Human colony-stimulating factor-1 (CSF-1) was purified from the serum-free conditioned medium of a human pancreatic carcinoma cell line. The four-step procedure included chromatography on DEAE Sepharose, Con A Sepharose and HPLC on phenyl column and reverse-phase C-3 column. The purity of human CSF-1 was demonstrated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS—PAGE) as a single diffuse band with a molecular weight (Mr) of 42,000-50,000 and was further confirmed by a single amino-terminal amino acid residue of glutamate. Under reducing conditions, purified CSF-1 appeared on SDS-PAGE as a single protein band with a Mr of 21,000-25,000 and concurrently lost its biological activity, indicating that human CSF-1 consists of two similar subunits and that the intact quaternary structure is essential for biological activity. When treated with neuraminidase and endo-8~D~N—acetylglucosaminidase D, the Mr of CSF-1 was reduced to 36,000-40,000 and to a Mr of 18,000-20,000 in the presence of mercaptoethanol.

Lecithin:cholesterol acyltransferase (LCAT) was isolated from hog plasma and basic physicochemical properties and functionally important regions were investigated. Approximately one milligram of the enzyme was purified to apparent homogeneity with approximately a 20,000-fold increase in specific activity. In the plasma, hog LCAT was found to associate with high-density lipoproteins (HDL) probably through hydrophobic interactions with apolipoprotein A-I. HDL was the preferred lipoprotein substrate of the enzyme as its macromolecular substrate. The enzyme was found to contain 4 free sulfhydryl groups; at least one of these appeared to be essential for catalytic activity. The enzyme had a tendency to aggregate at high concentrations. More than half of the tryptophan and none of the tyrosine residues of the enzyme were shown to be exposed to the aqueous environment based on fluorescence and absorbance studies, respectively.

Isotope partitioning experiments were carried out with the adenosine 3',5'-monophosphate-dependent protein kinase catalytic subunit (cAPK) from bovine hearts to obtain information on the order of addition of reactants and the relative rates of reactant release from enzyme compared to the catalytic step(s). A value of 100% trapping for both ErMgATP-[γ-32P] and E:3H-Serpeptide at low Mgf indicates that MgATP and Serpeptide dissociate slowly from the enzyme compared to the catalytic step(s). The K_Serpeptide for MgATP trapping is 17 μM, while the K_MgATP for Serpeptide trapping is 0.58 mM. The latter data indicate that the off-rate for MgATP from the E:MgATP complex is 14 s^-1 while that for Serpeptide from the E: Serpeptide complex is 64 s^-1. At high Mg^, 100% trapping is obtained for the E:MgATP-[γ-32P] complex but only 40% is obtained for the E:Serpeptide complex. Thus, the off-rate for Serpeptide from the E:MgATP:Serpeptide complex becomes significant at high Mg_f. Data suggest a random mechanism in which MgATP is sticky. The V for the cAPK reaction increases 1.5-1.7 fold in the presence of the R_II in the presence of saturating cAMP at a stoichiometry of R:C of 1:1. No change is obtained with the type-I complex under these conditions. At higher …

A method has been developed for the rapid, quantitative separation of normal and abnormal phosphoglucose isoemrase allozymes from individuals heterozygous for genetic variant forms of the enzyme. The method utilizes a substrate gradient elution of the enzyme from carboxymethyl Biogel and is far superior in terms of resolution and recovery to methods based on electrophoresis and isoelectric focusing. Four different genetic variant forms of the enzyme were isolated and subjected to a systematic comparison of their physical, catalytic and stability properties. The physical and catalytic properties of the variants were similar; however, clear differences in the stability of the allozymes were apparent.

The temperature-dependent catalytic activity of rat liver 3-hydroxy-3 -methylglutaryl coenzyme A reductase (HMG-CoA reductase) displays the nonlinear Arrhenius behavior characteristic of many membrane-bound enzymes. A two-conformer equilibrium model has been developed to characterize this behavior. In the model, HMG-CoA reductase undergoes a conformational change from a low specific activity to a high specific activity form. This conformation change is apparently driven by a temperature-dependent phase transition of the membrane lipids. It has been found that this model accurately describes the data from diets including rat chow, low-fat, high-carbohydrate, and diets supplemented with fat, cholesterol or cholestyramine. The effects characterized by the model are consistent with the regulation of HMG-CoA reductase by enzyme-lipid interactions.

Calcium/phospholipid-dependent protein kinase (PKC) was partially purified from P1798 lymphosarcoma. Phospholipid-dependence was specific for phosphatidylserine. PKC phosphorylated Histone 1, with an apparent K_m of 14.1 μM. Chlorpromazine, a lipid-binding drug, inhibited PKC activity by 100%. Further studies were undertaken to establish analytical conditions which could be applied to the study of PKC in intact cells. The conditions included (1) determining optimum cell concentration for measuring PKC activity, (2) recovering PKC into the soluble fraction of cell extracts, (3) evaluating calcium and phospholipid requirements of PKC in this fraction, and (4) inhibiting PKC in this fraction. Final studies involved treatment of intact cells with potential activators. Both phytohaemagglutinin and a phorbol ester increased PKC activation.

Huian plasma high-density lipoprotein (HDL) were isolated by a procedure employing polyanion precipitation and column chromatography. Lipid and protein composition of the HDL isolated by this method was found to be similar to another HDL preparation isolated by ultracentrifugation. However, minor differences were noted, including a higher phospholipid and apoproteinE content and lower triglyceride content of the HDL isolated by column chromatography. Four subfraction of HDL were obtained following chromatography on an anion exchange column. The subfraction four had the highest esterified to free cholesterol ratio, the second highest phospholipid to unesterified cholesterol, and the lowest molecular weight. In addition it was consistently coincided with lecithin: cholesterol acyltransferase (LCAT) activity and found to be the best substrate for the enzyme.

Recent studies have shown that the Sudden Infant Death Syndrome (SIDS) is related to abnormal control of respiration (Ischemic degeneration of the brainstem may play an important role in altered respiratory control leading to death). In our studies we have examined brainstem ganglioside compositions in samples derived from SIDS victims and appropriate controls. Gangliosides are acidic glycosphingolipids that contain sialic acid. The high concentration of gangliosides in the central nervous system (CNS) implies that these lipids play an important role in CNS function. Some studies have indicated that gangliosides may function as receptor site determinants or modifiers, and in neural transmission. In our studies we used the Tettamanti, et al methodology to extract gangliosides, and High Performance Thin Layer Chromatography (HPTLC) and laser densitometry techniques for ganglioside analysis. The results of these analyses are being employed to establish lipid profile patterns to determine if there are significant variations in these lipid patterns between SIDS and control groups.

The objectives of these investigations were (1) the purification of MG reductase from sheep liver and (2) studies of some of its characteristics. MG reductase was purified 40 fold and showed a single band on SDS-PAGE. Molecular weight estimations with SDS-PAGE showed a molecular weight of 44,000; although gel filtration with Sephadex G-150 gave a molecular weight of 87,000 indicating that the enzyme might be a dimer. The Km for MG is 1.42 mM and for NADH it is 0.04 mM. The pH optimum for the purified enzyme is pH 7.0. Isoelectric focusing experiments showed a pI of 9.3. In vivo experiments involving rats treated with 3,3',5-triiodothyronine (T_3) and 6-n-propyl-2-thiouracil (PTU) indicated that MG reductase was depressed by T_3 and elevated by PTU.

A Differentiation Factor (DF) was purified from rat lung conditioned medium by a four-steps procedure. The DF has a molecular weight of 27000, and an isoelectric point of 4.70. Although DF is stable up to 60°C, it is sensitive to digestion by trypsin, chymotrypsin and subtilisin. DF forms granulocyte colonies in soft agar. Studies using anti-NRK CSF antibody demonstrated that DF is distinct from GM-CSF.

In this study, the production of monoclonal antibodies directed against the activating enzyme for an S6 kinase is examined and described. Evidence is presented for the association of an Mr. 55,000 abd Mr. 95,000 protein with the s6 kinase. These proteins are phosphorylated in the presence of Activating Enzyme. A sequence of regulatory events for insulin-stimulated phosphorylation of ribosomal protein S6 in cells is postulated as follows: insulin activates the receptor tyrosine kinase, which phosphorylates the Mr 116,000 subunit of Activating Enzyme. The Activating Enzyme then activates the S6 kniase by phosphorylation, and phosphorylation of the ribosomal protein s6 is promoted.

Conditioned media prepared from umbilical cord (UC) segments or endothelial cells (EC) contain colony stimulating activity, Both UCCM and ECCM were partially purified by DEAE-Sepharose and ACA44 gel filtration chromatography. The molecular weights were estimated as 25,000 and 31,000 for UC-CSF and EC-CSF, respectively. UC-CSF was further fractionated by Con A Sepharose, IEF and HPLC on a hydrophobic phenyl column. The highly purified CSF stimulates human macrophage and granulocyte colony formation, indicating it is GM-CSF in nature. Characterization studies have revealed that both CSFs are heat stable at 60°C for 30 min. They are sensitive to digestion by protease and to periodate oxidation but are stable to treatment with sulfhydryl reagents. The synthesis of CSF in endothelial cells is inhibited by actinomycin D, cycloheximide and puromycin, indicating that protein and RNA synthesis are required for CSF production. Among the mitogens tested, only LPS exhibited stimulatory activity on the production of CSF. Metabolic modulators such as dibutyryl cAMP, isobutylmethylxanthine, PGE2 and lactoferrin inhibit CSF production, while PGF2 enhances CSF production.