Alkaline phosphatase is an ectoenzyme that acts on micromolar concentrations of natural substrates at physiologic pH in human osteosarcoma (SAOS-2) cells

Alkaline phosphatase (ALP) was examined in cultured human osteosarcoma cells (SAOS-2) with respect to isoenzyme form, kinetic properties toward two natural substrates, and topography and nature of attachment to the plasma membrane. ALP in SAOS-2 homogenates is the tissue-nonspecific (TNS) isoenzyme and a phosphoethanolamine (PEA) and pyridoxal 5'-phosphate (PLP) phosphatase, as demonstrated by heat and inhibition profiles and electrophoretic mobility. Kinetic studies indicate that TNSALP in SAOS-2 cells has both a low- and a high-affinity activity. The high-affinity activity (showing the greater catalytic efficiency) is active at physiologic pH toward physiologic concentrations (microM) of PEA and PLP. TNSALP was shown to be an ectoenzyme in SAOS-2 cells by our findings in intact cell suspensions, where (i) PEA and PLP degradation in the medium nearly equaled that of whole cell homogenates, (ii) greater than 85% of ALP activity was inactivated by acid treatment, and (iii) ALP activity was quantitatively released by phosphatidylinositol-specific phospholipase C. Our findings indicate that, in SAOS-2 cells, TNS (bone) ALP functions as an ectoenzyme to degrade physiologic concentrations of extracellular natural substrates at physiologic pH.     

Determination of equilibrium binding affinity of distamycin and netropsin to the synthetic deoxyoligonucleotide sequence d(GGTATACC)2 by quantitative DNase I footprinting

A new method for determining the equilibrium binding constant of antitumor drugs to specific DNA sequences by quantitative DNase I footprinting is presented. The use of a short synthetic DNA oligomer to define a homogeneous population of DNA binding sites enables the calculation of the free drug concentration and the fraction of DNA sites complexed with drug in solution and is described for the first time. Since a 1:1 stoichiometry is observed for each drug-oligomer DNA complex, it becomes possible to calculate equilibrium binding constants in solution. By use of this technique, the binding affinities of the nonintercalating drugs netropsin and distamycin to the synthetic oligonucleotide d(GGTATACC)2 are determined to be Ka (25 degrees C) = 1.0 X 10(5) and 2.0 X 10(5) M-1, respectively. Quantitation of the temperature dependence associated with complex formation results in a determination of standard enthalpies of -3.75 and -8.48 kcal mol-1 for the binding of netropsin and distamycin, respectively. Calculation of other thermodynamic parameters are found to be in agreement with previous studies and indicate that the DNA binding process for these compounds is predominantly enthalpy driven. This method of quantitative DNase I footprinting is demonstrated to be a useful technique for the measurement of drug affinities to specific binding sites on DNA oligomers which are designed and synthesized expressly for this purpose. Applications of the technique to the determination of drug binding affinities at specific sites within native DNA sequences are discussed.     

Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow

Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and the major pools of each containing the amino acid sequence Gly339-Met423 were isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometry identified tryptic peptides (found also in normal antithrombin treated in the same way) that corresponded to amino acid sequences Gly339-Lys370 and Val400-Met423. The tryptic peptides, corresponding to amino acid sequences Ala371-Arg393 and Ser394-Arg399 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+ 2976 and 2952 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Ala371-Arg399, but with single amino acid substitutions at the reactive site, Arg393 replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).     

Recombinant interleukin-2-induced polyclonal proliferation of in vitro unstimulated human peripheral blood lymphocytes

Human peripheral blood mononuclear cells as well as T-cell-enriched or T-cell-depleted populations were found to proliferate in response to recombinant interleukin-2 (IL-2) in vitro in the absence of a lectin preactivation signal. This proliferative response was detected at Day 2, peaked at Day 5, and was dependent on the concentration of IL-2 used. At the initiation of culture, these cells did not appear to be activated as determined by the expression of Tac antigens. In cultures of unfractionated T-cell-enriched suspensions, high concentrations of IL-2 resulted in preferential expansion of the OKT8+ population, although both OKT4+ and OKT8+ cells proliferated in response to IL-2 when cultured alone. These studies demonstrate that human lymphocytes obtained by standard fractionation procedures from peripheral blood are capable of proliferation in response to IL-2 without in vitro preactivation signals given by the addition of mitogens or antigens to cultures. These findings suggest that in vivo IL-2, in the absence of other exogenous stimuli, may directly influence immune responses and thus may have a potential role as a clinical immunopharmacologic agent.     

Structural studies on H+,K+-ATPase: determination of the NH2-terminal amino acid sequence and immunological cross-reactivity with Na+,K+-ATPase

The NH2-terminal amino acid sequence of the 100 kilodalton subunit of porcine gastric H+,K+-ATPase has been determined to be YKAENYELYQVELGPGP. Although the NH2-terminal region of this protein is not similar to the same region of the lamb kidney Na+,K+-ATPase catalytic subunit, other regions of these ATPase proteins appear to be homologous. Both monoclonal and polyclonal antibodies raised to lamb kidney Na+,K+-ATPase and its alpha, but not beta, subunit cross-react with the 100 kilodalton protein of H+,K+-ATPase.     

Development of the prepupal Verson's gland of the tobacco hornworm, Manduca sexta, and its hormonal control

The segmentally arranged Verson's glands are epidermal derivatives comprised of three cells: the duct, saccule, and secretory cells. The development of these glands was followed through the 5th instar and larval-pupal transition of Manduca sexta. The glands are relatively small during the feeding stage, begin to grow at wandering, and undergo about a 50-fold increase in size during the prepupal period. The increase in size is due mainly to the hypertrophy of the secretory cell which synthesizes a heterogeneous set of proteinaceous secretory products. Three prominent 11 to 12 kiloDalton (kD) polypeptides are made by the pharate fifth larval gland, whereas the pupal gland produces polypeptides ranging from 14 to 75 kD with a major complex at 30 to 34 kD. The secretory product is poured out onto the surface of the new cuticle at the time of ecdysis and contains all of the major proteins detected in extracts of the whole gland. The accumulation of secretory products by the gland occurs during the prepupal peak of ecdysteroid and is blocked if this rise is prevented by abdominal isolation. Infusion of 30 micrograms 20-hydroxyecdysone (20-HE) into such isolated abdomens caused synthesis of the pupal products. Treatment with the juvenile hormone mimic, methoprene, during the fifth instar showed that the commitment of the glands to produce the pupal proteins is independent of and occurs before the overlying epidermis becomes committed to make pupal cuticle.     

Identification of an essential sulfhydryl group in the ouabain binding site of (Na,K)-ATPase

Ellman's reagent 5,5'-dithiobis-(2-nitrobenzoic acid) inhibits sodium- and potassium-stimulated ATPase, p-nitrophenyl phosphatase activity, and [3H]ouabain binding to lamb kidney (Na,K)-ATPase. The inactivation of [3H]ouabain binding follows pseudo-first order reaction kinetics at pH values less than or equal to 8.2. The inactivation of [3H]ouabain binding, but not of enzymatic activity, can be blocked by preincubation with ouabagenin, a rapidly reversible aglycone derivative of ouabain. The reduction in [3H]ouabain binding is due to a decrease in the number of binding sites rather than an alteration of the affinity of the enzyme for ouabain. Differential labeling at pH 8.2 with 1.0 mM 5,5'-dithiobis-(2-nitrobenzoic acid), preincubated with or without 5 microM ouabagenin, followed by tryptic digestion and reverse-phase high performance liquid chromatography of the generated soluble peptides reveals a single peptide labeled by the sulfhydryl probe that is protected by ouabagenin. From these results it is concluded that there is a single sulfhydryl group, essential for ouabain binding, presumably located in the ouabain binding site of lamb kidney (Na,K)-ATPase.     

Insulin-dependent phosphorylation of the insulin receptor-protein kinase and activation of glucose transport in 3T3-L1 adipocytes

Insulin stimulates hexose transport and phosphorylation of the insulin receptor in monolayer cultures of intact 3T3-L1 adipocytes. To assess the phosphorylation state of the receptor in situ, cells were equilibrated with [32P]orthophosphate and then disrupted under denaturing conditions which preserved the phosphorylation state of the receptor established in the cell. The insulin receptor, isolated by lectin adsorption and two-dimensional nonreducing/reducing polyacrylamide gel electrophoresis, occurred as a single oligomeric species with an apparent alpha 2 beta 2 subunit composition. This oligomeric structure was not altered by treating cells with insulin. Only the beta-subunit of the receptor was phosphorylated; [32P]phosphoserine and [32P] phosphotyrosine were both identified in the beta-subunit from cells in the unstimulated state, but only [32P] phosphotyrosine increased in cells stimulated with insulin. Neither insulin-like growth factors I nor II stimulated insulin receptor beta-subunit phosphorylation, although both activated hexose transport. Upon the addition of insulin, [32P]orthophosphate incorporated into the beta-subunit increased 4.5-fold (7-fold with respect to [32P]tyrosine) and was complete within 1 min (t1/2 = 8 s). Following the removal of insulin from the monolayers, [32P]beta-subunit fell to the basal level (t1/2 = 2.5 min); there was no lag phase before either transition. The tyrosine protein kinase activity, measured in vitro with a model substrate, was higher with immunoaffinity-purified insulin receptor from insulin-stimulated cells than from cells in the basal state. Hexose transport rate, measured using 3-O-[methyl-14C]glucose, was half-maximally stimulated at 2 nM insulin. A 1-min latency period followed insulin addition, after which a 7-fold increase in the steady-state rate of hexose uptake was achieved within 5 min. Upon the removal of insulin, hexose transport continued at the stimulated steady-state rate for 2.5 min and then declined to the basal rate with a half-time of 8 min. These kinetic experiments in situ and protein kinase activity measurements in vitro support the hypothesis that beta-subunit phosphorylation is an intermediate step linking insulin binding to the increased glucose transport rate.     

Tissue specific expression of p422 protein, a putative lipid carrier, in mouse adipocytes

The differentiation of 3T3-L1 preadipocytes leads to the expression of a new protein, p422, and its mRNA. This protein has 70% and 20-30% amino acid sequence homology to myelin P2 and the fatty acid binding proteins of liver and intestine, respectively. Investigation of the distribution in mouse tissues of p422 protein by immunoblotting and of p422 mRNA by cDNA hybridization indicates that they are expressed only in adipose tissue. Liver and intestinal fatty acid binding protein mRNA's were not detectable in mouse adipose tissue or in 3T3-L1 adipocytes. It is suggested that p422 functions as an adipocyte fatty acid binding protein.     

Thrombin-induced fibrinopeptide release from a fibrinogen variant (fibrinogen Sydney I) with an Aalpha Arg-16----His substitution

Fibrinogen, purified from a recently identified case of dysfibrinogenaemia, fibrinogen Sydney I, was shown by thrombin digestion, high-performance liquid chromatography (HPLC) and amino acid analysis to be a heterozygous case of an A alpha Arg-16----His substitution. Kinetic studies have been carried out on the thrombin-induced release of fibrinopeptide A (FPA), fibrinopeptide B (FPB) and the variant peptide [His16]FPA. When thrombin was added to fibrinogen Sydney I at a concentration of 0.2 U/ml release of FPA was rapid and there was a 79-fold reduced rate of release of [His16]FPA, but the rate of release of FPB was not appreciably reduced. In contrast, at lower thrombin concentrations the rate of FPB release was reduced in proportion to the rate of total FPA release, supporting the view that release of fibrinopeptides is a sequential process. The second-order kinetic constant kcat/Km for hydrolysis of the abnormal A alpha chain by thrombin was calculated from Lineweaver-Burk plots to be 16-30-fold less than that for the normal A alpha chain. Molecular modelling studies, using a refined model of the trypsin-pancreatic-trypsin-inhibitor complex have been used to suggest how the histidine at the P1 site can be accommodated within the enzyme hydrophobic active-site pocket.