100,000 g (F-100) was selected as a crude plasmalemma fraction because of their highest specific activity for both enzymes. Chemical analysis of F-100 revealed a protein: cholesterol: phospholipids ratio of 37:9:29 as percent of membrane dry weight. RNA and DNA were 0.72 and 0.07%, respectively. Assays of mitochondrial and endoplasmic reticulum enzymes indicated that rare contamination of these structures was present in F-100. Negatively stained electron micrographs of F-100 revealed vesicles formed by concentric membranes. Further purification of F-100 was achieved by linear gradient centrifugatiou which separated two types of membranes. ATPase and NADH oxidoreductase were found in a first protein peak at 37% sucrose (PM 1 ), but mainly ATPase was found in the second peak at 27% sucrose (PM 2 ). Sections of osmium-fixed membranes studied under the electron microscope showed striking density of plasma membranes, 70–80 A in thickness. Proteins from F-100, PM 1 , and PM 2 were solubilized by various procedures and resolved either by sucrose-gradient centrifugation or acrylamide-gel electrophoresis. PM 1 and PM 2 differed in their number of protein bands, as well as in their uv spectrum and pH titration curve. These data together with the EM and the enzymic profile indicate that two types of plasma membranes chemically and structurally different were purified from the retinal axons of the squid. Their probable cellular origin is discussed.

The molecular organization of nerve membranes : IV. The lipid composition of plasma membranes from squid retinal axons.

SummaryThe lipid content and composition from an axolemma-rich preparation isolated from squid retinal axons was analyzed.The lipids, which accounted for 45.5% of the dry weight of this membrane, were composed of 22% cholesterol, 66.7% phospholipids and 5.2% free fatty acids. The negatively charged species phosphatidyl ethanolamine (37%), phosphatidyl serine (10%) and lysophosphatidyl ethanolamine (4%) made up 51% of the phospholipids. The amphoteric phosphatidyl choline and sphingomyelin accounted for 39% and 4%, respectively.The relative distribution of fatty acids in each of the isolated phospholipids was studied. The most remarkable feature of these phospholipids was the large proportion of long-chain polyunsaturated fatty acids. The 22∶6 acyl chain accounted for 37% in phosphatidyl ethanolamine, 21.7% in phosphatidyl choline, 17.5% on phosphatidyl serine and 20.3% in sphingomyelin (all expressed as area %).The molar fraction of unsaturated fatty acids reached 65% in phosphatidyl ethanolamine and 42.0 and 44.8% in phosphatidyl choline and phosphatidyl serine, respectively. The double bond index in these species varied between 1.0 and 2.6.The lipid composition of the axolemma-rich preparation isolated from squid retinal axons appears to be similar to other excitable plasma membranes in two important features: (a) a low cholesterol/phospholipid molar ratio of 0.61; and (b) the polyunsaturated nature of the fatty acid of their phospholipids.This particular chemical composition may contribute a great deal to the molecular unstability of excitable membranes.

Role of Dietary Substrates on Intestinal Disaccharidases, Digestibility, and Energetics in the Insectivorous Mouse-Opossum (Thylamys elegans)

In the insectivorous Chilean mouse-opossum, Thylamys elegans (Didelphidae), acclimated to eating pulp of fruits with sucrose added and live insects, we studied the activity of intestinal disaccharidases, digestive efficiency, and the effect of each diet on the energy budget. We observed activity modulation of the enzymes sucrase, maltase, and trehalase as well as changes in substrate affinities correlated with dietary carbohydrates. Although this species is able to hydrolyze and assimilate sugars such as sucrose from fruits, it is nevertheless unable to obtain sufficient energy to satisfy its euthermic daily energy requirements and to survive with an exclusive diet of fruits. This situation contrasts with that observed in individuals feeding on insects, for they were able to satisfy their maintenance energy requirements with an exclusive diet of insects. The preferences observed in the mouse-opossum for insects over fruits is explained in terms of trade-offs between factors such as digesta transit time, food digestion (including sugars hydrolysis), and food characteristics.

Sulfatide role in the sodium pump

SummarySodium efflux was studied in22Na-loaded red blood cells in the presence of arylsulfatase, an enzyme that specifically hydrolyzes sulfatide. Sodium efflux was inhibited in proportion to the amount of arylsulfatase present. Maximum inhibition was almost as high as the efflux obtained in medium with K+ absent. At maximum inhibition 83.2% of the sulfatide content of the fragmented red blood cell membranes was hydrolyzed and ouabain-sensitive (Na++K+)-ATPase activity was inhibited by 100%. Sodium efflux, sulfatide content, and (Na++K+)-ATPase activity were unaffected with arylsulfatase in the presence of a high concentration of sulfatide. These results indicate that sulfatide plays a specific role in sodium and potassium ion transport. They also suggest that most sulfatide is localized externally in the red blood cell membrane.

Sulfatide content and (Na++K+)-ATPase activity of skin and gill during larval development of the chilean frog,Calyptocephalella caudiverbera

SummaryThe sulfatide content, phospholipid concentration, and (Na++K+)-ATPase activity from skin and gills of different stages of larval development ofCalyptocephalella caudiverbera (a Chilean frog) were analyzed. Additionally, the short-circuit current in skin was studied. When skin and gills, depending on the stage of larval development, present (Na++K+)-ATPase activity, they have a high ratio of sulfatide to amount of membrane and the phosphatidylserine concentration remains unchanged. Sulfatide content and (Na++K+)-ATPase activity in skin are in direct relationship with the level of sodium flux present during development. The specific enzymatic hydrolysis of sulfatide with partially purified arylsulfatase of pig kidney inhibits 100% of the ouabain-sensitive (Na++K+)-ATPase. The ouabain-insensitive ATPase remains virtually unchanged with the treatment, even with a high concentration of arylsulfatase or with ouabain present in the medium. These experiments strongly suggest a role of sulfatides in the (Na++K+)-ATPase activity and, as a consequence, in sodium ion transport.

Role of sulfatide on phosphoenzyme formation and ouabain binding of the (Na+ + K+) ATPase☆

A microsomal fraction rich in (Na+ + K+)ATPase activity has been isolated from the outer medulla of pig kidney. The ability of this preparation to form phosphoenzyme on incubation with [gamma-32P]ATP and to bind [3H]ouabain was studied when its sulfatide was hydrolyzed by arylsulfatase treatment. The K+-dependent hydrolysis of the Na+-dependent phosphorylated intermediate as well as the ouabain binding were inactivated in direct relation to the breakdown of sulfatide. Both characteristics of the (Na+ + K+)ATPase preparation, lost by arylsulfatase treatment, were partially restored by the sole addition of sulfatide. These experiments indicate that sulfatide may play a role in sodium ion transport either in the conformational transition of the K+-insensitive phosphointermediate, E1P, to the K+-sensitive intermediate, E2P, or in the configuration of the high-affinity binding site for K+ of the E2P form. In addition, this glycolipid may have a specific role in the proteolipidic subunit that binds ouabain.

Possible role of sulphatide in the K+-activated phosphatase activity

A microsomal fraction rich in (Na+ + K+)-ATPase has been isolated from the outer medulla of pig kidney. (Mg2+ + K+)-activated ouabain-sensitive phosphatase activity was studied in this preparation treated with arylsulphatase, an enzyme that specifically hydrolyzes ceramide galactose-3-sulphate. The activity of phosphatase was inactivated in proportion to the amount of sulphatide hydrolyzed. A maximum inactivation of ouabain-sensitive activity was obtained with 60% of the sulphatide content hydrolyzed. The inactivation caused by arylsulphatase was partially reversed by the sole addition of sulphatide. The evidence offered in this paper about sulphatide function in the sodium pump mechanism supports the idea that sulphatides are involved in the K+-activated phosphatase, a partial reaction of the (Na+ + K+)-ATPase.

Sulphatide content in a membrane fraction isolated from rabbit gastric mucosal: its possible role in the enzyme involved in H+ pumping.

The sulphatide content of vesicular membrane fraction from rabbit mucosal gastric microsomes was analyzed. This vesicular membrane fraction, in addition to a high sulphatide content, was enriched in an ouabain-insensitive (H+ + K+)-ATPase, a (Mg+2 + K+)-activated phosphatase, and a H+ pumping activity. The enzyme system involved in the process of acid secretion and the translocation of K+ was studied in these membrane preparations treated with arylsulphatase A, an enzyme that specifically hydrolyzes sulphatide. The results indicate that the breakdown of sulphatides of the vesicular membrane fraction inactivated both the (H+ + K+)-ATPase activity and the H+ pumping. Both activities were partially restored by the sole addition of sulphatide. The K+-stimulated ouabain-insensitive phosphatase activity, suggested as a partial reaction of the (H+ + K+)-ATPase sequence, was unaffected by arylsulphatase. These results suggest that sulphatides may play a function in the high activity binding site for K+ of the enzyme involved in H+ pumping.

Arsenic contamination: Metabolic effects and localization in rats

1. 1. Arsenic content was measured in perfused organs, blood and hemoglobin of rats drinking arsenic trioxide solutions (4–20 μg ml−1) for 2–3 months. 2. 2. Almost all the arsenic was accumulated in the blood, with 88% in the isolated hemoglobin. 3. 3. The hematin fraction containing 99% of the toxic element. 4. 4. Little or no arsenic was detected in tissues. 5. 5. A negative correlation was found between maximum oxygen consumption and concentration of arsenic in the red blood cells of the contaminated rats. 6. 6. The results suggest that after arsenic ingestion the element affects the hemoglobin molecule, diminishing the amount of oxygen delivered to the tissues, especially under high energy demands.

Golgi Complex Function in the Excretion of Renal Kallikrein

Abstract A Golgi complex rich-fraction containing both N-acetylglucosamine galactosyltransferase and kallikrein activity has been isolated from kidney of rats previously treated with colchicine, a secretion inhibitor, followed by the administration of high-sodium solutions, to stimulate biosynthesis or activation of renal kallikrein. After the treatment, N-acetylglucosamine galactosyltransferase and kallikrein activities were increased in the Golgi complex, about 18- and 24-fold, respectively, as compared to the homogenate. Low kallikrein activity was found in the crude light mitochondrial fraction from treated animals, whereas a high level of activity was observed in the microsomal fraction. The inverse situation was found in rats treated only with high-sodium solution. Results suggest that kallikrein is probably transported by microsomal elements, particularly by the Golgi complex. Furthermore, the evidence seems to indicate that the kallikrein activity reported in the plasma membrane and/or in the lysosomal fraction is due to kallikrein secretion, in the form of intact granules, which have sedimented with these two fractions.