Low ionic strength solubility of myosin in sea urchin egg extracts is mediated by a myosin-binding protein

We identify a novel myosin-binding protein, designated 53K, which appears to mediate the low ionic strength solubility of myosin in extracts of unfertilized sea urchin eggs. The protein possesses a subunit molecular mass on SDS-PAGE of 53 kD, an S value of 7, may be organized into disulfide-linked oligomers, and is associated with myosin in egg extracts. Both myosin and 53K co-precipitate from extract upon the addition of nucleoside triphosphates and co-sediment with an S value of 24 by sedimentation velocity centrifugation. Myosin in extracts not associated with 53K has an S value of 10. Further, myosin can be immunoprecipitated from extract with antibody to 53K and the 53K in extracts binds to a myosin affinity column. When extract is depleted of 53K, a majority of the myosin precipitates out of extract in a nucleotide-independent manner. Whereas purified myosin precipitates in the absence of nucleotide when recombined with dialysis buffer or myosin-depleted extract, reconstituting 53K and myosin before addition to buffer or myosin-depleted extract partially restores the low ionic strength solubility demonstrated by myosin in fresh egg extracts. The 53-kD protein may represent a new class of authentic myosin-binding proteins that may regulate the supramolecular organization of myosin in nonmuscle cells.     

In vitro mutagenesis of trypsinogen: role of the amino terminus in intracellular protein targeting to secretory granules

The mouse anterior pituitary tumor cell line, AtT-20, targets secretory proteins into two distinct intracellular pathways. When the DNA that encodes trypsinogen is introduced into AtT-20 cells, the protein is sorted into the regulated secretory pathway as efficiently as the endogenous peptide hormone ACTH. In this study we have used double-label immunoelectron microscopy to demonstrate that trypsinogen colocalizes in the same secretory granules as ACTH. In vitro mutagenesis was used to test whether the information for targeting trypsinogen to the secretory granules resides at the amino (NH2) terminus of the protein. Mutations were made in the DNA that encodes trypsinogen, and the mutant proteins were expressed in AtT-20 cells to determine whether intracellular targeting could be altered. Replacing the trypsinogen signal peptide with that of the kappa-immunoglobulin light chain, a constitutively secreted protein, does not alter targeting to the regulated secretory pathway. In addition, deletion of the NH2-terminal "pro" sequence of trypsinogen has virtually no effect on protein targeting. However, this deletion does affect the signal peptidase cleavage site, and as a result the enzymatic activity of the truncated trypsin protein is abolished. We conclude that neither the signal peptide nor the 12 NH2-terminal amino acids of trypsinogen are essential for sorting to the regulated secretory pathway of AtT-20 cells.     

Enzymatic transformation of PGH2 to PGF2 alpha catalyzed by glutathione S-transferases

Glutathione S-transferases (GSTs) purified from both rat liver cytosol and microsomes catalyzed the direct reduction of PGH2 to PGF2 alpha. As much as 40% of the substrate was transformed into a prostanoid whose Rf value corresponded to that of PGF2 alpha. The identification of the reaction product as PGF2 alpha was confirmed by TLC and reverse-phase HPLC as well as by mass spectral analysis. In the absence of GSTs, PGH2 was found to be primarily converted to PGE2 and PGD2. Also, PGF2 alpha formation was completely abolished by decylglutathione, a potent inhibitor of both peroxidase and transferase activity associated with GSTs. These results indicate that the direct reduction of endoperoxide moiety of PGH2 to form PGF2 alpha is an enzymatic process. Interestingly, selenium-dependent glutathione peroxidase (Se-GSH-Px) showed very little PGF2 alpha formation from PGH2. However, this enzyme was very active in the reduction of PGG2 to PGH2. In contrast, GSTs were very poor in the conversion of PGG2 to PGH2. Therefore, it is possible that the relative tissue distribution of Se-GSH-Px and GSTs might play an important role in the tissue specific synthesis of PGF2 alpha.     

Major histocompatibility (B) complex and sex effects on the phytohaemagglutinin wattle response

The influence of the major histocompatibility (B) complex and sex on the phytohaemagglutinin (PHA) wattle response was studied in 136 segregants (B2/B2, B2/B5 and B5/B5) of a fourth generation cross between inbred lines 6(1) and 15(1). At 6 weeks of age, chickens were injected with 100 micrograms purified PHA-P. Wattle thickness measurements were taken 4, 24, 48, 72 and 96 h after injection. Analysis of variance showed that 4 h after injection, males had a significantly higher response than females but the sex-genotype interaction was also significant. Females had higher responses than males 24 and 48 h after injection as a consequence of more rapid development and earlier resolution of the reaction in males. B2/B2 chickens had significantly lower responses than B5/B5 chickens 72 and 96 h after injection, signifying a faster late resolution phase in the B2/B2 genotype. The developmental and early resolution phases of the PHA wattle response were influenced by sex while the late resolution phase was influenced by B genotype.     

Allometric Root/Shoot Relationships and Predicted Water Uptake for Desert Succulents

Root morphology, shoot morphology, and water uptake for Agavedeserti and Ferocactus acanthodes of various sizes were studiedusing allometric relationships (y = ax^b) and a previously developedwater uptake model. Shoot surface area increased with shootvolume with an exponent b of 0.75 for both species. Root lengthand the ground area explored by the roots increased with shootsurface area with b's of 0.72 for A. deserti and 0.92 for F.acanthodes. Various sized individuals had about the same ratioof root length to explored ground area, with higher values occurringfor A. deserti. Predicted water uptake averaged over the exploredground area was approximately constant over a 10⁴-fold rangein shoot surface area, suggesting that shoot size confers nointraspecific competitive advantage for water uptake. For theroot lengths per explored ground area observed in the field,water uptake was predicted to be 85 per cent of maximal; wateruptake could be increased by the production of more rain roots.When differences in shoot volume were accounted for by allometry,small plants had relatively less shoot surface area and relativelymore root length per shoot volume than did large plants, whichmay be important for the water relations of seedling establishment. Agave deserti, Ferocactus acanthodes, allometry, desert succulents, root distribution, root length, seedling growth, seedling establishment, shoot surface area, shoot volume, water uptake     

A dimerization model for the concentration dependent photophysical properties of diphenylhexatriene and its phospholipid derivatives. DPHpPC and DPHpPA.

We have investigated the reason for the sensitivity of the fluorescence excited-state lifetime of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its phospholipid derivatives, 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl)carbonyl)-3-sn-phosphatidylcholine (DPHpPC) and 1-palmitoyl-2-[2-[4-(6-phenyl-trans-1,3,5- hexatrienyl)phenyl]ethyl)carbonyl)-3-sn-phosphatidic acid (DPHpPA), to the concentration of these probes in dipalmitoylphosphatidylcholine (DPPC) multilamellar membranes (Barrow, D. A., and B. R. Lentz, 1985. Biophys. J. 48:221-234; Parente, R. A., and B. R. Lentz. 1985. Biochemistry. 24:6178-6185). We have interpreted self-quenching data, excitation and emission spectra, and phase and modulation lifetime data in terms of a model that envisions dimerization of these probes in a membrane bilayer. It is proposed that dimerization alters the symmetry of the DPH excited state so as to allow more rapid decay via the normally symmetry-disallowed route from the 1Ag* state. Global analysis of fluorescence phase shift and modulation ratio data for DPHpPC in terms of the dimerization model provided a good fit of these data as a function of both modulation frequency and probe concentration. Global analysis of a similar set of data for the charged phosphatide DPHpPA predicted that this probe was much less prone to dimerize than was the uncharged DPHpPC. This physically reasonable result provides support for the assumptions made in the development of our model. We conclude that the dimerization model allows rationalization of many of the anomalous photophysical properties of DPH and its derivatives in membranes.     

Characterization and cDNA cloning of phospholipase C-gamma, a major substrate for heparin-binding growth factor 1 (acidic fibroblast growth factor)-activated tyrosine kinase.

Heparin-binding growth factors (HBGFs) bind to high-affinity cell surface receptors which possess intrinsic tyrosine kinase activity. A Mr 150,000 protein phosphorylated on tyrosine in response to class 1 HBGF (HBGF-1) was purified and partially sequenced. On the basis of this sequence, cDNA clones were isolated from a human endothelial cell library and identified as encoding phospholipase C-gamma. Phosphorylation of phospholipase C-gamma in intact cells treated with HBGF-1 was directly demonstrated by using antiphospholipase C-gamma antibodies. Thus, HBGF-1 joins epidermal growth factor and platelet-derived growth factor, whose receptor activation leads to tyrosine phosphorylation and probable activation of phospholipase C-gamma.