Phenol-soluble Protein Research Articles

Multiple γ-glutamylation: A novel type of post-translational modification in a diapausing Artemia cyst protein

A highly hydrophilic, glutamate-rich protein was identified in the aqueous phenol extract from the cytosolic fraction of brine shrimp (Artemia franciscana) diapausing cysts and termed Artemia phenol soluble protein (PSP). Mass spectrometric analysis revealed the presence of many protein peaks around m/z 11,000, separated by 129 atomic mass units; this value corresponds to that of glutamate, which is strongly suggestive of heterogeneous polyglutamylation. Polyglutamylation has long been known as the functionally important post-translational modification of tubulins, which carry poly(l-glutamic acid) chains of heterogeneous length branching off from the main chain at the γ-carboxy groups of a few specific glutamate residues. In Artemia PSP, however, Edman degradation of enzymatic peptides revealed that at least 13, and presumably 16, glutamate residues were modified by the attachment of a single l-glutamate, representing a hitherto undescribed type of post-translational modification: namely, multiple γ-glutamylation or the addition of a large number of glutamate residues along the polypeptide chain. Although biological significance of PSP and its modification is yet to be established, suppression of in vitro thermal aggregation of lactate dehydrogenase by glutamylated PSP was observed.

35] Interferon induction with microbial products

This chapter describes two approaches to obtain extracorporeal photopheresis (ECP) by either aqueous or phenol extraction of heat-killed bacterial cells. These preparations have been shown to induce interferon and provide antiviral resistance in animals. The phenol extraction, provides a phenol-soluble protein (PSP) for which the starting material is an envelope suspension composed of the 10,000g pellet referred to above suspended in saline to 0.5 g/ml. The PSP may be concentrated while still in the dialysis bag by removing water through contact with polyethylene glycol flakes (20,000 MW polymer, Carbowax) and made isotonic by further dialysis, then diluted to the desired volume (usually equal to that of the 0.5 g per milliliter of envelope suspension) with saline and sterilized by filtration. After intravenous injection of 0.2 ml of ECP or PSP into the tail vein of adult female mice, samples of blood are taken from the retroorbital venous sinus in CO 2 -narcotized mice, usually 2 hour after injection, the time of maximum titer (about 1000 units/ml).

Phosphorylation of chromatin proteins in cerebral anoxia and ischemia.

Phosphorylation of nuclear protein was investigated in cerebral anoxia up to 30 min with rabbit brain and in cerebral ischemia up to 6 h with gerbil brain in vitro. Isolated nuclei were incubated in the presence of [gamma-32P]ATP and were then fractionated into the NaCl-soluble, HCl-soluble, and phenol-soluble protein fraction. Each protein fraction was further separated by gel electrophoresis, and profiles of 32P incorporation were evaluated in these pathophysiological conditions. 32P incorporation of the acidic phenol-soluble nonhistone chromatin protein became significantly suppressed in cerebral anoxia after 15 min, and there were decreases of 32P incorporation in protein with high molecular weight and increase in protein with low molecular weight on gel electrophoresis. With gerbil brain nuclei, 32P incorporation into the NaCl-soluble and HCl-soluble fraction was increased without significant decrease in the phenol-soluble fraction after an ischemic period of 3 h. However, further separation of the phenol-soluble fraction demonstrated decrease of 32P incorporation in protein with high molecular weight and increase in protein with low molecular weight. At present, the significance of these findings, particularly in relation to chromatin template activity or irreversibility of these pathophysiological conditions, is not clear.

Developmental changes in the non-histone proteins of chromatin from the salivary glands of Calliphora stygia

The non-histone protein of salivary gland chromatin from Calliphora stygia can be separated into two groups: phenol-soluble and phenol-insoluble. Each group consists of about 20 protein species. As development of the third instar larva proceeds, the amount of phenol-soluble proteins in the chromatin decreases, but the amount of phenol-insoluble proteins remains about the same. Electrophoretic analysis of the non-histone proteins revealed quantitative and qualitative changes amongst the phenol-soluble protein species and the phenol-insoluble protein species.

Membrane modifications in nutritionally induced filamentous Escherichia coli B.

Nutritionally induced filamentous cell forms of Escherichia coli B were examined for their morphological and biochemical lesions. The filamentous forms showed no significant alteration in total DNA concentration, RNA synthesis, ability to form beta-galactosidase in response to isopropylthiogalactoside, or insensitivity to actinomycin D as compared to the normal cell form. The filamentous cells showed a marked decrease in the ability to incorporate N-acetylglucosamine-UL-(14)C into a phenol-soluble glycoprotein fraction relative to the normal cell form or relative to strain E-26 of E. coli grown in the filament-inducing medium. The filaments yielded an envelope-specific phenol-soluble protein fraction markedly reduced in or lacking three proteins as determined by acrylamide gel electrophoresis. Amino acid analysis, and chemical and enzymatic treatments of the envelope-specific phenol-soluble proteins showed striking differences between the fractions obtained from normal and filamentous cells. Electron microscope studies of divalent cation-induced aggregates of the envelope proteins showed different aggregation patterns dependent upon the cell form yielding the protein fraction.

Immunologic Cross-Reactivity of Escherichia Coli B Envelope Glycoproteins with Some Animal and Plant Cell Membrane Proteins

Abstract During the past few years a number of laboratories have been investigating the antigenic character of various components of the bacterial envelope. The goals are to develop vaccines against pathogenic organisms (1) and to understand more completely the structural organization of the cell surface (2). We recently reported that rabbit antiserum prepared against isolated envelopes of Escherichia coli B had immunologic reactivity with certain animal cell membrane proteins (3). Specifically, the anti-envelope serum reacted with a “structural protein” preparation from beef heart mitochondria and a phenol soluble protein fraction from the same type of mitochondria. The phenol solubilization technique has been shown to be extremely effective in isolating membrane specific proteins (3), solubilizing membranes such as erythrocyte ghosts which are normally quite resistant to most procedures (4) and preferentially solubilizing glycoproteins (5).