Membrane alterations in cellular aging: Susceptibility of phospholipids in density (age)-separated human erythrocytes to phospholipase A2

Human erythrocytes were separated into four density (age) groups representing the top 10% (young), bottom 10% (old), and two middle fractions of 40% each (intermediary ages). When these erythrocytes of different age groups were treated with the low levels of a purified basic phospholipase A₂ from Agkistrodon halys blomhofii, under conditions where little or no hemolysis occurred, the optimum extent of phosphatidylcholine (PC) hydrolysis in all age groups was the same, but interestingly, the rate of its hydrolysis was two to three times faster in the older cells compared to younger erythrocytes. On the other hand, hydrolysis of phosphatidylethanolamine (PE) of younger erythrocytes by the phospholipase A₂ was negligible under the particular experimental conditions. However, in erythrocytes of older age groups, both the rate and extent of PE hydrolysis by the enzyme increased in a distinctive fashion. Concomitant with the above pattern of PC and PE hydrolysis, the shape changes in the erythrocytes also were different; whereas all older erythrocytes became echinocytic only two-thirds of the younger erythrocytes showed a similar shape change. These observations firmly establish that during in vivo aging of normal erythrocytes in circulation significant changes in the structural organization of membrane phospholipids take place. Importance of this phenomenon in membrane phospholipid asymmetry studies and in the elimination of senescent cells also is discussed.     

Bovine follitropin. Isolation and characterization of the native hormone and its alpha and beta subunits

1) A reproducible procedure was developed for the purification of bovine follitropin. 2) The method involved ammonium sulfate precipitation, ion exchange and adsorption chromatography, concanacaline-A-Sepharose chromatography and gel filtration. 3) A specific radioligand receptor assay was used to monitor each chromatographical step. 4) The potency of highly purified bovine follitropin as measured by Steelman and Pohley bioassay was 62 times the NIH-FSH-B1 standard preparation. 5) Contaminations of bovine follitropin by other glycoprotein hormones such as thyrotropin and lutropin amounted to 3 and 0.45 per cent by weight respectively as measured by specific radioimmunoassays and radioligand receptor assays. 6) The subunits alpha and beta of bovine follitropin were obtained by incubation in acidic urea, the chains being then separated by anion exchange chromatography. The subunits were subjitted to complete characterization. The amino-terminal residue of the alpha subunit is phenylalanine while a half cystine residue was found at the aminoterminal end of the beta chain. 8) Cross-contamination of the alpha and beta subunit preparations was measured by specific radioimmunoassays and amounted to 0.02 and 0.1 per cent by weight respectively.     

Ca++ binding properties of human prothrombin.

The binding of Ca++ to human prothrombin has been investigated by equilibrium dialysis. The protein exhibited a positive cooperativity phenomenon for the first three Ca++ bound. Eleven to twelve Ca++ binding sites have been found. They could be differentiated in terms of two classes of sites with respect to their Ca++ affinity: 5 strong binding sites (log Kassoc = 3.9) and 7 weak binding sites (log Kassoc = 2.9). We attempted to determine the Hill coefficient of the strong binding sites responsible for cooperativity. Results have been compared to data previously reported for bovine prothrombin.     

Ultrastructural and lectin binding changes during the formation of the animal dimple in oocytes of Discoglossus pictus (Anura)

The numbers of spores, stalk cells, and basal disk cells in fruiting bodies of Dictyostelium discoideum were estimated by direct cell counting. It was found that the ratios of differentiated cells varied with the number of cells in the fruiting body. Hence, this invalidates, in D. discoideum at least, an assumption used in many theories of differentiation that proportions do not vary with size. Simple statistical analysis showed that a semilogarithmic equation could describe the relationship of spore to stalk cell number and spore to basal disk cell number, whereas a double-logarithmic equation described the basal disk and stalk cell number relationship. Studies under different environmental conditions and with different strains suggest that the basic equations describing the relationships are conserved. However, quantitative differences in the proportioning of the cell types have been observed. Previous papers concerning the proportions of D. discoideum are reviewed, and the implications of the results, in regard to theories of differentiation, are analyzed.     

Temperature-dependent vesiculation of human erythrocytes caused by hypertonic salt: A phenomenon involving lipid segregation

Treatment of human erythrocytes with 4 M NaCl causes hemolysis with concomitant release of microvesicles from the membrane. The microvesicles have an average diameter of 200–300 nm and reveal an in creased lipid content in particular of cholesterol and sphingomyelin. Phosphatidylcholine and phosphatidylserine contents remain unaltered whereas the phosphatidylethanolamine content is lowered in comparison with the erythrocyte membrane.Decreasing the temperature at which the microvesicles are produced causes an increase in the cholesterol/phospholipid ratio, the sphingomyelin/phosphatidylethanolamine ratio, and the amino-phospholipids, which contain low amounts of arachidonic acid.The total protein content of the vesicles is further decreased when the temperature is lowered. This is due to a reduced content of spectrin and several integral membrane proteins. The results indicate that a significant, temperature-dependent segregation of membrane constituents occurs during the vesiculation process.     

Anion binding to oxidized type 2 depleted and native laccase: a spectroscopically effective model for exogenous ligand binding to the type 3--type 2 active site

Xanthine oxidase, a mammalian nitroreductase, catalyzed the binding of [³H]1-nitropyrene to DNA. The binding was dependent on the presence of hypoxanthine and was inhibited by allopurinol, a specific xanthine oxidase inhibitor. These data support the hypothesis that nitroreduction is a necessary step in the metabolic activation of 1-nitropyrene to a bacterial mutagen.     

Tight binding of oxaloacetate to succinate dehydrogenase

[¹⁴C]Oxaloacetate forms a stable complex with succinate dehydrogenase which withstands repeated Sephadex filtration. Oxidized glutathione, 2-thenoyltrifluoroacetone, KCN and ageing at +4° at neutral pH do not prevent the enzyme to bind oxaloacetate. The binding is prevented by succinate or malonate but the complex, once formed, can not be split by these compounds, although the enzyme activity can be restored; the reconstitutive property of succinate dehydrogenase is, however, irreversibly lost. Bound oxaloacetate does not exchange with added oxaloacetate, but can be released by perchloric acid. Sonic particles of beef heart mitochondria can also bind oxaloacetate. However, this complex can be split by succinate or malonate.     

A fluorometric-HPLC assay for quantitating the binding of benzo[a]pyrene metabolites to DNA

A nonradiometric method is presented for quantitating low levels of benzo[a]pyrene (BP) derivatives that are covalently bound to the DNA of BP-treated mice. This method consists of hydrolyzing the DNA with acid to liberate the BP-adducts in the form of the isomeric tetrols of BP. These tetrols have fluorescence quantum yields of ～0.7 in deoxygenated solution at 298 K. Hence they are easily quantitated, following HPLC separation, by means of fluorescence detection. The sensitivity of the method is such that one bound BP residue per 10⁷ bases can be detected in 100 μg of DNA.     

Biochemical properties of the 1 alpha, 25-dihydroxyvitamin D3 cytoplasmic receptors from human and chick parathyroid glands

Cytoplasmic receptors for 1α, 25-dihydroxyvitamin D₃ from human parathyroid adenoma tissue and rachitic chick parathyroid glands have been characterized with regard to a number of physical, chemical, and ligand binding properties. Both receptors are 3.6–3.7 S proteins with molecular weights of approximately 75,000 and Stoke's molecular radii of 36 Å. It was found that the receptors possess a cysteine residue in or near the 1α, 25-dihydroxyvitamin D₃ binding site which is critical for ligand binding activity. The receptors both have equilibrium dissociation constants for 1α, 25-dihydroxyvitamin D₃ in the range of 2 to 5 × 10^?10m at 4 °C and second-order association rate constants for their seco-steroid ligand of 1 × 10⁷, m^?1 min^?1 (0 °C). The dissociation rate constants were found to be 5.3 × 10^?4 min^?1 (4 °C) for the human receptor and 1.3 × 10^?5 min^?1 (4 °C) for the chick receptor. The great deal of similarity which exists between the cytoplasmic 1α, 25-dihydroxyvitamin D₃ receptors from avian and mammalian parathyroid glands suggests a homologous function for these molecules in the two tissues.     

Photoaffinity labeling of beta-adrenergic receptors: identification of the beta-receptor binding site(s) from turkey, pigeon, and frog erythrocyte

The β-adrenergic receptors in the erythrocyte membranes from turkey, pigeon, and frog have been identified $\text{in situ}$ utilizing the photoaffinity label ±[¹²⁵I]-iodoazidobenzylpindolol, ±[¹²⁵I]IABP. The molecular weights determined by SDS-polyacrylamide gel electrophoresis are the following: turkey, 43,500; pigeon, 53,500, 46,000, and 45,000 [labeled in a ratio of 5 (53,500):2 (46,000 plus 45,000)]; and frog, a broad 60,000 to 67,000 dalton band. The data identify the binding site subunit(s) of these β-adrenergic receptors and suggest that the receptor structure from different β-receptor subtypes and different sources may be different. These biochemical differences may contribute to the pharmacologically observed distinction of β-receptor subtypes.     

Reexamination of the carbohydrate binding stoichiometry of lima bean lectin

The carbohydrate binding stoichiometry of lima bean lectin component III was reexamined using equilibrium dialysis and quantitative affinity chromatography following limited chemical modification. Equilibrium dialysis employing methyl[2-14C]benzamido-2-deoxy-alpha-D-galactopyranoside as ligand demonstrated that the lectin tetramer bound 4 mol of sugar with Kassoc = 1.44 +/- 0.13 X 10(3) M-1 (T = 5 degrees C, pH 7.0, ionic strength 0.1). The previous report of two sites/tetramer [Bessler, W. and Goldstein, I. J. (1974) Arch. Biochem. Biophys. 165, 444] appears to be the result of partial inactivation of the lectin due to oxidation of essential thiol groups. Following limited chemical modification of the thiol groups by methyl methanethiosulfonate, multiple intermediate forms with reduced affinity for Synsorb A were obtained. The number and hemagglutinating activities of these intermediates provided further support for the presence of four carbohydrate binding sites on lima bean lectin component III.     

Human serum hemopexin: direct evidence for change of its isoelectric point upon heme binding. A new serum protein fractionation

Human serum was submitted to a one step displacement-ligand exchange chromatography. Displacement removed serum albumin and part of gamma-globulins. Ligand exchange furnished an enriched heme-hemopexin fraction. An original, non denaturing human heme-hemopexin preparation is proposed.     

The chick intestinal cytosol binding protein for 1,25-dihydroxyvitamin D3: A study of analog binding

The structural features of 1,25-dihydroxyvitamin D₃ that permit its high affinity binding to a 3.7 S protein from chick intestinal cytosol were determined in a series of binding and competition experiments analyzed by sucrose density gradient centrifugation. Optimal binding to the 3.7 S protein was achieved when both 1α- and 25-hydroxyls were present in the vitamin D₃ molecule. Modification of the side chain by the introduction of a methyl on C-24 and a double bond on C-22,23 (1,25-dihydroxyvitamin D₂) did not alter the binding of 1,25-dihydroxyvitamin D₃, but significantly diminished the binding of 25-hydroxyvitamin D₃. However, introduction of a hydroxyl on C-24 decreased the ability of either 1,25-dihydroxyvitamin D₃ or 25-hydroxyvitamin D₃ to compete, especially when the 24-hydroxyl was in the S configuration. These results reveal that the 3.7 S protein requires specific ligand structural features for binding and suggest that metabolite discrimination by the chick intestinal receptor system is likely located in the 3.7 S cytosol protein.     

Sulfhydryl groups in urocanase: Relationship to nicotinamide adenine dinucleotide binding

This study concerned the role of the sulfhydryl groups in urocanase of Pseudomonas putida. When p-chloromercuribenzoate was added to the enzyme, two sulfhydryl groups reacted at once with little inhibition; the enzyme slowly became inhibited while further sulfhydryls reacted. After the p-chloromercuribenzoate inhibition occurred, if a thiol was subsequently added, most of the original activity was recovered. As the incubation time with p-chloromercuribenzoate was increased, the thiol became less effective in reversing the inhibition. However, if NAD⁺ (10 μm) was added with the thiol, 60–90% of the initial activity was restored even after long p-chloromercuribenzoate incubations. Restoration of activity by NAD⁺ was concentration dependent and specific for NAD⁺. Radioactive NAD⁺ could be bound to urocanase. These results confirm the coenzyme role for NAD⁺ in urocanase. In urea, p-chloromercuribenzoate titration of urocanase measured 11.9 -SH groups per molecule. Sulfite-modified enzyme treated with p-chloromercuribenzoate and dialyzed was substantially photoactivated in the presence of a thiol; that is, NAD⁺ was not required to restore activity. From these results, it is proposed that this enzyme contains two reactive —SH groups and that an essential —SH group is involved in NAD⁺ binding. Forces present in the sulfite-modified enzyme prevent the release of the NAD⁺ in the presence of mercurials.     

Purification of radiolabeled and native polypeptides by gel permeation high-performance liquid chromatography

Some results and observations concerning the use of protein columns are presented. The combined use of four protein columns having different fractionation ranges together with a volatile triethylamine formate buffer allowed the sieving of various polypeptides according to their molecular weights over a range of 500 to 150,000. The addition of 4 or 6 m guanidine-HCl permitted the reduction of aggregation with no sacrifice in resolution or linearity. With that denaturant, rapid separation, and molecular weight determination in the range 500–90,000 is easily accomplished. Moreover, sample recoveries as determined with radiolabeled proteins always exceeded 70% while radioimmunoassay techniques can be directly applied to the column eluate. Applications to quick identification of natural fragments of a serine protease, tonin, analysis of maturation products of pro-opiomelanocortin in an in vitro pulse experiment and finally quantitation by radioimmunoassays of pituitary peptides and elution of their ¹²⁵I-labeled derivatives are described.     

Single-stranded bacteriophage T5 stO DNA as template for in vitro fMet-tRNA binding to ribosomes and protein synthesis.

Good evidence is provided that fMet-tRNA binding and aminoacid incorporation, when single-stranded DNA is used instead of mRNA in an E. coli cell-free system, are strictly dependent on the magnesium concentration. Ten sites homologous to the initiation sites of translation can be detected on denatured T5 stO DNA when using ribosomes and initiation factors from uninfected E. coli F. In S-30 extracts, at high magnesium concentrations and in the presence of neomycin, initiation of the translation of denatured T5 stO DNA begins anywhere on the molecule, and yet high molecular weight polypeptides are synthesized. The template potentiality of the denatured T5 stO DNA decreased when using ribosomes plus initiation factors and crude extracts from T5 stO-infected bacteria. By in vitro formation of initiation complexes sites analogous to initiation sites of translation were localized on T5 stO DNA molecules using single-stranded fragments separated by sedimentation in alkaline sucrose gradient.     

Opiate binding in rat hearts: modulation of binding after hemorrhagic shock

[3H] Diprenorphine was used to measure binding in sectioned rat hearts. Saturable binding for concentrations up to about 20 nM was obtained in the right atrium and ventricle. Unlabeled diprenorphine displaced bound [3H] diprenorphine most effectively in the right atrium (up to 55%), as compared to less than 27% in the right ventricle and the remaining parts of the heart. Scatchard analysis of the binding in the right atrium revealed cooperative binding. The delta agonist [D-Ala2,D-Leu3] enkephalin, the kappa agonist ethylketocyclazocine, and levorphanol, but not the mu agonist [D-ala2,MePhe4,Gly-(ol)5] enkephalin or dextrophan competed variably with [3H]diprenorphine for the binding in the right atrium and ventricle. A significant decrease in binding was observed in the right atrium (-66%) and ventricle (-45%) of hearts removed from rats 2 h after hemorrhagic shock; 24 h after shock, recovery of binding was found. This novel observation suggests that the diprenorphine binding sites in the heart may be physiologically active receptors, involved in regulation of peripheral cardiovascular processes.     

Carbohydrate binding studies on the lectin from Datura stramonium seeds

The carbohydrate-binding properties of the Datura stramonium seed lectin were studied by equilibrium dialysis, quantitative precipitation of natural and synthetic glycoproteins, and hapten inhibition of precipitation. The dimeric lectin (M_r = 86,000) possesses two carbohydrate-binding sites for N,N′,N′',N?-tetraacetylchitotetritol/mol protein, with an apparent K_a = 8.7 × 10³M^?1 at 4 °C. Whereas fetuin and orosomucoid reacted poorly with the Datura lectin, the asialo derivatives of these glycoproteins gave strong precipitation with the lectin. Carcinoembryonic antigen, type 14 pneumococcal capsular polysaccharide, and bovine serum albumin, highly substituted with N,N′- diacetylchitobiose units, also precipitated the lectin. Of the homologous series of chitin oligosaccharides tested, N,N′,N?-triacetylchitotriose was over 6-fold more potent than the disaccharide (N′,N′-diacetylchitobiose) which, in turn, was 90 times more reactive than N-acetyl-d-glucosamine.N-Acetyllactosamine [β-d-Gal-(1 → 4)-d-GlcNAc] was also a potent inhibitor of Datura lectin being equivalent to N,N′-diacetylchitobiose. The requirement for an N-acetyl-d-glucosaminyl unit linked at the C-4 position was established. The biantennary pentasaccharide (penta-2,6) was a 500-fold more potent inhibitor than N-acetyllactosamine, suggesting that it might interact with both saccharide-binding sites of the Datura lectin simultaneously.