The two-stranded alpha-helical coiled-coil is an ideal model for studying protein stability and subunit interactions.

We have designed de novo a two-stranded alpha-helical coiled-coil which consists of two identical 35-residue polypeptide chains arranged in a parallel and in-register alignment. Their structure is stabilized by interchain hydrophobic interactions from hydrophobes at positions "a" and "d" of a repeating heptad sequence. The formation and stability of the coiled-coil is dependent on peptide concentration due to the monomer-dimer equilibrium. In contrast, that coiled-coil containing an inter-helical disulfide bond does not show any concentration dependence in the guanidine hydrochloride denaturation experiments as expected. Replacement of one large hydrophobic Leu residue in each chain with Ala significantly decreases coiled-coil stability in both the reduced and oxidized coiled-coils [decreases in transition midpoint of 1.6M (2.3-0.7) and 2.4M (5.3-2.9), respectively]. A large pH dependence on coiled-coil stability is observed over the pH range 4 to 7 (transition midpoints at pH 4, 5, 5.5, 6 and 7 were 3.8, 3.2, 2.0, 1.2 and 0.7M, respectively). The increasing stability with decreasing pH correlates with the protonation of the Glu acid side-chains and reduction of intrachain repulsions between Glu-Glu side-chains in positions i, i + 3 or i, i + 4 along each alpha-helix of the coiled-coil. In addition, coiled-coil stability increases with increasing ionic strength.     

Continuous hydrolysis of olive oil by immobilized lipase in organic solvent

Lipase (EC 3.1.1.3) from Candida rugosa was immobilized with DEAE-Sephadex A50, Sephadex G50, Sephadex LH-20, Amberlite IRA94, and Amberlite XAD-7. The enzye immobilized with DEAE-Sephadex A50 was found to be most effective for continuous hydrolysis of olive oil in isooctane. For the continuous reaction, 0.2 g of dry immobilized enzyme was swollen with predetermined amount of water, and packed in a glass column reactor. When the organic solvent (Isooctane) containing olive oil substrate was cocurrently fed with aqueous buffer, the two phases were evenly distributed throughout the packed bed without surfactant supplement or prior mixing of the two phases. A small amount of the surfactant (AOT) was used only in packing procedure, and no additional surfactant was necessary thereafter. Effects of initial water content of the swollen gel, buffer types, and strength were examined in the continuous reaction. Our results suggest that the operational half-life was affected by desorption of the bound enzyme. Under the conditions of 20% olive oil in isooctane and 25 mM triethanolamine buffer (pH 7.0), operational half life was 220 h at 30 degrees C. The reactor was also operable with n-hexane, but the operational stability of the immobilized enzyme in n-hexane was only half of that in isooctane. Our results indicate that various enzyme carrier having hydrophilic or amphiphilic properties could be used for two-phase continuous reaction in packed-bed column, reactor without any surfactant supply or prior dispersion of the two immiscible phases. (c) 1992 John Wiley & Sons, Inc.     

Measurement and simulation of the morphological development of filamentous microorganisms

Growth of Streptomyces tendae was investigated in submerged culture. Images of several mycelia were analyzed by means of an image-processing system. The studies revealed that tip growth angles and branching outgrowth angles could be regarded as normally distributed. Based on these results, a random model for directional growth of hyphal tips as well as directional growth of branches is proposed. This model shows curved elongation of hyphal tips, so that the morphological development of a mycelium up to the formation of a pellet is predicted, similar to that observed in nature.     

Enhancement of cellular immune function during cold adaptation of BALB/c inbred mice.

The cell-mediated immune function of cold-adapted BALB/c inbred mice was studied in experiments of splenic lymphocyte blastogenesis, indicated by tritium-labeled deoxythymidine incorporation and SDS-PAGE autoradiography of synthetic proteins in lymphocytes. Male BALB/c inbred mice were randomly divided into two groups: control (living at 25 degrees C) and cold-exposed (living at 2 degrees C). Results are as follows: in contrast with the control group, there was an obvious fluctuation of cell-mediated immune function in the cold-exposed group at initial cold exposure because of transient stress to cold; then cell-mediated immune function gradually recovered to control level. From Day 15, the cell-mediated immune function of the cold-exposed group was remarkably enhanced. On Day 15, the lymphocyte blastogenesis rate was increased by 20.66% (P less than 0.05), which implies the onset of cold adaptation; on Days 21 and 31, the rates increased by 80.15% (P less than 0.05) and 40.36% (P less than 0.05), respectively. Two to six months later, with continuing cold exposure, the murine lymphocyte blastogenesis rate in the cold-exposed group remained higher than that in the control group. The lymphocyte protein synthesis of the cold-exposed group, indicated by tritium-labeled leucine incorporation, apparently increased on Day 15 and the stimulated rate was 101.47% (P less than 0.05). SDS-PAGE autoradiography of synthetic proteins in lymphocytes demonstrated that after 2 weeks of cold exposure, protein bands were enriched in both quantity and quality. These results are identical to the results obtained from lymphocyte blastogenesis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Lack of in vivo transcription of Acetabularia mediterranea 1175 bp ctDNA fragment homologous to the Drosophila per locus.

The period (per) locus of Drosophila melanogaster has a fundamental role in the expression of biological rhythms. A DNA sequence homologous to a short region of the Drosophila per locus was detected in the chloroplast of Acetabularia mediterranea. A 1175 bp DNA fragment containing the sequence was used as a probe in 'Northern' hybridization experiments. It was found that this DNA was not transcribed or only marginally transcribed in A. mediterranea, at least at the developmental stage just prior to cap formation. It seems that the 1175 bp ctDNA fragment is not involved in the Acetabularia biological rhythm mechanism.     

Alteration of folate analogue transport inward after induced maturation of HL-60 leukemia cells. Molecular properties of the transporter in an overproducing variant and evidence for down-regulation of its synthesis in maturating cells.

Studies are described examining further the decline in folate analogue influx mediated by the one-carbon reduced-folate transport system in HL-60 cells following induction of maturation by cytodifferentiation agents. To facilitate the investigation of the underlying basis of this phenomenon, we derived a variant (HL-60/LCV) with 4-5-fold elevated influx capacity (Vmax) for folate analogues. A commensurate increase in the putative transporter for this system was documented by affinity labeling of these cells with N-hydroxysuccinimide-[3H]aminopterin. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the affinity labeled plasma membrane in HL-60/LCV cells delineated a protein peak at Mr = 75,000-80,000. This was substantially greater than the analogous transporter (Mr = 45,000-47,000) we had delineated (Yang, C.-H., Sirotnak, F.M., and Mines, L.S. (1988) J. Biol. Chem. 263, 9703-9709) with the same methodology in the L1210 cell plasma membrane. In addition, the rate of translocation of the Mr = 75,000-80,000 transporter in HL-60 and HL-60/LCV cells was 2-fold lower than the rate of translocation determined for the Mr = 45,000-47,000 transporter in L1210 cells. During induced maturation of HL-60/LCV cells toward the granulocyte pathway, [3H]methotrexate (MTX) influx capacity and the amount of the affinity labeled transporter decreased rapidly in a parallel fashion. The decrease in [3H]MTX influx and in affinity labeling and in the amount of the Mr = 75,000-80,000 transporter was 5-fold following exposure to 210 mM dimethyl sulfoxide (Me2SO) for 5 days during growth in culture. Moreover, during cycloheximide treatment, the decay in [3H]MTX influx at 37 degrees C and in amount of affinity labeled transporter was the same (t1/2 = 144-155 min) for both control and Me2SO-treated HL-60/LCV cells. These results, which reveal no difference in metabolic turnover for control and Me2SO-treated cells, suggest that the decline in folate analogue influx in HL-60/LCV influx cells is a very early event in the program of differentiation and probably occurs by down-regulation of synthesis of the transporter for the one-carbon reduced-folate transport system.     

N-myristoylation of the rod outer segment G protein, transducin, in cultured retinas.

Bovine retinas incubated with [3H]myristic acid incorporated detectable radiolabel into only a few proteins. The most heavily labeled was the alpha subunit of the rod outer segment G protein transducin (Gt alpha). The radiolabeled protein was specifically eluted from illuminated membranes in the presence of GTP, displaying the unique solubility properties of Gt alpha. It comigrated with Gt alpha in electrophoresis and chromatography and was immunoprecipitated by Gt alpha-specific antibodies. The radiolabel was confirmed by hydrolysis, chemical derivatization, and chromatography to be amide-linked myristic acid. The solubility of the myristoylated Gt alpha indicates that myristoylation is not sufficient to cause tight membrane association of this normally membrane-bound subunit. Incorporation of [3H]myristate was blocked by the protein synthesis inhibitor cycloheximide, suggesting that that fatty acid group is introduced during or soon after translation in the rod inner segment.     

Glycan components in the glycoinositol phospholipid anchor of human erythrocyte acetylcholinesterase. Novel fragments produced by trifluoroacetic acid.

Inositol glycans were prepared from reductively radiomethylated human erythrocyte acetylcholinesterase by sequential treatment with Proteinase K, methanolic KOH, and phosphatidylinositol-specific phospholipase C. Four glycans denoted alpha-delta were resolved by anion exchange high performance liquid chromatography (HPLC). Each glycan was subjected to hydrolysis in 4 M trifluoroacetic acid, and their hexose and hexose phosphate compositions were determined by anion exchange HPLC. The predominant glycan alpha showed a relative stoichiometry of 2 mannoses, 1 mannose 6-phosphate, 1 radiomethylated glucosamine, 1 radiomethylated ethanolamine, and 1 inositol. In contrast, the stoichiometry of glycan beta was 1 mannose, 2 mannose 6-phosphates, 1 radiomethylated glucosamine, 2 radiomethylated ethanolamines, and 1 inositol. Glycans alpha and beta were analyzed by electrospray ionization-mass spectrometry, and respective parent ions of m/z 1266 and 1417 were observed. The fragmentation pattern produced by collision-induced dissociation mass spectrometry of these parent ions was consistent with a common linear core glycan sequence prior to radiomethylation of ethanolamine-phosphate-mannose - mannose - mannose - glucosamine - inositol. Glycan alpha contained a single additional radiomethylated phosphoethanolamine branching from the mannose adjacent to glucosamine, whereas glycan beta contained two additional radiomethylated phosphoethanolamines, one branching from each of the mannoses nearest to glucosamine. Trifluoroacetic acid hydrolysis did not cleave within the N,N-dimethylglucosamine-inositol-phosphate moiety in these glycans, and this component was resolved by anion exchange HPLC and structurally confirmed by mass spectrometry. Dephosphorylation of this component by treatment with 50% HF produced N,N-dimethylglucosamine-inositol, and this conjugate was shown to have a characteristic elution time on cation exchange chromatography in an amino acid analyzer. Both of these fragments involving an intact radiomethylated glucosamine-inositol bond are proposed as new diagnostic indicators in the search for minor glycoinositol phospholipids in cells and tissues.     

Conformational interconversion in protein crystals.

We present evidence that the structure of carbonmonoxy myoglobin crystals can be altered by lowering the pH. This structural change is monitored by the characteristic Fe-CO Raman modes at 508 and 491 cm-1 and is thought to involve a localized distal pocket transition from a "closed" conformation at pH 7 to a more "open" conformation at pH 4. These changes take place in the crystal without loss of intensity of a conformationally sensitive Raman mode at 252 cm-1 that signals a partial unfolding of the globin structure in solution. Quantitative studies, which monitor the open and closed populations as a function of laser photolysis, demonstrate that the interconversion rates (k+/-) in solution at 298 K are fast compared to the photolysis and CO entry rates (i.e. k+/- much greater than 10(3) s-1), while in frozen samples the interconversion is much slower than the experimental time scale (minutes). Since the open conformation is a minority species at pH 7, rapid exchange in aqueous solution is a necessary condition for this species to play a functional role. In the crystal, the interconversion rates are slowed compared to solution and begin to approach the photolysis rate (i.e. k+/- approximately 10(3) to 10(4) s-1). This indicates that the barriers for conformational exchange are increased in the crystal environment, compared to the solution, apparently due to the packing forces of the surrounding molecules. X-ray and neutron diffraction studies of MbCO crystals at high and low pH are needed to characterize the details of the structural changes and to test the hypothesis that closed and open distal pocket structures are associated with the 508 and 491 cm-1 Fe-CO modes.