Purification of a phospholipase C from rat liver cytosol that acts on phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 4-phosphate

A soluble phospholipase C from rat liver was purified to homogeneity using phosphatidylinositol 4,5-bisphosphate (PIP2) as substrate. After ammonium sulfate fractionation, the purification involved chromatography on phosphocellulose, DEAE-Sepharose CL-6B, hydroxylapatite, Reactive Blue 2 dye-linked agarose, and Mono S cation exchanger. Under the conditions of the assay, the pure enzyme had a specific activity of 407 mumol/mg protein/min. It migrated as a single band with a molecular mass of 87 kDa on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The water-soluble product formed during the hydrolysis of PIP2 by the purified enzyme was inositol 1,4,5-trisphosphate. The enzyme shows one-half of maximum velocity at 2 microM Ca2+ with PIP2 as substrate. Between 0 and 100 microM Ca2+, the enzyme shows approximately the same activity with phosphatidylinositol 4-phosphate (PIP) as it does with PIP2, and very low activity with phosphatidylinositol. The enzyme is activated by low concentrations of basic proteins; for example, with PIP2 as substrate, 1 microgram/ml histone activates the enzyme 3.6-fold. The enzyme shows an almost absolute requirement for monovalent salts which can be met by different alkali metal halides. A second, minor peak of PIP2-hydrolyzing phospholipase C activity was resolved during chromatography of the enzyme on hydroxylapatite. The substrate specificity suggests that PIP and PIP2 are normal substrates of this enzyme. Under physiological conditions of activation, the enzyme may therefore generate inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate in amounts determined by the ratio of PIP and PIP2 present in the cellular membranes.     

Step reductions in extracellular Ca2+ activate a transient inward current in chick dorsal root ganglion cells.

We investigated whether transient step reductions in divalent cations would produce detectable changes in neuronal excitability similar to those reported in the total absence of divalent cations. Using cultured chick dorsal root ganglion cells as a model system, our results indicate that a step reduction in divalent cations induces a transient inward current. This response is mediated by a tetrodotoxin-resistant, Na+-permeable, cation channel that is blocked by cadmium. This, and our observation that the response is abolished by verapamil, suggests that the current passes through calcium channels. This transient inward current was estimated to be activated by decreases in extracellular calcium ([Ca2+]o) as small as 0.5-0.8 mM and thus represents a different response from the one previously observed when steady-state [Ca2+]o levels were reduced to micromolar levels.     

An Alternative Interpretation of Nuclear Magnetic Resonance Observations in the Gel State of Lipid Bilayers

In the established interpretation of nuclear magnetic resonance (NMR) spectra of phospholipid bilayers in the gel state, the molecules are assumed to perform rotational diffusion about their long axis. Here we present an alternative model of the molecular mobility in this phase, which considers the positions of the lipid molecules in the two-dimensional bilayer lattice as fixed within the NMR timescale. Instead we assume an intramolecular two-site hopping of the hydrocarbon chains about their long axis. It is shown that deuterium NMR spectra of chain-labeled compounds are very sensitive to the precise angle of this flip-flop motion near 90°, so that the diversity of these gel-phase spectra is easily explained by slight variations of this angle. In addition, it is argued that the axial symmetry of ¹³C spectra of carbonyl-labeled phospholipids might also result from this intramolecular mobility.     

Complete amino acid sequence of steer liver microsomal NADH-cytochrome b5 reductase

The complete covalent structure of liver microsomal NADH-cytochrome b5 reductase from steer liver microsomes was determined. Cleavage at methionyl bonds gave 10 peptides accounting for all the residues of the protein. Acid cleavage of the reductase at the Asp-Pro bonds gave three peptides accounting for all the CNBr peptides in the molecule. Subfragmentation of these peptides by chemical and enzymatic cleavage provided overlaps which established all the fragments in an unambiguous sequence of 300 residues, corresponding to Mr 34,110. Limited tryptic digestion cleaved reductase at residues 28 and 119, yielding a preparation having two noncovalently linked peptides having a conformation which binds flavin and retains the structural features essential for NADH-cytochrome b5 activity. A model for the secondary structure of cytochrome b5 reductase is proposed that is based on computer-assisted analysis of the amino acid sequence. In this model the beta-turns are predominant and there is some 25% alpha and 30% beta structure.     

Parallelophoridae - isolierte Analfelder eozäner Schaben (Insecta: Blattodea)

As actuopalaeontological experiments clearly show, the family ParallelophoridaeHaupt 1956 (Blattodea incertae sedisRohdendorf 1962) from the Geiseltal near Merseburg (Middle-Eocene) are not a new family but isolated analfields of the forewings of other cockroach-families. During the process of fossilisation they get isolated from the wing in a rather early stage of destruction by fungi and other microorganisms, during the time the insect corpses are still swimming on the surface of a lake. The reasons, why only two specimens of such isolated analfields have been described till now, are briefly discussed.     

Establishment and Growth Kinetics of the Mutant Ehrlich-Lettré Ascites Cell Strain Hd33 In Permanent Suspension Culture1,2

Cells of a mutant in vivo subline of the Ehrlich-Lettré mouse ascites tumour (ELAT) were converted to growth in suspension culture. Kinetic analysis revealed the selective character of the conversion process; without a detectable adaptation period, a fraction of about 2 × 10^-5 of the explanted cells continued to grow in vitro. the resulting, mutant Ehrlich-Lettré ascites cell strain was designated HD33 and propagated uninterruptedly from 1974 on. the corresponding in vivo ELAT subline HD33 was derived from the HD33 ascites cell strain by intraperitoneal retransplantation. In HD33 cell suspension cultures, the population doubling time, the average intermitotic interval, as determined by videomonitoring, and the average duration of the cell cycle, as determined from percentage of labelled mitoses (PLM) data, were all measured at 15 hr. Cell loss and quiescent compartments were insignificant. the duration of the G₁ phase was effectively zero. Both PLM data and [³H]/[¹⁴C] thymidine double-labetling measurements revealed an S-phase duration of between 11 and 12 hr. the G₂ phase lasted 3–5 hr. The HD33 strain differs from comparable suspension strains of wild-type Ehrlich ascites cells in the insignificant role of density-dependent inhibition in growth, and the striking prolongation of the S phase which is associated with an excessive, cytoplasmic storage of glycogen by the mutant cells.     

Plant tumor reversal associated with the loss of marker chromosomes in tobacco cells

Summary The determination of partial genotype, B/b and D/d, for coat colour in dogs, from phenotypic observations, is discussed. It is shown that the probability of a given genotype can be reliably determined where multiple observations on the mating of a given dog are available.     

Antigen-induced lymphomagenesis: identification of a murine B cell lymphoma with known antigen specificity

CH12, a murine B cell lymphoma derived in B10 H-2aH-4bp/Wts mice after transfer of SRBC hyperimmunized spleen cells into an adult-thymectomized, sublethally irradiated, syngeneic recipient, is demonstrated to bear surface IgM specific for a determinant found on SRBC and ChRBC. The Ig specificity has been demonstrated by rosetting assays and complement-dependent hemolysis. The removal of CH12 surface IgM by capping with anti-mu or with anti-CH12 idiotype, but not with anti-gamma or with irrelevant anti-idiotype, eliminated the formation of rosettes between CH12 and SRBC or ChRBC. The absorption of CH12 Ig produced in vitro, with either SRBC or ChRBC but not with HRBC, removed all hemolysin activity against SRBC, demonstrating that only one CH12 product was responsible for the reactivity with both SRBC and ChRBC. CH12 has a surface phenotype of a relatively mature B cell expressing surface Ig (IgM-mu,kappa) and la antigens, but lacking Thy-1 or detectable Fc or C3 receptors. CH12 also expresses the antigen Lyt-1. Growth of CH12 in vivo or in vitro results in the generation of up to 3% direct PFC and serum hemolysin, which shows that CH12 is not irretrievably "frozen". The generation of PFC and serum hemolysin is associated with increased population density, and the rate of PFC and serum hemolysin accumulation cannot be explained by simple cell division. A continuously secreting hybridoma derived from CH12 was used to purify the CH12 IgM to facilitate studies of protein sequence and idiotype.