Production of superoxide radicals and hydrogen peroxide by NADH-ubiquinone reductase and ubiquinol-cytochrome c reductase from beef-heart mitochondria.

Complex I (NADH-ubiquinone reductase) and Complex III (ubiquinol-cytochrome c reductase) supplemented with NADH generated O₂^? at maximum rates of 9.8 and 6.5 nmol/min/mg of protein, respectively, while, in the presence of superoxide dismutase, the same systems generated H₂O₂ at maximum rates of 5.1 and 4.2 nmol/min/mg of protein, respectively. H₂O₂ was essentially produced by disproportionation of O₂^?, which constitutes the precursor of H₂O₂. The effectiveness of the generation of oxygen intermediates by Complex I in the absence of other specific electron acceptors was 0.95 mol of O₂^? and 0.63 mol of H₂O₂/mol of NADH. A reduced form of ubiquinone appeared to be responsible for the reduction of O₂ to O₂^?, since (a) ubiquinone constituted the sole common major component of Complexes I and III, (b) H₂O₂ generation by Complex I was inhibited by rotenone, and (c) supplementation of Complex I with exogenous ubiquinones increased the rate of H₂O₂ generation. The efficiency of added quinones as peroxide generators decreased in the order Q₁ > Q₀ > Q₂ > Q₆ = Q₁₀, in agreement with the quinone capacity of acting as electron acceptor for Complex I. In the supplemented systems, the exogenous quinone was reduced by Complex I and oxidized nonenzymatically by molecular oxygen. Additional evidence for the role of ubiquinone as peroxide generator is provided by the generation of O₂^? and H₂O₂ during autoxidation of quinols. In oxygenated buffers, ubiquinol (Q₀H₂), benzoquinol, duroquinol and menadiol generated O₂^? with k₃ values of 0.1 to 1.4 m^? · s^?1 and H₂O₂ with k₄ values of 0.009 to 4.3 m^?1 · s^?1.     

Purification and partial characterization of bovine heart phosphorylase kinase

Bovine heart phosphorylase kinase has been isolated by a procedure involving precipitation with polyethylene glycol, DEAE-Sephacel chromatography and calmodulin-Sepharose affinity chromatography. The isolated enzyme had a specific activity of 8.3 IU/mg of protein at pH 8.2 at 30 degrees C in the presence of 1% glycogen. The native enzyme had a sedimentation coefficient of 23 S and the Mr of the alpha', beta, gamma, and delta subunits, were 140,000, 130,000, 46,000, and 18,000, respectively. Activation of the phosphorylase kinase by the catalytic subunit of bovine heart cAMP-dependent protein kinase increases the pH 6.8/8.2 activity ratio from 0.01 to 0.32-0.38. Glycogen (1%) decreased the Km of the activated phosphorylase kinase at pH 6.8 for phosphorylase b from 5.5 to 1.25 mg/ml. Trypsin treatment increased the pH 6.8 activity but decreased the pH 8.2 activity. During this process the alpha' subunit was converted to a Mr 110,000 polypeptide and the enzyme activity was converted essentially to a 5.9 S species having an apparent Mr of 100,000 as determined by gel filtration. On extended trypsin treatment only one major polypeptide corresponding to the beta subunit remained. The same polypeptide was present in the active fractions following gel filtration of the trypsinized kinase.     

The activation of bovine Factor X by bovine Factor Xa

A steady-state kinetic analysis of the activation of bovine Factor X, by bovine Factor X_a, was undertaken. The activation was found to be dependent on the presence of divalent cations; Ca²⁺ showing the greatest stimulatory effect and Mn²⁺ exhibiting a lower degree of activity for this reaction. Although Sr²⁺ and Mg²⁺ were ineffective when present alone, each contributed synergistically to the activation rate at suboptimal levels of Ca²⁺. The effect of phospholipid (phosphatidylcholine:phosphatidylserine, 4:1, w:w) on the rate of activation and on the activation pathway was investigated. Phospholipid (PL) concentrations of up to 40 μm had no effect on the activation rate; whereas, concentrations of 40–180 μm were slightly inhibitory. In the absence of PL, the major product of the activation was Factor α-X_a, while in the presence of PL, lower-molecular-weight forms of Factor X (Factor β-X) and Factor X_a (Factor β-X_a were produced. At saturating levels of Ca²⁺, the K_{m app} for the activation, at pH 7.4 and 37 °C, in the absence of PL, was found to be 0.6 ± 0.1 μm and the V was 1.7 ± 0.3 mol Factor X cleaved min^?1 mol^?1 Factor X_a. The corresponding values, in the presence of 90 μm PL, were 1.4 ± 0.2 μm and 2.2 ± 0.2 mol Factor X cleaved min^?1 mol^?1 Factor X_a.     

Bacteriophage P1 site-specific recombination: I. Recombination between loxP sites

We have studied P1 site-specific recombination by cloning a 6·5 × 10³ base EcoRI fragment (fragment 7) of P1 DNA into a λ vector and then asking whether that fragment can promote efficient recombination for λ markers that flank the fragment. Our results indicate that fragment 7 can reassort these markers very efficiently, and that this recombination can occur in the absence of the bacterial recA and recBC functions. The fragment 7 recombination system has been dissected by an analysis of deletion mutations into two components, a site (called loxP) that must be present in both partners in the recombination in order for recombination to occur, and a P1 gene (called cre), whose product is necessary for recombination. The location of the loxP site at the end of the P1 genetic map suggests that this site-specific recombination system is responsible for the lack of linkage between terminal P1 markers and therefore for the linearity of that map.     

Interaction of ferredoxin and ferredoxin-NADP reductase with thylakoids

Ferredoxin-NADP reductase accounts for about 50% of the NADPH diaphorase activity of spinach leaf homogenates. The enzyme is bound to thylakoid membranes, but can be slowly extracted by aqueous buffers. Ferredoxin-NADP reductase can be extracted from the membranes by a 1- to 2-min treatment with a low concentration of trypsin. This treatment completely inactivates NADP photoreduction but does not affect electron transport from water to ferredoxin. It is shown that the inactivation is due to solubilization of ferredoxin-NADP reductase: the activity can be restored by addition of a very large excess of soluble enzyme in pure form. When ferredoxin-NADP reductase is added as a soluble enzyme after extraction or inactivation (by a specific antibody) of the membrane-bound enzyme, NADP photoreduction requires a very large excess of this enzyme, and the apparent K_m for ferredoxin is also increased. These observations are discussed as related to the interactions of thylakoids with ferredoxin-NADP reductase.     

Multifunctionality of lipoamide dehydrogenase: Role of histidine residues in reductase reaction

Rose bengal sensitizes photoinactivation of lipoamide dehydrogenase from pig heart to a constant residual reductase activity resulting from specific destruction of histidine residues. The rate of sensitized photoinactivation is pH dependent and is associated with an ionizable group with pK 6.6 ± 0.2. All steady-state kinetic parameters are markedly reduced by photooxidation. Spectroscopic studies indicate the contribution of oxidized flavin/dithiol to the half-reduced form of the photooxidized enzyme. The proton magnetic resonance spectrum of lipoamide dehydrogenase shows resolved histidine C₂ proton peak at δ9.18 ppm and a shoulder at δ9.23 ppm. The shoulder protons are eliminated by the sensitized photooxidation and shifted upfield on deprotonation. At high pH, the characteristic Faraday A term also disappears. These observations suggest that the essential histidine stabilizes the nascent thiolate via the ion pair formation to facilitate the reductase reaction catalyzed by lipoamide dehydrogenase.     

Purification and characterization of a novel pyrazole-sensitive carbonyl reductase in guinea pig lung

Mouse Ehrlich ascites tumor cells incubated with the creatine analog, N-ethylguanidinoacetate (N-amidino-N-ethylglycine), accumulated up to 8 μmol/g packed cells of the creatine-P analog, N-ethylguanidinoacetate-P. Evaluation of N-ethylguanidinoacetate-P as a synthetic phosphagen under in vivo conditions was performed with Ehrlich cells loaded with equimolar amounts of a common reference phosphagen, cyclocreatine-P (1-carboxymethyl-2-imino-3-phosphonoimidazolidine) plus either N-ethylguanidinoacetate-P or creatine-P. It was concluded that N-ethylguanidinoacetate-P has a Gibbs free energy of hydrolysis equal to that of creatine-P and 2 kcal/mol greater than that of cyclocreatine-P. The relative rates of utilization of intracellular phosphagens by Ehrlich cells when their ATP pools were depleted with 2-deoxyglucose were in the order: creatine-P > N-ethylguanidinoacetate-P > cyclocreatine-P. Dietary N-ethylguanidinoacetate was nontoxic even at very high levels to all animal systems tested. Feeding of 2% N-ethylguanidinoacetate to mice or chicks resulted in equimolar replacement of natural by synthetic phosphagen to the following extents: heart, 75%; leg muscle, 50%; and brain 10–25%. N-Ethylguanidinoacetate-P is the most active synthetic phosphagen thus far found to be accumulated by animal tissues.     

The complete amino acid sequence of bovine liver catalase and the partial sequence of bovine erythrocyte catalase

The data upon which the sequence of the 506 residues in the subunit of bovine liver catalase (BLC) is based are presented in detail. A partial sequence of bovine erythrocyte catalase (BEC) which accounts for 493 residues shows complete concordance with the BLC data. On the other hand, BEC has at least 517 residues, that is, an extension beyond the C terminus of the BLC data. Although normally BLC has only 506 residues, there is evidence that, at some point in its history, it also had the C-terminal extension. It is speculated that this extension is lost in BLC either through a different processing of the molecule in liver than in erythrocytes or by partial degradation in the first stages of catabolism.     

Assay of ribonucleotide reductase activity in intact permeabilized hamster cells: an evaluation

An intact cell assay system, based on Tween-80 permeabilization can be used to investigate ribonucleotide reductase activity in a variety of mammalian cell lines. An important consideration in the use of intact cells is the presence of other nucleotide metabolizing activities. The influence of these activities on estimates of pyrimidine (CDP) and purine (ADP) reductase in permeabilized hamster cells has been examined. Studies on the incorporation of label from CDP and ADP into RNA indicated that a very small proportion of the reductase substrates was eventually incorporated into RNA during routine enzyme assays, and would have no detectable effect on activity estimates. The possibility that the products of the reaction (dCDP and dADP) were eventually phosphorylated and incorporated into DNA was also examined, and it was found that proper permeabilization of the cells eliminated or greatly reduced loss of deoxyribonucleotides to DNA. An analysis by HPLC of nucleotides present during CDP and ADP reductase reactions showed that various kinases and phosphatases were active in permeabilized cells, as all levels of phosphorylation of nucleotide substrates and allosteric effectors were detected. The base composition of the nucleotides added to the assay systems were not altered. Although movement of phosphates occurred during the assay, the concentrations of substrates quickly reached equilibrium (within 1 min) with their respective nucleosides and nucleotides, resulting in a relatively constant although reduced concentration of CDP or ADP substrates during the 20-min assay. Similarly the levels of allosteric effectors, ATP for pyrimidine and dGTP for purine reductase activities, declined within the first minute of the assays and quickly reached an equilibrium with their respective adenine or guanine containing nucleotides during most of the reaction time. Although useful approximations of intracellular reductase activity can be obtained without correcting for modified nucleotide concentrations, precise determinations can be calculated when these alterations are taken into consideration. For example, estimates of intracellular K_m values for CDP closely resembled those reported with highly purified mammalian enzyme preparations in other studies. Clearly, the intact cell assay system provides worthwhile information about mammalian ribonucleotide reductase in its physiologically relevant environment.     

Plasma fibronectin-binding glycosaminoglycans in the substratum adhesion sites of neural tumor cells

The metabolism of high-density lipoprotein (HDL) in cells of five human cancer cell lines maintained in monolayer culture was investigated. In cells of some of the lines there was evidence of high-affinity binding sites for HDL, whereas in others this could not be demonstrated. However, in one cell line, viz., HEC-B-296 (human endometrial carcinoma), degradation of the protein component of HDL was demonstrated. The proteolytic activity was specific for HDL in so far as human serum albumin was not degraded by these cells. However, this degradative process did not involve internalization of the HDL molecule and degradation was not mediated by lysosomal proteolytic enzymes. HDL, when present in the medium, did not affect the degradation of low-density lipoprotein and low-density lipoprotein did not affect the degradation of HDL. HDL did not affect significantly cholesterol biosynthesis or cholesteryl ester biosynthesis as estimated from the activity of the regulatory enzymes, 3-hydroxy-3-methylglutaryl coenzyme A reductase and acyl-CoA:cholesterol acyltransferase. The degradation of HDL by HEC-B-296 cells was inhibited, to various degrees, when trypsin inhibitor or a protease inhibitor such as leupeptin, was present in the culture medium. It is concluded that degradation of the protein component of HDL by human neoplastic cells of the HEC-B-296 line was the result of activity of a proteolytic enzyme that is present on the external surface of the cells.     

Mapping of DNA gyrase cleavage sites in vivo oxolinic acid induced cleavages in plasmid pBR322

We have developed a procedure which permits the mapping of DNA gyrase cleavage sites in vivo. Addition of oxolinic acid, an inhibitor of DNA gyrase, to growing cells of Escherichia coli containing the plasmid pBR322 resulted in double-strand cleavage of DNA, and allowed the isolation of significant quantities of linearized plasmid DNA after lysis of treated cells with sodium dodecyl sulfate. Initially the linear product was purified from agarose gels, cleaved by restriction endonucleases, and then subjected to Southern hybridization analysis using defined DNA probes. A number of distinct cleavage sites, used with varying degrees of efficiency, were identified within pBR322 using this simple procedure. To achieve greater resolution and to improve sensitivity, we then employed an electroblotting procedure to transfer DNA fragments from acrylamide gels onto nylon membranes. This alternative method does not require the isolation of the linearized product before performing the mapping procedure. The improved resolution obtained from acrylamide gels and the superior binding properties of the nylon membranes have allowed us to accurately map 74 distinct oxolinic acid-induced cleavage sites within pBR322. The significance of these findings in light of previously reported studies in vitro, as well as the possible role of such sites during illegitimate recombination, are discussed.     

Messenger RNAs from the transforming region of bovine papilloma virus type I

Messenger RNAs present in C127 mouse cells transformed by bovine papilloma virus type 1 (BPV-1) were studied by the S1 nuclease protection technique, Northern blotting, and electron microscopic heteroduplex analysis. The results revealed at least five classes of spliced mRNAs which we designate types 1 to 5. They had a common poly(A) addition site located at co-ordinate 53 and all mRNAs, except the type 3 mRNAs, contained an exon located between co-ordinates 41 and 53. In the type 1 mRNAs this exon was connected to a very short leader sequence located around co-ordinate 31. The type 2 mRNAs contained 220 to 400-nucleotide long leaders which were located approximately 1.5 X 10(3) base-pairs further upstream. Two different subclasses of type 2 molecules (2A and 2B) were identified and these had slightly different leaders. The type 4 mRNAs contained a bipartite leader, whereas the type 5 mRNAs carried an approximately 900-nucleotide long leader. The type 3 mRNAs consisted of a main exon located between co-ordinates 32 and 53, linked to the same leader as is present in the type 2A mRNAs. A cap site which presumably is utilized by the type 2A, type 3, type 4 and type 5 mRNAs was mapped at nucleotide 89 in the BPV-1 sequence. A putative cap site for the type 1 mRNAs was mapped at co-ordinate 31.     

Relationship between membrane and cytoplasmic domains in cytochrome c oxidase by electron microscopy in media of different density

We have investigated the relative location of the exposed cytoplasmic and membrane domains in cytoehrome c oxidase vesicle crystals by varying the density of the embedding medium in electron microscopy. This gives the connectivity of the domains, revealing a Y-shaped cytoehrome c oxidase monomer. A large domain, the stem of the Y, projects over 50 Å into solution from the side of the crystal membrane corresponding to the cytoplasmic face of the inner mitochondrial membrane. Two smaller domains are embedded in the bilayer and must be largely separated by lipid. The Y-shaped cytochrome c oxidase monomers are compactly paired as dimers.     

The interaction of substrate-related ketals with bacterial and viral neuraminidases

Arthrobacter sialophilus neuraminidase catalyzes the hydration of 5-acetamido-2,6-anhydro-3, 5-dideoxy-d-glycero-d-galacto-non-2-enonic acid (2,3-dehydro-AcNeu) with K_m and k_cat values of 8.9 × 10^?4m and 6.40 × 10^?4 s^?1, respectively. The methyl ester of 2,3-dehydro-AcNeu as well as 2,3-dehydro-4-epi-AcNeu are also hydrated by the enzyme. The product resulting from the enzymatic hydration of 2,3-dehydro-AcNeu is N-acetylneuraminic acid. A series of derivatives of 2,3-dehydro-AcNeu (K_I 1.60 × 10^?6m) including 2,3-dehydro-4-epi-AcNeu (2.10 × 10^?4m) and 2,3-dehydro-4-keto-AcNeu (K_I = 6.10 × 10^?5 m) were each competitive inhibitors of the enzyme. The methyl esters of these ketal derivatives were also competitive enzyme inhibitors. Dissociation constants for these ketals were determined independently by fluorescence enzyme titrations which gave values similar to those found kinetically. These six relatives of 2,3-dehydro-AcNeu were also competitive inhibitors for the influenza viral neuraminidases. For the viral neuraminidases, the dissociation constant for 2,3-dehydro-AcNeu and its methyl ester were 2.40 × 10^?6 and 1.17 × 10^?3m, respectively. The interpretation placed upon the K_I values determined for these ketals against the Arthrobacter versus influenza neuraminidases is that the bacterial enzyme has a more flexible glycone binding site.     

A quasi-elastic laser light scattering study of tubulin and microtubule protein from bovine brain

Quasi-elastic light scattering has been used to characterize the oligomeric properties of solutions of glycerol-cycled bovine microtubule protein, and the properties of the 30 S oligomeric species and 6 S tubulin heterodimer prepared by gel filtration on Sepharose 6B. It is shown that in dimer preparations, as little as 0.04% by number of 30 S rings would account for the difference between an observed mean diffusion coefficient D_{20, W} = 3.1 × 10^?7 cm² s^?1 and the value of D_{20, W} = 5.1 × 10^?7 cm² s^?1 calculated for tubulin dimer of M_rel 100,000. The 30 S ring has an observed diffusion coefficient of D_{20, W} = 0.49 × 10^?7 cm² s^?1. These values are not changed significantly by the presence of 4 m-glycerol, indicating the persistence of 6 S and 30 S forms for dimer and ring, respectively.Mixtures of ring and dimer components of this preparation behave as a non-interacting two-component system, indicating the absence of substantial re-equilibration between the species at 5 °C and pH 6.5.The effect of salt on ring and microtubule protein samples indicates partial dissociation, consistent with the formation of additional intermediate oligomeric forms.In quasi-elastic light scattering measurements adapted to kinetic studies, changes in the oligomeric composition of microtubule protein are detected in the early stages of the reversible assembly process at pH 6.5. A 25% decrease in scattered light intensity, without significant change in mean diffusion coefficient, indicates the lability of the ring oligomeric structures, which undergo partial transformation to alternative oligomeric species under these assembly conditions.     

NADH: (acceptor) oxidoreductase from bovine adrenal medulla chromaffin granules

The NADH:(acceptor) oxidoreductase from membranes of bovine adrenal medulla chromaffin granules has been purified by column chromatography. After solubilization of the membranes with emulphogen, a nonionic detergent, the enzyme was purified by dye-ligand chromatography and gel filtration. The oxidoreductase appeared essentially homogeneous on two gel electrophoretic systems. On polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, the enzyme revealed a dimeric structure with a combined molecular weight of about 55,000. The enzyme eluted as a detergent-lipid-protein aggregate with a Stoke's radius of 43 Å on gel filtration columns in the presence of emulphogen. The amino acid composition of the oxidoreductase was found to be distinct from that of similar enzymes from other organelles. Topographical experiments indicated that the enzyme is a transmembrane protein.     

Purification and properties of carnitine acetyltransferases from bovine spermatozoa and heart

To investigate the physical and kinetic properties of sperm carnitine acetyltransferase, the enzyme was purified from bovine spermatozoa and heart muscle. Carnitine acetyltransferase was purified 580-fold from ejaculated bovine spermatozoa to a specific activity of 85 units/mg protein (95% homogeneity). Sperm carnitine acetyltransferase was characterized as a single polypeptide of M_r 62,000 and pI 8.2. Heart carnitine acetyltransferase was purified 650-fold by the same procedure to a final specific activity of 71 units/mg protein. The kinetic properties of purified bovine sperm carnitine acetyltransferase were consistent with the proposed function of this enzyme in acetylcarnitine pool formation. Product inhibition by either acetyl-l-carnitine or CoASH was not sufficient to predict significant in vivo inhibition of acetyl transfer. At high concentrations of l-carnitine, bovine sperm and heart carnitine acetyltransferases were most active with propionyl- and butyryl-CoA substrates, although octanoyl-, iso-butyryl-, and iso-valeryl-CoA were acceptable substrates. Binding of one substrate was enhanced by the presence of the second substrate. Carnitine analogs that have significance in reproduction, such as phosphorylcholine and taurine, did not inhibit carnitine acetyltransferase. Bovine sperm and heart carnitine acetyltransferases were indistinguishable on the basis of purification behavior, pI, pH optima, kinetic properties, acyl-CoA specificity, and sensitivity to sulfhydryl reagents and divalent cations; thus there was no indication that bovine sperm carnitine acetyltransferase is a sperm-specific isozyme.     

Spectral changes induced in Cibacron Blue F3GA by salts,organic solvents,and polypeptides: Implications for Blue dye interaction with proteins

The interactions of Cibacron Blue F3GA with organic solvents, salts, oligopeptides, and polypeptides were studied by visible difference spectroscopy. The difference spectrum of the dye in an aqueous solution of NaCl (vs water) has a characteristic positive peak at 690 nm and negative double minima at 630 and 585 nm. Such a “salt-like” spectrum is also obtained for interaction of the dye with polycations such as oligolysines, polylysine, polyarginine, and protamine. In contrast, the difference spectrum of the dye in binary aqueous solvents containing dioxan or t-butyl alcohol at moderately high concentrations, measured against water, displays a positive peak and shoulder at 655 and 610 nm, respectively, with a small negative contribution below 550 nm. This spectrum is attributed to a nonpolar interaction of the dye with organic cosolvent molecules. The spectrum of the dye in 7 M urea is changed little from that in water, indicating similar interactions of the dye with water or urea molecules. The spectral characteristics described here for the interaction of the polyaromatic polysulfonate dye with positively charged groups, polar groups, and nonpolar moieties of neutral molecules provide a basis for describing the details of the interactions of Cibacron Blue F3GA with several proteins and for characterizing the dye binding environments in the proteins.     

Synthesis and accumulation of an extremely stable high-energy phosphate compound by muscle, heart, and brain of animals fed the creatine analog, 1-carboxyethyl-2-iminoimidazolidine (homocyclocreatine)

A new creatine analog, 1-carboxyethyl-2-iminoimidazolidine (homocyclocreatine), has been synthesized and compared with other synthetic analogs of creatine as a substrate for creatine kinase under both in vitro and in vivo conditions. Reactivity with rabbit muscle creatine kinase at 2 mM and pH 7.0 occurred in the order: creatine greater than cyclocreatine (1-carboxymethyl-2-iminoimidazolidine) greater than N-ethylguanidinoacetate greater than N-propylguanidinoacetate greater than guanidinoacetate greater than N-methyl-3-guanidinopropionate greater than 3-guanidinopropionate greater than homocyclocreatine. Homocyclocreatine was 10,000-fold less active than creatine. In the reverse direction at 0.2 mM and pH 7.0: creatine-P greater than N-ethylguanidinoacetate-P greater than cyclocreatine-P much greater than homocyclocreatine-P. Homocyclocreatine-P was 200,000-fold less active than creatine-P. The phosphoryl group transfer potential of homocyclocreatine-P was estimated to be 2 kcal/mol lower than that of creatine-P. Chicks fed 5% homocyclocreatine for 16 days synthesized and accumulated homocyclocreatine-P in breast muscle (32 mumol/g wet wt), leg muscle (24 mumol/g), heart (7 mumol/g), intestine (8.5 mumol/g), and brain (2.4 mumol/g). During ischemia homocyclocreatine-P was utilized by muscle much more slowly for the regeneration of ATP than was creatine-P or cyclocreatine-P. Our results suggest that in tissues of homocyclocreatine-fed animals subjected to a sudden large increase in work load or to ischemia, the residual creatine-P system would rapidly equilibrate with the adenylate system at the new lower cytosolic phosphorylation potential, whereas in the same cytosol the (homocyclocreatine-P)/(homocyclocreatine) ratio would exhibit a hysteresis or memory effect and reflect for a considerable period of time the earlier higher (ATP)/(free ADP) ratio rather than the actual lower (ATP)/(free ADP) ratio.     

Proteoglycans from the substratum adhesion sites of MSV-transformed Balb/c 3T3 cells

Kirsten murine sarcoma virus-transformed Balb/c 3T3 cells (KiMSV) are highly tumorigenic and metastatic in the appropriate murine host, are loosely adherent to the tissue culture substratum, and can be readily detached from the substratum by ethylene glycol bis(β-aminoethyl ether) N,N′-tetraacetic acid treatment leaving their adhesion sites as substratum-attached material. Both long-term culture-generated adhesion sites (L-SAM) of KiMSV cells and newly formed adhesion sites of reattaching cells (R-SAM) contain high levels of hyaluronate (HA) and chondroitin sulfate (CS) whereas the R-SAM of parental Balb/c 3T3 cells is enriched in heparan sulfate (HS). A sizable fraction of KiMSV L-SAM proteoglycans (PG) and a smaller fraction of R-SAM PG's aggregate into two size classes of supramolecular complexes, after extraction off the substratum with 4 m guanidine hydrochloride, as determined by chromatography on columns of Sepharose CL2B in several buffer systems. Isopycnic density gradient analyses under associative conditions of KiMSV L-SAM generated three classes of material—high-density G_A1 which contained some HA but principally CS and HS; intermediate-density G_A2 which contained only HA; and low-density G_A3 which contained some HA and principally glycoprotein. R-SAM gradients contained no G_A2 but a sizable amount of “low-density” HA in G_A3. When centrifuged under dissociative conditions, most of G_A1 and all of G_A2 from L-SAM shifted to the top of the gradient, whereas most of the HS-PG in R-SAM remained at the bottom of dissociative gradients. Comparison of these analyses with previous analyses of Balb/c 3T3 extracts demonstrates that (a) KiMSV cells generate adhesion sites with different PG contents than 3T3 sites; (b) the PG's of KiMSV sites have a reduced potential to aggregate into high-molecular-weight complexes but do form intermediate-size complexes not apparent in material from 3T3 sites; (c) these data support the hypothesis that HA is important in detachment of cells from extracellular matrices; and (d) HS-PG's in newly formed adhesion sites of KiMSV cells are considerably different from sites which have “matured”, indicating that there is metabolic activity in these sites during prolonged adherence and movement of transformed cells.