The human LT system. II. Immunological relationships of LT molecules released by mitogen activated human lymphocytes in vitro.

Materials with LT activity present in supernatants from PHA stimulated human lymphocytes in vitro are very heterogeneous and can be separated into multiple molecular weight classes, termed complex, α, β, and γ. Several of these classes can be further resolved into subclasses by other physical and chemical methods. The immunologic relationships of these materials one to another were examined employing various rabbit anti-humn LT sera which will neutralize LT activity on L-929 cells in vitro. These studies reveal: (a) LT activities are due to a distinct group of substances which are immunologically related one to another and can exist in several molecular weight forms; (b) a high MW class of molecules, termed complex, appears to contain all currently known LT classes and subclasses; (c) LT classes and subclasses both have common (public) and discrete (private) antigenic specificities; (d) human LT classes and subclasses do not appear to share Ag determinants with materials with LT activity released by lectin stimulated lymphoid cells from rabbit, rat, hamster, guinea pig, or mouse; and (e) human LT molecules are not immunologically related to cell toxins released by glass adherent human peripheral blood monocytes or PMN cells. These data indicate human LT molecules form a “discrete system” of lymphocyte derived cell toxins, which can associate together into various related but different MW forms in the supernatant.     

The human LT system. I. Physical-chemical heterogeneity of LT molecules released by mitogen activated human lymphocytes in vitro.

Molecules with LT activity which can be identified in supernatants from PHA stimulated human lymphocytes by lysis of murine L-929 cell in vitro are heterogeneous. They can be separated by MW on sephadex or ultrogel columns into four separate classes: (a) complex (>200,000 daltons); (b) α (70–90,000 d); (c) β (25–50,000 d); and (d) γ (10–20,000 d). The amount of activity in a supernatant due to each class varies but is approximately: Cx—5 to 20%, α—40 to 60%, β—20 to 40%, and γ—0 to 10%. These classes differ one from another in their stability and kinetics of appearance in culture. Furthermore, they may aggregate together with the complex class under conditions of low ionic strength. Each class, except γ, can be further separated into subclasses by ion exchange chromatography and polyacrylamide gel electrophoresis. Alpha class can be separated on DEAE into the three subclasses, termed, α₁ (rf 0.25), α₂ (rf 0.37), and α₃ (rf 0.50). Alpha₂ subclasses can be further separated on phosphocellulose at pH 6.6 into α_2a (rf 0.37) and α_2b (rf 0.30). However, α₂ contains additional subclasses, which were resolved on PC columns at pH 5.5. Beta class activity can be resolved by DEAE and PAGE into two subclasses, termed β₁ (rf 0.28) and β₂ (rf 0.49). Gamma class activity was not studied, because of its instability. The complex class of LT activity is a macromolecular aggregate greater than 200,000 daltons which appears to contain smaller MW LT class(es). This study demonstrates that materials with LT activity in supernatants from PHA activated human lymphocytes in vitro are very heterogeneous.     

The human LT system. V. A comparison of the relative lytic effectiveness of various MW human LT classes on 51Cr-labeled allogeneic target cells in vitro: enhanced lysis by LT complexes associated with Ig-like receptor(s).

The present studies examine the in vitro cell-lytic capacity of various molecular weight (MW) human lymphotoxin (LT) classes obtained from lectin-activated normal or immune lymphocytes on allogeneic target cells. The findings reveal that the high-MW complex class of LT is up to 100 times more effective than the smaller MW LT forms (α, β, and γ) in causing lysis of various allogeneic cell types including lymphoid cells in vitro. Moreover, the data suggest that lectin-stimulated alloimmune cells (MLC sensitized) release complex LT forms in association with a specific antigen-binding receptor(s), and that these complexes are from 3 to 10 times more effective on the sensitizing target cell than complexes obtained from lectin-stimulated nonimmune cells. Positive evidence that complex-induced lysis involved LT was indicated by the finding that lysis was completely neutralized by incubation with heterologous antisera directed against a refined human α₂-LT subclass (anti-α₂) and partially neutralized with anti-human Fab₂′ serum. These findings support the concept that LT molecules may represent a system of related cell-lytic molecules. While the smaller MW forms are only weakly lytic by themselves, they can be assembled into highly lytic complexes which may be focused or directed by an antigen-binding receptor(s).     

The human LT system. IV. Studies on the large MW LT complex class: association of these molecules with specific antigen binding receptor(s) in vitro.

The present studies demonstrate that a portion of lymphotoxin (LT) cell-lytic activity present in supernatants from: 1) lectin (Con A, PHA) stimulated nonimmune; or 2) antigen (soluble or cellular) stimulated immune human lymphocytes in vitro, is associated with immunoglobulin (Ig) or “Ig-like” receptor molecule(s). This concept was supported by three findings: 1) LT activity in these supernatants was partially inhibited by heterologous anti-human (IgG) Fab′₂ antisera; 2) LT activity present in soluble antigen stimulated immune human lymphocyte supernatants could specifically bind to and be eluted from Sepharose 4B columns to which the specific stimulating antigen was covalently attached; and 3) LT activity present in primary one-way mixed lymphocyte culture (MLC) supernatants could be removed by absorption on the specific stimulator cells. The amount of total LT activity found to be associated with “Ig” in these supernatants was variable, but ranged from 5 to 20% in lectin stimulated cell supernatants to 20 to 50% in antigen or MLC stimulated supernatants. Physical-chemical studies on the molecular weight class of LT molecules having reactivity with anti-Fab′₂ sera, as well as antigen binding capacity, revealed these properties reside in the large (>200,000) MW LT class, termed complex. The nature and biological significance of these “antigen specific” LT complexes, as they relate to mechanisms of cytotoxicity in vitro, will be discussed.     

Non-ferritin, non-heme iron pools in rat tissues

Concentrations of intracellular, low molecular weight (LMW) and desferrioxamine B (DF) chelatable Fe, in tissues of normal, Fe-deficient and Fe-loaded female rats, were determined. Ice cold, high speed supernatants were rapidly fractionated on Ultrogel AcA202 or by filter centrifugation. After correction for residual blood and DF effects on Fe proteins, liver, kidney, heart and spleen contained 3-8 micrograms/g LMW Fe, brain 20 micrograms/g, with DF; two-thirds of this was detected without DF. There was little variation with Fe status. MW standardization and fractionation on Sephadex G-25 indicated components of apparent MW 13,000, 1400 and 350; the latter two were rapidly labeled with in vivo 59Fe.     

Intracellular cyclic GMP-binding proteins in cellular slime molds.

The complexity of cyclic GMP-binding activity in the 48,000 X g supernatant of three species of the cellular slime molds (Dictyostelium discoideum, Dictyostelium rosarium, and Polysphondylium violaceum) was studied by gel filtration chromatography on AcA 34 Ultrogel. All these species have in common a cyclic GMP-binding protein of molecular weight of about 2.5 X 10(5) which specifically binds this nucleotide. In addition, Scatchard plots of assays carried out with the 48,000 X g supernatant of these species exhibit cyclic GMP-binding activity with an apparent dissociation constant of about 1 nM. None of the cyclic GMP-binding proteins separated by chromatography on AcA 34 Ultrogel was associated to protein kinase activity stimulation. In view of the cyclic GMP function during chemotactic transduction in the cellular slime molds, the possible molecular function for this 2.5 X 10(5)-dalton cyclic GMP-binding protein is discussed.     

The human LT serum. X. The initial form released by T-enriched lymphocytes is 150,000 m.w., associated with small nonlytic components, and can dissociate into the smaller alpha, beta, and gamma m.w. classes

The present studies examine the various lymphotoxin (LT) forms released in vitro by phytohemagglutinin- (PHA) activated T-enriched (Te) human peripheral blood lymphocytes. It is clear that Te cells rapidly released (24 to 48 hr) these molecules in vitro. The 1st cell-lytic form detected in these supernatants is a 140-160,000 m.w. molecule(s) termed precursor alpha heavy (P alpha H). This form does not express alpha-LT antigenic determinants but is neutralized by antisera from animals injected with serum-free PHA-activated unseparated lymphocyte supernatants (anti-WS). The P alpha H is converted into alpha H, which expresses alpha determinants, by passage through molecular sieving columns or by treatment with low levels of Nonidet P-40 or urea. These treatments dissociate a small nontoxic 10-20,000 m.w. molecule(s), termed precursor factor (Pf), which masks the alpha-LT determinant on the P alpha H molecule. The dissociation of Pf is reversible, since alpha H from the molecular sieving columns will reassociate with the Pf. The alpha H LT class can further dissociate into the smaller alpha, beta, and gamma LT forms upon chromatography on a molecular sieving column, and a certain small percentage of the alpha H forms appear capable of associating to form the high m.w. complex (Cx) LT class. These findings suggest P alpha H may represent an intermediate that requires additional processing in order to proceed down 1 of 2 pathways: a) formation of complexes that are highly cell-lytic, or b) degradation by dissociation into the smaller weakly cell-lytic molecules identified as LT forms.     

Purification and characterization of a xylobiose- and xylose-producing endo-xylanase from Aspergillus niger

A xylanase from a commercial Aspergillus niger pentoglycanase was purified to homogeneity by column chromatography on Ultrogel AcA 54, SP-Sephadex, Sephadex G-50, and SP-Sephadex. The enzyme hydrolyzed xylotriose slowly to xylose and xylobiose, and xylotetraose and higher xylo-oligosaccharides rapidly to mixtures of smaller xylo-oligosaccharides, with xylobiose and xylose being the preponderant final products. The anomeric configuration of the products was inverted, in contrast to the behavior of most other carbohydrases that initially produce mixtures of oligosaccharides. This enzyme is a glycoprotein having an amino acid composition high in acidic residues. Its molecular weight is 20,800 and its isoelectric point is at pH 6.7. Optimal pH values for activity and stability are between 4 and 6 and, in a 20-min assay, maximal activity is attained at 55°.     

Isolation of multiple forms of metleghemoglobin reductase and leghemoglobin components from lupin nodules

Method for simultaneous obtaining in homogeneous state of two main forms of metleghemoglobin reductase and main leghemoglobin components from lupine nodules was worked out. The method included steps of saturation with ammonium sulphate (40-80%), gel-filtration on Ultrogel AcA 44, isoelectric focusing and repeated isoelectric focusing. As result the forms of metleghemoglobin reductase with molecular weights 62 and 66 kDa were obtained purified 725 and 402 times respectively and obtained in homogeneous state. The total yield of activity was 37%. The form with 62 kDa molecular weight was more active.     

Purification of proteins similar to HPr and enzyme I from the oral bacterium Streptococcus salivarius. Biochemical and immunochemical properties

The phosphoenolpyruvate:sugar phosphotransferase system (PTS) is made of several proteins. Two of them are designated general proteins because they are required for the transport and phosphorylation of all sugars of the PTS. These two proteins are found in the soluble fraction of cellular extracts and are termed HPr and enzyme I (EI). We reported in this work the purification and the characterization of these two proteins from Streptococcus salivarius ATCC 25975. HPr was purified by DEAE-cellulose chromatography, molecular sieving on Ultrogel AcA44, and carboxymethylcellulose chromatography. Sodium dodecyl sulfate electrophoresis in the presence of urea revealed a single band with a molecular weight of 6700. The protein contained no tryptophan and had a pI of 4.8. The purification scheme of EI was as follows: DEAE-cellulose chromatography, hydroxylapatite chromatography, DEAE-Sephadex A-50 chromatography, preparative electrophoresis, and molecular sieving on Ultrogel AcA34. The five-step purification for EI produced a 199-fold purified preparation with a specific activity of 530 mumol of HPr phosphorylated per minute per milligram of protein at 37 degrees C. The fraction obtained after filtration on Ultrogel AcA34 gave one band (68 000) on sodium dodecyl sulfate - polyacrylamide gel electrophoresis. The molecular weight of the native enzyme determined by gel filtration at 4 degrees C was 135 000, suggesting that it was a dimer. Enzyme I had a pI of 4.2, a pH optimum of 6.7, a Km for HPr of about 27 microM, a Km for phosphoenolpyruvate of 0.48 mM, and kinetics that were consistent with a Ping-Pong mechanism. Evidence had been obtained which indicated that S. salivarius enzyme I was antigenically very similar to enzyme I from various strains of Streptococcus mutans, but not to the enzyme from Bacillus subtilis, Staphylococcus aureus, Streptococcus faecalis, and Escherichia coli.     

Multiple forms of hydroxycinnamate : CoA ligase in etiolated pea seedlings

A survey of a range of plant tissues showed that the hydroxycinnamate CoA ligase in crude extracts of pea shoots had a high relative activity towards sinapic and other methoxycinnamic acids, together with high activity with p-coumaric acid. The pea enzyme has been resolved by chromatography on DEAE-cellulose into two peaks which differ in their substrate specificity. The form which elutes at relatively low salt concentrations has a ratio activity towards p-coumaric and sinapic acids of about 1.8:1 while the form eluting at higher salt concentrations, although showing very high activity with p-coumaric acid, is inactive towards sinapic acid. The pattern of elution of these forms following gel filtration on Ultragel AcA 34 and Sephadex G100 suggests that these two isoenzymes which differ in ionic properties and substrate specificity can exist in two or three molecular weight forms and there is evidence that these forms are under certain circumstances interconvertible.     

The aggregation state of bovine heart cytochrome c oxidase and its kinetics in monomeric and dimeric form

The monomeric and dimeric forms of bovine cytochrome c oxidase (EC 1.9.3.1) were obtained from gel filtration chromatography on Ultrogel AcA 34 and analyzed. Both species contained all 12-13 subunits described for this enzyme. In the dimer 320 molecules [3H]dodecyl-beta-D-maltoside were bound per heme aa3 and in the monomer 360 molecules per heme aa3. The monomers contained 10 mol of tightly bound phospholipid/mol heme aa3 and the dimers 14. Sedimentation coefficients of 15.5-18 S for the dimer and 9.6 S for the monomer were calculated from sucrose density centrifugation analysis and analytical centrifugation. By the laser beam light-scattering technique a Stokes radius of 70 A for the dimeric detergent-lipid-protein complex was measured. From those parameters and the densitometric determined partial specific volumes of the detergent and the enzyme, the molecular weights of 400,000 for the protein moiety of the dimer and 170,000-200,000 for the monomer were calculated. Under very low ionic strength conditions the monomer/dimer equilibrium was found to be dependent on the protein concentration. At low enzyme concentrations (10(-9) M) monomers were predominant, whereas at concentrations above 5 X 10(-6) M the amounts of dimers and higher aggregates were more represented. The cytochrome c oxidase activity, measured spectrophotometrically and analyzed by Eadie-Hofstee plot, was biphasic as a function of cytochrome c concentration for the dimeric enzyme. Pure monomers gave monophasic kinetics. The data, fitting with a homotropic negative cooperative mechanism for the dimer of cytochrome c oxidase, are discussed and compared with other described mechanisms.     

IMP dehydrogenase. II. Purification and properties of the enzyme from Yoshida sarcoma ascites tumor cells

The preceding paper showed that IMP dehydrogenase [IMP:NAD+ oxidoreductase, EC 1.2.1.14] tended to form a precipitable complex(es) through ionic and hydrophobic interactions. On the basis of these observations, a method was developed for purification of IMP dehydrogenase from Yoshida sarcoma ascites cells. On SDS-polyacrylamide gel electrophoresis, the purified preparation (1.19 U/mg protein) appeared homogeneous and its minimum molecular weight was estimated to be 68K daltons. Amino acid analyses indicated a subunit molecular weight of 68,042. Molecular sieve chromatography in the presence of 10% (NH4)2SO4 showed that the molecular weight of the native enzyme was 127K daltons. These values indicate that the native enzyme is composed of two identical subunits. However, the purified enzyme gave 4 protein bands on polyacrylamide gel electrophoresis under non-denaturing conditions, and appeared as a single fraction in the vicinity of the void volume on Ultrogel AcA 34 column chromatography at low salt concentration, indicating that its molecular weight exceeded 200K daltons. These findings indicate that the enzyme tends to aggregate owing to its own physicochemical characteristics. The Km values for IMP and NAD were calculated to be 12 and 25 microM, respectively, and the Ki values for XMP, GMP, and AMP to be 109, 130, and 854 microM, respectively. The purified enzyme showed full activity in the presence of K+, and K+ could be partially replaced by Na+. PCMB inactivated the enzyme, but the activity was completely restored by the addition of DTT. Cl-IMP also inactivated the enzyme and IMP prevented this inactivation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Purification and Characterization of an Iminopeptidase from the Primary Leaf of Wheat (Triticum aestivum L.)

Iminopeptidase (EC 3.4.11.5) was substantially purified from the primary leaves of 7-day-old wheat seedlings (Triticum aestivum L.). The purification procedure consisted of five steps: acid precipitation, molecular exclusion chromatography on Sephacryl S-200, Ultrogel AcA 44, Sepharose 2B and ion-exchange chromatography on DEAE-cellulose. Iminopeptidase isolated in this manner was only active against the β-naphthylamides of proline and hydroxyproline. For each substrate, the pH optimum was 7.4 and activity was sensitive to sulfhydryl group inhibitors. The iminopeptidase hydrolyzed the dipeptides Pro-Leu, Pro-Gly, Hyp-Gly, and Pro-Tyr. Iminopeptidase activity against the dipeptide Pro-Gly was higher than against Hyp-Gly. The molecular weight was estimated to be about 400,000. Evidence was obtained for the existence of endogenous inhibitors of iminopeptidase activity.     

Substrate-bonded hyaluronic acid exhibits a size-dependent stimulation of chondrogenic differentiation of stage 24 limb mesenchymal cells in culture

Extracellular matrix molecules including glycosaminoglycans have been implicated in several differentiative and morphogenetic processes including cell aggregation and migration. Previous reports have shown that plating of stage 24 limb mesenchyme cells onto hyaluronic acid (HA) bonded to the culture substrate causes an increase in the number of cells exhibiting chondrogenesis. This increased chondrogenesis is now shown to be dependent upon the source of the HA. When limb mesenchymal cells are plated onto HA from bovine vitreous humor, human umbilical cord, or large molecular weight HA (Healon), increased chondrogenesis is observed only on the bovine vitreous humor HA. Unsulfated chondroitin, which has a structure and charge density similar to those of HA, is capable of enhancing chondrogenesis, while cells plated onto sulfated glycosaminoglycan substrates are indistinguishable from controls. The evidence in this report suggests that the differentiation response is related to the molecular size of the HA bound to the culture substrate. Healon and human umbilical cord HA are ineffective because their molecular weight is too large, while smaller HA derived from these larger molecules or normally present in bovine vitreous humor preparations stimulates the chondrogenic differentiation of stage 24 limb mesenchymal cells in culture. The most active size class of HA elutes from a Sepharose CL-2B column with a Kav between 0.6 and 0.7 and, thus, has a molecular weight of approximately 200,000-400,000. These observations reinforce the hypothesis that local cues have an informational effect on the differentiation of chick limb mesenchymal cells.     

Role of hydration in the binding of lac repressor to DNA

The osmotic stress technique was used to measure changes in macromolecular hydration that accompany binding of wild-type Escherichia coli lactose (lac) repressor to its regulatory site (operator O1) in the lac promoter and its transfer from site O1 to nonspecific DNA. Binding at O1 is accompanied by the net release of 260 +/- 32 water molecules. If all are released from macromolecular surfaces, this result is consistent with a net reduction of solvent-accessible surface area of 2370 +/- 550 A. This area is only slightly smaller than the macromolecular interface calculated for a crystalline repressor dimer-O1 complex but is significantly smaller than that for the corresponding complex with the symmetrical optimized O(sym) operator. The transfer of repressor from site O1 to nonspecific DNA is accompanied by the net uptake of 93 +/- 10 water molecules. Together these results imply that formation of a nonspecific complex is accompanied by the net release of 165 +/- 43 water molecules. The enhanced stabilities of repressor-DNA complexes with increasing osmolality may contribute to the ability of Escherichia coli cells to tolerate dehydration and/or high external salt concentrations.     

A solid phase assay for galactosyl transferases. Evidence for an active site of less than 14 kDa

Galactosyltransferase (GalTase) prepared from human milk was found to exist as a complex with e-lactalbumin as demonstrated by crossed immunoelectrophoresis against specific antibodies raised against the complex. GalTase activity was stable to proteolysis and, when subjected to gel filtration on Ultrogel AcA54, the enzyme activity eluted as a single peak. A second peak of activity was found to be adsorbed to the column matrix and was eluted with buffer containing 1 M NaC1. The hydrophobic fraction represented 5% of the total GalTase activity in human milk. After polyacrylamide gel electrophoresis the main enzyme activity peak was represented by polypeptides of 67kDa molecular weight and of 14kDa molecular weight. Electroblotting of these peptides onto a nitrocellulose membrane followed by determination of GalTase activity showed activity for 45–55 kDa and for 14 kDa peptides. The hydrophobic fraction from the AcA54 column was resolved into polypeptides of 110 kDa-45 kDa molecular weight, all of which contained GalTase activity after blotting. It is supposed that the GalTase from non-proteolyzed milk is composed of a 14 kDa polypeptide containing the active site together with another part of the polypeptide backbone which is involved in the regulation of GalTase activity by -lactalbumin, a third part of the polypeptide is responsible for the membrane insertion.Abbreviations UDP-Gal uridine diphosphatidyl galactose - GlcNAc N-acetylglucosamine - Glc glucose - PAGE polyacrylamide gel electrophoresis - GalTase galactosyl transferase (EC 2.4.1.22) - -ovo pronosac digest fraction of hen ovomucoid To whom correspondence should be addressed.     

Purification and characterization of a Corynebacterium ulcerans bacteriocin (ulceracin 378)

Corynebacterium ulcerans strain 378 produces a bacteriocin (ulceracin 378) and a toxin when grown on semi-solid medium. Ulceracin 378 was purified 360-fold by dialysis and chromatography on DEAE-cellulose and Sephadex G-200. On the basis of Ultrogel AcA22 gel filtration its molecular weight was about 900 000. It could be dissociated by 2-mercaptoethanol and sodium dodecyl sulphate into smaller subunits of 25 000. The bactericidal activity was associated with this subunit which contained no carbohydrate or lipid. Ulceracin 378 was thermostable and stable over a wide pH range. Purified ulceracin 378 did not have a toxic effect (lethal) on guinea-pigs and rabbits and was immunologically distinct from the toxin.     

Isolation of a plasma protein elevated during hypertension in the rat

Plasma from male rats contains a protein that is elevated during essential hypertension. This protein, termed hypertension associated protein (HAP), can be detected as a peptide that has a molecular weight of 14,000 daltons on high resolution SDS-gradient polyacrylamide gels. The native protein has now been isolated by elution from DEAE-Sepharose, carboxymethyl cellulose and by gel permeation on Ultrogel AcA44. The procedure yields 102 mg of highly purified protein from 5 ml (250 mg) of plasma in 72 h. The native protein has a molecular weight of 28,000 daltons.     

Soluble calcium-binding proteins: parvalbumins and calmodulin from eel skeletal muscle

1. Eel skeletal muscle contains three parvalbumin isoforms. The overall parvalbumin concentration in the muscle is 0.5 mmol kg-1 wet weight. 2. Calmodulin (0.1 mumol kg-1 wet weight) was purified by extraction with ethylenediamine tetraacetate-containing buffer, fractionation with trichloroacetic acid and separation by ion exchange chromatography on DEAE-cellulose and by molecular sieving on Ultrogel AcA 54. 3. Troponin-C-free calmodulin was obtained by fast protein liquid chromatography on a Mono Q column.