Externally disposed plasma membrane proteins. I. Enzymatic iodination of mouse L cells

The enzymatic iodination technique has been utilized in a study of the externally disposed membrane proteins of the mouse L cell. Iodination of cells in suspension results in lactoperoxidase-specific iodide incorporation with no loss of cell viability under the conditions employed, less than 3% lipid labeling, and more than 90% of the labeled species identifiable as monoiodotyrosine. 90% of the incorporated label is localized to the cell surface by electron microscope autoradiography, with 5-10% in the centrosphere region and postulated to represent pinocytic vesicles. Sodium dodecylsulfate-polyacrylamide gels of solubilized L-cell proteins reveals five to six labeled peaks ranging from 50,000 to 200,000 daltons. Increased resolution by use of gradient slab gels reveals 15-20 radioactive bands. Over 60% of the label resides in approximately nine polypeptides of 80,000 to 150,000 daltons. Various controls indicate that the labeling pattern reflects endogenous membrane proteins, not serum components. The incorporated 125-I, cholesterol, and one plasma membrane enzyme marker, alkaline phosphodiesterase I, are purified in parallel when plasma membranes are isolated from intact, iodinated L cells. The labeled components present in a plasma membrane-rich fraction from iodinated cells are identical to those of the total cell, with a 10- to 20-fold enrichment in specific activity of each radioactive peak in the membrane.     

Proton magnetic resonance spectra or porcine muscle adenylate kinase and substrate complexes.

Porcine muscle adenylate kinase with a molecular weight of 22,000 has 2 histidine, 5 phenylalanine, 7 tyrosine, and no tryptophan residues. The effect of pH, substrate, and the paramagnetic manganous ion on the proton magnetic resonance spectrum of the enzyme, particularly the aromatic region, has been investigated at 220 MHz. The well resolved C2 proton peaks of the 2 histidine residues have been individually assigned to His-36 and His-189 by comparison with the spectrum of the carp muscle enzyme which has only one C2 proton peak and only 1 histidine residue, 36. The chemical shift of the peak designated C2-H of His-36 in the porcine enzyme has a normal titration curve with a pKalpha = 6.3 but the peak for His-189 is not titratable in the pH range 5.8 to 8.1. The pKalpha of the single His-36 of the carp enzyme is similar to that of His-36 of the porcine enzyme. Changes in pH, particularly at low pH, also affect the chemical shifts of the tyrosine residues. Occupation of either the monophosphate site by AMP or the triphosphate site by ATP or GTP causes a downfield shift of the C2-H of His-36, and the equilibrium mixture causes an even greater shift, but no shift in the C2-H of His-189. The substrates also induce changes in the chemical shifts in the phenylalanine-tyrosine region of the spectrum. Tentative assignments of the highest and lowest field peaks in this region have been made based on the three-dimensional structure determined by x-ray crystallography. On the basis of these assignments, it is concluded that Phe-183 is unperturbed by substrate binding but that Tyr-153 or -154 at the hinge of the molecule, are perturbed. The C2-H of adenine and C8-H of adenine or guanine of the bound substrates were also observed; those of AMP are unperturbed but C2-H of ATO is shifted downfield and the C8-H of ATP and GTP are shifted upfield. The paramagnetic manganous ion had no effect on the spectrum at Mn(II) to enzyme ratios below 1:10; above this ratio, a general broadening was observed...     

Glutathione metabolism in resting and phagocytizing peritoneal macrophages

The steady state GSH content of cultured mouse resident peritoneal macrophages was 34 +/- 5 pmol/microgram of cell protein. Intracellular GSH content decreased concomitantly with zymosan ingestion. The half-life of GSH decreased from 1.9 h in resting cells to 0.58 h during phagocytosis as determined by inhibition of GSH synthesis with buthionine sulfoximine. The decrease in GSH half-life was directly related to the extent of particle uptake. In cytochalasin D-treated cells, attachment of zymosan to the macrophage plasma membrane in the absence of particle interiorization was sufficient to stimulate GSH turnover. Efflux was the major route of GSH loss in [35S]cystine-labeled macrophages, and was enhanced 3-fold by a zymosan challenge. GSH was lost intact since resident macrophages lack gamma-glutamyl transpeptidase (less than 1 pmol of L-gamma-glutamyl-p-nitroanilide/microgram of protein . h). Macrophages obtained from mice challenged in vivo with Corynebacterium parvum maintained higher intracellular GSH levels (50 +/- 5 pmol/microgram of cell protein) than did resident cells. The half-life of GSH in buthionine sulfoximine-treated C. parvum-elicited macrophages was 3.8 +/- 0.2 h while resting and 1.3 +/- 0.2 h during phagocytosis. C. parvum-elicited macrophages, in contrast to resident cells, contained sufficient levels of gamma-glutamyl transpeptidase activity to hydrolyze 55 pmol of L-gamma-glutamyl-p-nitroanilide/microgram of cell protein . h. These studies indicate that phagocytosis and cellular activation have profound effects on GSH metabolism in macrophages.     

Characterization of Sialyl and Galactosyl Residues on the Body Wall of Different Plant Parasitic Nematodes

The plant parasitic nematodes Helicotylenchus multicinctus, Meloidogyne javanica, Tylenchulus semipenetrans, and Xiphinema index, differing in their host specificity and parasitic habits, were analyzed as to their cuticle surface sialyl, galaclosyl, and/or N-acetylgalactosaminyl residues. The procedure involved the selective oxidation of sialic acid and galactose/N-acetylgal-actosamine residues using periodate and galactose oxidase, respectively, to form reactive aldehyde groups. These functional groups were coupled directly with a new hydrazide-containing compound, the fluorescent reagent lissamine rhodamine-β-alanine hydrazide, or they were utilized to introduce DPN-groups to the nematode cuticle. The distribution of the DNP-tagged glycoconjugates was visualized by treating the nematodes with rabbit anti-DNP antibody and staining with fluorescein isothiocyanate (FITC)-labeled goat antirabbit IgG. Sialo residues were observed along the entire outer body wall of the first three aforementioned nematodes, but there were some differences in reaction among the various life stages within the species. In X. index, sialo residues were sited in the tail and head areas, mainly on the lips, oral opening, amphid apertures and stylet. Galactose oxidase treatments revealed galactose on N-acytylgalactosamine residues on T. sentipenetrans and X. index, but there were no indications that their presence was dependent on the developmental stage. Trypsin, pronase, and neuraminidase pretreatment completely abolished the fluorescence in T. semipenetrans but did not alter the sialo residue binding reaction in H. multicinctus or M. javanica, indicating possible differences in the outer body wall saccharide structure and composition between these nematodes. The existence and nature of sugar residues on the cuticle surface of nematodes could contribute to an understanding of the specific recognition by phytophagous nematodes of their host, and perhaps also of the virus transmission mechanism in those nematodes which serve as vectors.     

PINOCYTOSIS IN FIBROBLASTS : Quantitative Studies In Vitro

Horseradish peroxidase (HRP) was used as a marker to determine the rate of ongoing pinocytosis in several fibroblast cell lines. The enzyme was interiorized in the fluid phase without evidence of adsorption to the cell surface. Cytochemical reaction product was not found on the cell surface and was visualized only within intracellular vesicles and granules. Uptake was directly proportional to the administered concentration of HRP and to the duration of exposure. The rate of HRP uptake was 0.0032–0.0035% of the administered load per 10⁶ cells per hour for all cells studied with one exception: L cells, after reaching confluence, progressively increased their pinocytic activity two- to fourfold. After uptake of HRP, L cells inactivated HRP with a half-life of 6–8 h. Certain metabolic requirements of pinocytosis were then studied in detail in L cells. Raising the environmental temperature increased pinocytosis over a range of 2–38°C. The Q₁₀ was 2.7 and the activation energy, 17.6 kcal/mol. Studies on the levels of cellular ATP in the presence of various metabolic inhibitors (fluoride, 2-desoxyglycose, azide, and cyanide) showed that L cells synthesized ATP by both glycolytic and respiratory pathways. A combination of a glycolytic and a respiratory inhibitor was needed to depress cellular ATP levels as well as pinocytic activity to 10–20% of control values, whereas drugs administered individually had only partial effects. In spite of the availability of an accurate quantitative assay for fluid and solute uptake, the function of pinocytosis in tissue culture cells remains unknown.     

STUDIES IN THE PHYSICAL CHEMISTRY OF THE PROTEINS : I. THE SOLUBILITY OF CERTAIN PROTEINS AT THEIR ISOELECTRIC POINTS.

1. Two proteins of the globulin type, serum globulin and tuberin, and the protein of milk, casein, have been purified (a) of the other proteins and (b) of the inorganic electrolytes with which they exist in nature. The methods that were employed are described. 2. All three proteins were found to be only very slightly soluble in water in the pure uncombined state. The solubility of each was accurately measured at 25.0° ± 0.1°C. The most probable solubility of the pseudoglobulin of serum was found to be 0.07 gm. in 1 liter; of tuberin 0.1 gm. and of casein 0.11 gm. The methods that were employed in their determination are described. 3. Each protein investigated dissolved in water to a constant and characteristic extent when the amount of protein precipitate with which the solution was in heterogeneous equilibrium was varied within wide limits. The solubility of a pure protein is therefore proposed as a fundamental physicochemical constant, which may be used in identifying and in classifying proteins. 4. The concentration of protein dissolved must be the sum of the concentration of the undissociated protein molecule which is in heterogeneous equilibrium with the protein precipitate, and of the concentration of the dissociated protein ions. 5. The dissociated ions of the dissolved protein give a hydrogen ion concentration to water that is also a characteristic of each protein.     

INTENSIVE X-RAY SURVEY FOR TUBERCULOSIS IN A RURAL COUNTY

In an intensive fast-tempo tuberculosis case-finding survey in a rural county 34,345 residents (73 per cent of all persons 15 years of age or over) had miniature x-ray films of the chest taken. In 256 instances, x-ray findings were consistent with pulmonary tuberculosis. Sixty-eight persons were ultimately reported as having active tuberculosis (one case of active tuberculosis for every 505 persons covered by the survey). Within one year, 57 of them had been hospitalized for treatment. Only four of the 68 cases had been known to the health department before the survey.The cost of the survey (80 cents per person surveyed and $444.58 per case of active tuberculosis) compares favorably with that of other surveys.     

Rotational relaxation times of 1,6-diphenyl-1,3,5-hexatriene in phospholipids isolated from LM cell membranes. Effects of phospholipid polar head-group and fatty acid composition.

Phospholipids were isolated from mitochondrial, microsomal, and plasma membranes of LM cells and fractionated into individual phospholipid classes on silicic acid columns. The fatty acid composition and the rotational relaxation time of 1,6-diphenyl-1,3,5-hexatriene (DPH) were determined for each phospholipid class. Sphingomyelin was the only phospholipid isolated from LM cell membranes that showed a phase transition within the temperature range investigated, 5-40 degrees C. The rotational relaxation times for DPH were lowest in phosphatidylcholine in all the membrane fractions. Phosphatidylcholine isolated from the three membrane fractions of choline-supplemented cells had similar rotational relaxation times and phosphatidylcholine isolated from microsomal membranes of linoleate-supplemented cells had lower rotational relaxation times. The results indicate that the differences in the rotational relaxation times of DPH between mitochondrial, microsomal, and plasma membrane phospholipids could be explained primarily by differences in the polar head-group composition, while differences in the fatty acid composition had only a minor effect. This provides a basis for understanding how the different lipid components in these cells contribute to membrane fluidity.