Studies on thyroid cell surface glycoproteins: Isolation of plasma membranes and characterization of carbohydrate units

Plasma membranes were isolated from calf thyroid microsomes and further resolved into two subfractions by sucrose density gradient centrifugations. The lighter and major membrane fraction was obtained in a yield of 10 mg/100 g of thyroid and was enriched 38-fold with respect to 5′-nucleotidase activity compared to the homogenate. It differed from the denser plasma membrane fraction in containing greater amounts of phospholipid and cholesterol but had a similar total carbohydrate content (16 mg/100 mg protein) and monosaccharide composition. The membranes were found to retain most (80%) of their carbohydrate after delipidation. The major protein-bound sugars present in the lighter membrane fraction expressed as micromoles per 100 mg of peptide were: galactose 24, mannose 17, fucose 3, glucosamine 23, galactosamine 4, and sialic acid 9. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate of the lipid-free membranes revealed at least 18 protein bands and 3 periodic acid-Schiffreactive glycoprotein components. Incubation of the delipidated membranes with Pronase resulted in the solubilization of 95% of the saccharide portion which upon filtration through Bio-Gel P-6 and P-10 columns yielded several glycopeptide fractions. While some of the carbohydrate was found in glycopeptides which appeared to contain the well-known complex and polymannose asparagine-bound oligosaccharides, as well as small O-glycosidically linked units, approximately half was recovered in high molecular weight components which contained galactose and glucosamine as their principal sugar constituents, and which were similar in composition to glycopeptides recently isolated (T. Krusius, J. Finne, and H. Rauvala, 1978, Eur. J. Biochem.92, 289–300) from human erythrocyte membranes.     

Basement membrane glycosaminoglycans: Examination of several membranes and evaluation of the effect of sonic treatment

The glycosaminoglycans of various basement membranes (human and bovine renal glomerular and tubular basement membranes as well as calf and cow anterior and posterior lens capsules) have been isolated by DEAE-cellulose chromatography after protease digestion. On the basis of composition, ion-exchange elution, electrophoretic mobility, and susceptibility to nitrous acid treatment heparan sulfate was identified as the predominant glycosaminoglycan component of each membrane. Quantitation of the heparan sulfate was achieved by a DEAE-cellulose microcolumn procedure and indicated that the amount of this component present in basement membranes spanned a wide range, extending from 0.3% of peptide weight in bovine and human tubular membranes to 6% in calf posterior lens capsule. Comparison of the heparan sulfate content of calf and cow anterior lens capsules indicated that it underwent a pronounced decrease with increasing age. Analyses of the glycosaminoglycan-peptide fractions from calf anterior and posterior lens capsules indicated hexuronic acid to xylose ratios of 29 and 37, respectively, and relatively low degrees of N-sulfation (0.2 N-sulfate, 0.6 total sulfate groups per repeating disaccharide). The composition of the lens capsule heparan sulfate was in many ways similar to that from bovine glomerular basement membrane (N. Parthasarathy and R. G. Spiro, 1981, J. Biol. Chem.256, 507–513). The present study also indicated that the heparan sulfate content of bovine glomerular basement membrane (0.8 mg/100 mg peptide) was not appreciably altered even by prolonged sonic treatment.     

Cleavage of dolichyl pyrophosphoryl oligosaccharides by endo-beta-N-acetylglucosaminidase H: comparison of enzymatic and acid hydrolysis techniques for saccharide release

Endo-beta-N-acetylglucosaminidase H (endo H) was found to bring about the complete hydrolysis of dolichyl pyrophosphoryl oligosaccharides. Both glycosylated and unglucosylated polymannose oligosaccharides were released by the enzyme through cleavage of the di-N-acetylchitobiose sequence. The action of the endo H on the oligosaccharide-lipids was facilitated by the inclusion of Triton X-100 (maximal stimulation at concentrations greater than 0.03%) or small amounts of a variety of other detergents; however, sodium dodecyl sulfate (0.1%) was strongly inhibitory. Although incubations were routinely carried out at pH 5.2, the enzyme was noted to be equally effective at pH 6.5 and to retain 75% of its activity toward oligosaccharide-lipid at pH 7.4. While these results broaden the known specificity of the endo H for the aglycon moiety, it was observed that even under optimal conditions the rate of hydrolysis of lipid-linked Glc3Man9GlcNAc2 was substantially slower than that of the same oligosaccharide attached to asparagine in a peptide sequence. The use of endo H, an enzyme which can be obtained free of exoglycosidases, appears to have a number of advantages over mild acid hydrolysis as a tool for cleaving oligosaccharide-lipids. It was found that the latter procedure causes a small but detectable degradation of the sugar chains and, when carried out in the presence of methanol, leads to the release of about 10% of the oligosaccharide as its beta-methyl glycoside. Furthermore, the oligosaccharides released by the endo H can be directly compared to those liberated by this enzyme from glycoproteins; this may prove to be useful in metabolic studies dealing with oligosaccharide-lipid assembly and their involvement in the N-glycosylation of proteins.     

Change in kinetic property of M1-type pyruvate kinase from hyperbolic type to sigmoidal type by limited proteolysis with an acid fraction from rabbit liver

An acid extract of rabbit liver contained M1-type pyruvate kinase inactivating activity, and was separated to three fractions. The optimal inactivation of the enzyme with Fraction II (Mr 42,000) was observed at pH 5.5, and this inactivation was completely prevented by leupeptin and antipain, but not by pepstatin. With Fraction III (Mr 22,000), on the other hand, optimal inactivation of the enzyme was observed at pH 8-9, and was not prevented by these inhibitors. The kinetic properties, with phosphoenolpyruvate, of the enzyme were changed from hyperbolic type to sigmoidal type by the limited proteolysis with Fractions II and III. The subunit molecular weight of the enzyme (57,300) was decreased to 55,800 via 56,400 in the former case and to 56,400 in the latter case.     

Studies on the rat glomerular basement membrane: Age-related changes in composition

To examine the effect of age on the glomerular basement membrane, compositional analyses were performed on membranes isolated in highly purified form from rats at different stages of their growth (35 to 200 days old). Substantial age-related changes were observed in the amino acid composition of the basement membranes. A significant correlation with age (P < 0.01) was evident in the contents of 3- and 4-hydroxyproline, threonine, serine, alanine, valine, half-cystine, hydroxylysine, and lysine. Of these amino acids, hydroxylysine and both isomers of hydroxyproline demonstrated a progressive increase with age, while the others were found to decline. The direct relationship of hydroxylysine content with age (P < 0.001) was associated with an inverse correlation of lysine with age (P < 0.001) so that the ratio of hydroxylysine to lysine increased in a highly significant manner from 0.92 at 35 days to 1.33 at 200 days. This elevation in the hydroxylysine content was accompanied by an augmentation in the number of saccharide units linked to it so that the percentage glycosylation of this amino acid was not significantly affected by age. The relative differences in the hydroxylysine and lysine levels between young and older rats were maintained in sodium dodecyl sulfateextracted membranes. These results suggest that the compositional changes observed during the aging process reflect an alteration in the subunit makeup of the basement membrane, possibly due to an increased synthesis or decreased degradation of the more collagen-like polypeptide components.     

Regulation of natural killer cell activity by anti-I-region monoclonal antibodies

The effects of a monoclonal antibody directed against immune response gene products on mouse NK activity were examined. In vivo administration of an anti-I-A^k antibody to C3H/He (H-2^k) mice modulated their peritoneal cell (PC) and spleen cell (SC) natural killer (NK) activity against YAC-1 lymphoma target cells in vitro. No such effect was observed when BALB/c (H-2^d) mice were treated with this antibody. Administration of anti-I-A^k antibody to mice before and after infection with Toxoplasma or treatment with poly(I:C) leads to suppression of NK activity in comparison to NK activity of mice infected with Toxoplasma or injected with poly(I:C) alone. A similar treatment regimen with M5/114 antibody which reacts with I-A^b, I-A^d, I-E^d, and I-E^k molecules resulted in decreased NK activity in B10.D2 (H-2^d) but not in B10.BR (H-2^k) mice. Serum and cell culture supernatant interferon (IFN) concentrations were not altered as a result of anti-I-A^k treatment. Removal of adherent cells did not restore NK activity of anti-I-A^k-treated Toxoplasma-infected mice to levels obtained with mice infected with Toxoplasma. In contrast, depletion of Ly 2.1⁺ cells from nylon-wool nonadherent SC of mice treated with anti-I-A^k antibody, before and after infection with Toxoplasma, resulted in restoration of NK activity to the same level as that observed in Toxoptasma-infected mice.     

Isolation of the fibrinogen-binding region of platelet thrombospondin

Purified platelet thrombospondin binds to immobilized fibrinogen if both Ca++ and Mg++ are present. Digestion of the purified molecule with thermolysin results in a limited number of discrete proteolytic fragments. When such digests are subjected to affinity chromatography on immobilized fibrinogen, only the fragments with Mr of 120,000 and 140,000 are specifically bound and subsequently eluted by the addition of EDTA to the column buffer. Examination by SDS-PAGE under both reducing and nonreducing conditions reveals that the fibrinogen-binding domain is derived from the region of the thrombospondin molecule containing the interchain disulfide bonds. The requirement for Ca++ and Mg++ for optimal binding to fibrinogen is also manifest by the Mr 120,000/140,000 thermolytic fragments.     

Rat intestinal brush border membrane dipeptidyl-aminopeptidase IV: kinetic properties and substrate specificities of the purified enzyme

The kinetic properties and substrate specificities of dipeptidyl-aminopeptidase IV (EC 3.4.14.—) detergent-solubilized and purified from the brush border membrane of rat small intestinal mucosal cells were investigated. Kinetic analysis of purified dipeptidyl-aminopeptidase IV was carried out with a variety of oligopeptides and β-napthylamide derivatives as substrates. In general, peptides with proline penultimate to the amino terminus (XPro, X= amino acid) are more favored substrates while those with alanine (XAla) are hydrolyzed at a slower rate. There is some activity toward substrates having leucine at both the penultimate position and amino terminus (LeuLeu). The activity of the purified enzyme toward GlylProβ-napthylamide derivative is maximal at pH 8.4 in Tris-HCl buffer, with an activation energy of 7.98 kcal/mol. There is no requirement for metal ion. The ability of various dipeptides to inhibit Gly-l-Pro-β-napthylamide derivative hydrolysis was used to determine the binding specificity of the enzyme for the amino-terminal amino acid. These data show that a free amino acid group is necessary for enzymatic activity and increased hydrophobicity of the amino acid at the amino terminus enhances binding.     

Analysis of 2',5'-oligoadenylates in cells and tissues

Complex mixtures of 2',5'-oligoadenylates are formed in cells and tissues under several different circumstances, and methods for analyzing such mixtures are reviewed. Separation is achieved by high-performance liquid chromatography and quantitation by competition-binding assays, using three different types of antibodies or a specific binding protein, or by functional assay, using preparations of an endonuclease specifically activated by some of the 2',5'-oligoadenylates. Representative results from three different biological systems are presented. The function of 2',5'-oligoadenylates as activators of intracellular RNA degradation is discussed, along with the possibility that these compounds may serve as signals for other intracellular regulatory processes.     

Fibronectin expression during the processes leading to axis formation in the chick embryo

Fibronectin expression was studied and found not to be present during the shedding process of stage VII chick embryos which indicates that fibronectin is not relevant during the implementation of the gravity-determined process of symmetrization. Fibronectin was detected, however, at the later stage XIII just prior to streak formation as a thin fluorescent sheet on the epiblastic side facing the hypoblast suggesting that it might be involved in the specific interactions that occur between epiblast and hypoblast and that lead to axis formation. Cultures of either epiblastic or hypoblastic chick cells indicate that both types of cells are capable of autonomous expression of fibronectin under in vitro conditions.     

Heme complexes of rabbit hemopexin,human hemopexin and human serum albumin: Electron spin resonance and Mőssbauer spectroscopic studies

The electron spin resonance (ESR) spectra of human and rabbit ferriheme-hemopexin complexes at 30^oK show an ESR absorption characterized by g_x = 1.60, g_y = 2.25 and g_z = 2.86, characteristic of low-spin ferriheme-proteins. The low-spin nature of the heme-iron in heme-hemopexin is corroborated by the observation of nuclear hyperfine splitting in M?ssbauer spectra at 4.2^oK. The similarity of the ESR spectra of ferriheme-hemopexin with those of low-spin cytochromes which coordinate heme via two histidine residues points to a similar coordination mechanism in hemopexin. In contrast, the ESR spectra of the 1:1 and 2:1 complexes of heme with human serum albumin display signals at g = 6.0 and g = 2.0, characteristic of high-spin ferrihemeproteins.     

Natural killer-like activity mediated by activated T lymphocytes

Diverse types of lymphocytes mediate in vitro cytotoxic activity. In addition to CTLs (cytotoxic T lymphocytes) and NK (natural killer) cells which differ in their activation requirements, target specificities, and lytic mechanisms, a natural killer-like activity of activated cells (A-NK) has recently been described. The data presented here suggest that an activated T lymphocyte can mediate A-NK activity. A-NK activity can be separated from resting NK activity by its requirement for activation and an effector phenotype (T12+,Ia+,Mol-) which includes the presence of the T12 and Ia antigens and the absence of the Mol antigen. In contrast, resting NK activity is mediated by T12-,Ia-,Mol+ cells. Cells that mediate A-NK activity can be differentiated from CTLs by their differing kinetics of activation and susceptibility to inhibition by monoclonal antibodies. An additional distinguishing feature is the fact that A-NK cells are predominantly Ia+ and are derived from either the T4+ or T8+ T-cell subsets whereas CTLs generated under similar conditions are predominantly T8+,T4-,Ia-. The in vivo relevance of this newly defined T-cell cytolytic activity remains to be defined.     

Effects of calcitonin and parathyroid hormone on the metabolism of chondrocytes in culture

Rabbit articular chondrocytes in suspension culture synthesize Type II colagen [3α₁(II)] in the absence of extracellular Ca²⁺ and Type Icollagen [2α₁?(I)·α₂] in the complete medium. As a result of pre-treatment in monolayer culture with calcitonin or parathyroid hormone in the complete medium, an influx of Ca²⁺ into the cells occurs. These cells produce mainly Type I collagen when transferred to suspension cultures in the medium devoid of CaCl₂. If added directly to the suspension culture medium containing no CaCl₂, calcitonin stimulates an active efflux of Ca²⁺ from the cells into the medium and leads the cells to synthesize Type I collagen. Under similar conditions, parathyroid hormone does not change the collagen-phenotype.     

Yeast RNA polymerase III: A zinc metalloenzyme

Atomic absorption spectroscopy has been used to demonstrate that zinc is associated with yeast RNA polymerase III. The enzyme purified by DNA-Sepharose chromatography gives a single predominant protein band in polyacrylamide gel electrophoresis and contains 0.7 gram-atoms of zinc per 100,000 grams of protein. The zinc is tightly associated with the enzyme and cannot be removed by passing the protein through a column of Chelex-100 resin under conditions where free zinc is quantitatively removed. Inhibition by the chelating agent 1,10-phenanthroline demonstrates that the zinc is essential to the catalytic process. The enzyme is inhibited 50% at 0.1 mM and 100% at 1 mM 1,10-phenanthroline.     

Theoretical considerations regarding the cell membrane function during cold-induced preservation of tissues and organs: new possibilities for optimizing the process

An analysis of the effects of temperature variation on the function of the mammalian cell membrane strongly suggests that during the cold-induced preservation of living tissues and organs the passive flow of micromolecules and ions across the cell membrane is the main factor in determining the state of the cellular biological system. Practically, the optimal organ (tissue) storage could be obtained at low temperatures (exceeding the freezing point) by using preservation fluids with constitutive substances in concentrations equal to those existing in the normal cytoplasm. Consequently, the formulae for adequate preservation fluids must be calculated according to the peculiar cytoplasmic constitution of the cells of each organ and tissue.     

Evidence of ubisemiquinone radicals in electron transfer at the cytochromes b and c1 region of the cardiac respiratory chain

A highly purified reduced ubiquinone-cytochrome c reductase preparation (the b-c₁III complex) has been made. The b-c₁III complex is not reconstitutively active with succinate dehydrogenase. When the complex at about 10 mg/ml is reduced by succinate in the presence of catalytic (nanomolar) amounts of SDH and a ubiquinone protein (required in the succinate dehydrogenase region i.e, OP-S), a ubisemiquinone radical(s) has been detected using EPR measurements. The formation of the radical(s) is concurrent with the reduction of cytochrome b after the complete reduction of cytochrome c₁. All these rates are dependent on the amounts of succinate dehydrogenase and QP-S used. The maximal concentration of the radical formed is independent of the amounts of succinate dehydrogenase and QP-S added but dependent on the amount of succinate present. The formation of the radical and the reduction of b and c₁ by succinate requires the presence of phospholipids. Addition of thenoyltrifluoroacetone not only prevents the formation of the ubisemiquinone but also abolishes the prior formed radical and causes the reoxidation of b. Antimycin A also diminishes the radical intensity but causes only slight reoxidation of prior reduced cytochrome b. Treatment of the b-c₁III complex with α-chymotrypsin results in the diminishing of the radical formation. Consideration of all these results presented collectively indicates the existence of a ubiquinone binding protein in the b-c₁III complex preparation.     

Thermodynamic and EPR characterization of mitochondrial succinate-cytochrome c reductase-phospholipid complexes

Succinate-cytochrome c reductase can be easily solubilized in a phospholipid mixture (1:1, lysolecithin:lecithin) in the absence of detergents. The resulting solution contains two b cytochromes with half-reduction potentials of 95 ± 10 mV (b₅₆₁), and 0 ± 10 mV (b₅₆₆) and cytochrome c₁ (E_{m 7.2} = +280±5 mV). The oxidation-reduction midpoint potentials obtained by optical potentiometric titrations are identical to those determined by the EPR titrations and are 40–60 mV higher than the corresponding midpoint potentials of these cytochromes in intact mitochondria. In contrast to detergent-suspended preparations, no CO-sensitive cytochrome b can be detected in the phospholipid-solubilized preparation or intact mitochondria. The half-reduction potential of cytochrome b₅₆₆ is pH-dependent above pH 7.0 (?60 mV/pH unit) while that of b₅₆₁ is essentially pH-independent from pH 6.7–8.5, in contrast to its pH dependence in intact mitochondria. EPR characterizations show the presence of three oxidized low-spin heme-iron signals with g values of 3.78, 3.41 and 3.37. The identification of these signals with cytochromes b₅₆₆ (b_T), b₅₆₁ (b_K) and c₁ respectively is made on the basis of redox midpoint potentials. No significant amounts of oxidized high-spin heme-iron are detectable. In addition, the preparation contains four distinct types of iron-sulfur centers: S₁ and S₂ (E_{m 7.4} = ?260 mV and 0 mV), and two iron-sulfur proteins which are associated with the cytochrome b-c₁ complex: Rieske's iron-sulfur protein (E_{m 7.4} = +280 mV) and Ohnishi's Center 5 (E_{m 7.4} = +35 mV).     

ω-Hydroxylation of acyclic monoterpene alcohols by rat lung microsomes

Rat lung microsomes were shown to ω-hydroxylate acyclic monoterpene alcohols in the presence of NADPH and O₂. NADH could neither support hydroxylation efficiently nor did it show synergistic effect. The hydroxylase activity was greater in microsomes prepared from β-naphthoflavone (BNF)-treated rats than from phenobarbital (PB)-treated or control microsomal preparations. Hydroxylation was specific to the C-8 position in geraniol and has a pH optimum of 7.8. The inhibition of the hydroxylase activity by SKF-525A, CO, N-ethylmaleimide, ellipticine, α-naphthoflavone, cyt. $\text{c}$ and p-CMB indicated the involvement of the cyt. P-450 system. However, NaN₃ stimulated the hydroxylase activity to a significant level. Rat kidney microsomes were also capable of ω-hydroxylating geraniol although the activity was lower than that observed with lungs.     

Neocarzinostatin: spectral characterization and separation of a non-protein chromophore.

Chromatographically purified neocarzinostatin exhibits absorption, fluorescence, magnetic circular dichroic and circular dichroic spectral characteristics above and below 300 nm atypical for a protein with its reported aminoacid composition, indicating the presence of a non-protein chromophore. The drug complex, stable at acidic pH, can be dissociated by treatment with reducing or denaturing agents at neutral or basic pH. Chromatography of the dissociated complex, or more conveniently, methanol extraction of the lyophilized drug, separates a protein with an amino-acid composition identical to neocarzinostatin and a highly fluorescent chromophore free of amino-acids.