Endogenous phosphorylation of retinal photoreceptor outer segment proteins by calcium phospholipid-dependent protein kinase

A calcium phospholipid-dependent protein kinase (C-kinase) activity was detected in the soluble fraction of rod outer segments (ROS) of the bovine retina. The enzyme required calcium, phosphatidylserine (PS) and diacylglycerol for maximal activity. In the presence of calcium and PS, C-kinase endogenously phosphorylated proteins with molecular weights of 95,000, 91,000, 31,000, 21,000, 19,000, 18,000, 16,000, 14,000 and 11,000. Addition of diolein in the reaction mixture further enhanced the endogenous phosphorylation of these proteins. Retinal was found to inhibit the phosphorylation of endogenous proteins by C-kinase in a concentration dependent manner. Half-maximal inhibition of enzyme activity was obtained at a retinal concentration of about 12μM. These results suggest that calcium, phospholipids and the C-kinase enzyme may play an important role in the functional regulation of rod photoreceptors and, with retinal, perhaps in the visual process as well.     

Identification of albumin-synthesizing polysomes from mouse liver and a mouse hepatoma cell line.

Albumin-synthesizing polysomes from mouse liver and mouse hepatoma cells in in tissue culture have been localized on sucrose gradients with ¹²⁵I-labeled antimouse serum albumin used as a marker. Competition studies show that the ¹²⁵I-labeled antibody binds specifically to albumin-synthesizing polysomes from both tissues. The ¹²⁵I-labeled polysomes from liver and hepatoma cells have identical sedimentation properties on sucrose gradients, which indicates that the polysomes range in size from 9–14 ribosomes. This is comparable in size to polysomes from rat liver and Morris hepatoma. One significant difference between these albumin-synthesizing polysomes is that those extracted from hepatoma cells bind 70% less antibody than equivalent amounts of polysomes from liver cells. Since the level of albumin synthesis in the hepatoma cells is comparable to the level of albumin synthesis $\text{in vivo}$, this difference in antibody-binding capacity is not likely to be due to differences in polysomal content, but appears to be a characteristic difference between hepatoma and normal mouse liver cells.     

Mitochondrial phosphate transport and the N-ethylmaleimide binding proteins of the inner membrane

Mitochondria have been prepared from the flight muscles of mature blowflies (Sarcophaga bullata). Phosphate transport by these mitochondria, determined by rates of passive swelling in ammonium phosphate, is sensitive to inhibition by N-ethylmaleimide. 20 nmol of N-ethylmaleimide/nmol cytochrome a inhibit the swelling by 90%. When the mitochondria are inhibited by $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide}$, then solubilized in dodecyl sulfate/mercaptoethanol at 100°C and then electrophoresed on dodecyl sulfate-polyacrylamide gels, many labeled protein bands can be detected, including a large labeled peak that has the same mobility as the tracking dye, bromophenol blue. Sonic submitochondrial particles that are prepared from the $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimidelabeled}$ mitochondria, solubilized, and electrophoresed on dodecyl sulfatepolyacrylamide gels, possess only seven major labeled protein bands with no radioactive peak at the tracking dye. These labeled proteins have molecular weights of 71, 68, 64, 45, 32, 30, and approx. 10 · 10³. The nmol $\text{N-[}{}^3\text{H}\text{]-}\text{ethylmaleimide}$ bound to each of these proteins per nmol cytochrome a are 0.15, 0.19, 0.35, 0.45, 0.87, 0.10, and 0.17, respectively, when the mitochondria are inhibited with 21.5 mol $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide/mol}$ cytochrome a at 10 μM cytochrome a. Coty and Pedersen ((1975) J. Biol. Chem. 250, 3515–3521) sensitized rat liver mitochondria to $\text{N-[}{}^3\text{H}\text{]}\text{ethylmaleimide}$ and identified five labeled proteins. Only the labeled 32 · 10³ dalton and the 45 · 10³ dalton proteins are common to both systems     

Comparison of Proteoglycans Extracted by Saline and Guanidinium Chloride from Cultured Chick Retinas

Abstract: To compare the loosely associated sulfated proteoglycans with those tightly bound to membranes, retinas from 14-day chick embryos were subjected to progressively disruptive techniques. The most easily removed proteoglycans were isolated from the medium in which the tissue was labeled with [³⁵S]sulfate. On the average, 25% of the glycosaminoglycans were in the labeling medium, 39% were in proteoglycans extracted from the tissue in the balanced salt solution, 32% were in a 4 m -guanidinium chloride (GuCl) fraction, and 4% remained unextracted. These glycosaminoglycans contained, respectively, 28, 28, 40, and 4% of the incorporated [³⁵S]sulfate. On the basis of electrophoretic mobility and TLC of chondroitinase digests, the ratio of ³⁵S in chondroitin sulfate to that in heparan sulfate was 4–7 times higher in the medium and balanced salt extracts than in the GuCl extracts. In both extracts there was more ³⁵S in chondroitin-6-sulfate than in chondroitin-4-sulfate. Dialysis of the extracts against 0.5 M-NaCl resulted in the precipitation of about 12% of the glycosaminoglycans in the saline extracts and about 40% in GuCl extract. These subfractions, which were relatively enriched in heparan sulfate, were largely soluble in dithiothreitol in 8 m -urea (DTT). Similarities between the proteoglycans in the medium and those extracted by balanced salt solutions suggest that the saline-extracted proteoglycans were for the most part loosely associated with cell surfaces or extracellular matrices, whereas the GuCl-extracted proteoglycans probably were bound to membranes.     

Structure and Synthesis of Dopastin,an Inhibitor of Dopamine β-Hydroxylase

Dopastin, an inhibitor of dopamine β-hydroxylase of microbial origin, was shown to be N-[2(S)-nitrosohydroxylamino-3-methylbutyl] crotonamide based on chemical, spectroscopic and synthetic studies. The total synthesis of dopastin was completed in 8 steps starting from l-valinol. N-Nitrosohydroxylamino function was introduced through an oxaziran with retention of the absolute configuration in the final product. Thus, the 2S-configuration of dopastin was proved by the total synthesis. Racemic dopastin was also synthesized from isobutyraldehyde in 7 steps.     

Intracellular Distribution and Some Properties of β-Glucosidases of a Cellulolytic Pseudomonad

Two distinct forms of β-glucosidase, A and B, were found to occur in the cells of Pseudomonas fluorescens var. cellulosa : A was membrane-bound, while B cytosolic. They differed also from each other in some properties, such as molecular size, kinetic parameters, and susceptibility to various compounds. β-Glucosidase B was partially purified and studied especially of its substrate specificity. The results indicated that it may be an atypical β-glucosidase which possesses a certain character of exo-cellulase.     

Mutual structural effect of bilirubin and model membranes by vibrational circular dichroism

In this study, vibrational circular dichroism (VCD) spectroscopy was employed for the first time to study the bilirubin (BR) interaction with model membranes and models for membrane proteins. An enantioselective interaction of BR with zwitterionic 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and sphingomyelin (SPM) liposomes was observed by VCD and electronic circular dichroism (ECD) complemented by absorption and fluorescence spectroscopy. The M-form of BR was preferentially recognized in the BR/DMPC system at concentration above 1 × 10^− 4 M, for lower concentrations the P-form of BR was recognized by the DMPC liposomes. The VCD spectra also showed that the SPM liposomes, which represent the main component of nerve cell membrane, were significantly more disturbed by the presence of BR than the DMPC liposomes—a stable association with a strong VCD signal was observed providing the explanations for the supposed BR neurotoxicity. The effect of time and pH on the BR/DMPC or SPM liposome systems was shown to be essential while the effect of temperature in the range of 15–70 °C was negligible demonstrating the surprisingly high temperature stability of BR when interacting with the studied membranes. The influence of a membrane protein was tested on a model consisting of poly-l-arginine (PLAG) bound in the α-helical form to the surface of 1,2-dimyristoyl-sn-glycero-3-phospho-(1′-rac-glycerol) liposomes and sodium dodecyl sulfate micelles. VCD and also ECD spectra showed that a variety of BR diastereoisomers interacted with PLAG in such systems. In a system of PLAG with micelles composed of sodium dodecyl sulfate, the M-form of bound BR was observed.     

Role of p53/FAK association and p53 phosphorylation in staurosporine-mediated apoptosis: Wild type versus mutant p53-R175H

A novel survival role of focal adhesion kinase (FAK) that involves its nuclear translocation and direct association with p53 has been demonstrated. Here we examined the relationship between the p53/FAK interaction and Ser46 phosphorylation of p53 (p-p53^Ser46) in the apoptotic regulation of human esophageal squamous cell carcinoma (HOSCC) cell lines, expressing either wild type (wt) p53 or mutant (mt) p53-R175H. In contrast to the wt p53 cell lines, the mt p53-R175H cell line was resistant to staurosporine (STS)-mediated detachment and caspase-3 activation. Furthermore, despite the resistance of mt p53-R175H to Ser46 phosphorylation, both wt and mt HOSCC cells translocate FAK into the nucleus and maintain the p53/FAK interaction post STS treatment. These findings provide unique insight into how tumor cells harboring the R175H mutant may resist chemotherapeutic intervention.

Structured summary

MINT-7294020: FAK (uniprotkb:Q05397) physically interacts (MI:0915) with p53 (uniprotkb:P04637) by anti-bait coimmunoprecipitation (MI:0006)     

The influence of electrostatic interactions on partition in aqueous polyethylene glycol/dextran biphasic systems: Part II

In aqueous polyethylene glycol/dextran two-phase systems, the hydrophobicity, free volume, surface tension, and interfacial tension of the phases in equilibrium were measured as a function of pH and ionic strength. These parameters were found to change with pH, but the pattern and magnitude cannot explain the unusual partition of charged macromolecules, observed previously. The electrostatic potential difference was determined by a new experimental approach based on the measurement of the pH difference between the phases at equilibrium. In polyethylene glycol/dextran systems containing sodium chloride as ionized species, the electrostatic potential is not constant in the pH range 2 to 11. The partition behavior of charged macromolecules and its dependence on pH can be explained by the combined action of charge and phase potential. This conclusion was tested with poly-L-glutamate, which partitioned as predicted and in a pattern opposite to positively charged macro- molecules. (c) 1995 John Wiley & Sons, Inc.