The gelation kinetics of platelet extracts

The kinetics of the gelation process that occurs upon warming cold platelet extracts were studied using a sensitive rheometer. At micromolar or less free Ca²⁺ concentrations and in the presence of 1 mM ATP, the gel rigidity curves showed several peaks, indicating that platelet extract proteins went through network assembling/disassembling cycles during gelation. The gelation kinetics were accelerated by increasing the free Ca²⁺ concentration up to about 2 μM. At 4–15 μM free Ca²⁺, the gelation cycles were completely abolished except for the first peak. The gelation process became one of monotonically increasing elastic modulus at millimolar free Ca²⁺ concentrations. Trifluoperazine (50 μM), a calmodulin inhibitor, did not affect gelation at micromolar free Ca²⁺ concentrations. Except for the first gelation step, which was completed within 5 min after warming, the rest of the gelation process was found to be affected by K⁺, ATP, cytochalasin E and colchicine. K⁺ at concentrations higher than 10 mM retarded the gelation kinetics. Extracts prepared with low (0.1 mM) ATP content showed impaired gelations, and this was partially reversed by adding 1 mM ATP, but not 1 mM adenylylimidodiphosphate (p[NH]ppA). Both cytochalasin E (1 μM) and colchicine (1 mM) interfered with the gelation process.     

The action of organic mercurials on the erythrocyte membrane

The solubilisation of proteins from erythrocyte membranes by treatment with organic mercurials has been studied with different species. The marked solubilisation previously reported for human membranes does not seem to be a general phenomenon. All of the other species examined showed less than 50% of the solubilisation shown by human membranes. The protein-solubilising effect seems to be dependent on hydrophobic mercury derivatives carrying a net negative charge. Uncharged compounds like phenylmercuric acetate blocked the effect, although $\text{N-}\text{ethylmaleimide}$ and iodoacetamide did not. With the aid of radioactively labelled compounds, and of atomic absorption spectrophotometry, the proteins reactive towards the mercurials were identified. The major integral protein, band 3, was the major protein capable of binding the mercurial. Reaction with the mercurial appears to disrupt interaction of band 3 with bands 2.1 and 4.2, allowing dissociation of the cytoskeleton from the membrane. In addition, band 4.9 was also found to react with the mercurials, possibly resulting in disruption of the cytoskeleton.     

Plasma membrane protein kinase activity in normal and Rous sarcoma virus-transformed chick embryo fibroblasts

A preliminary study has been carried out to investigate the effect of Rous sarcoma virus transformation on plasma membrane protein kinase activity in chick embryo fibroblasts. Enzyme activity was measured using an in vitro phosphorylation method employing [γ-³²P]ATP with isolated plasma membranes serving as the source of both protein kinase and protein substrate. In general, the enzymatic properties observed were similar to those of other known protein kinases. However, for maximal activity a marked dependence on high Mg²⁺ concentrations was noted. Evidence was obtained which showed that cyclic nucleotide-dependent protein kinases were present in membranes from normal cells, but none could be measured in preparations from transformed cells. In addition, transformation appeared to result in a slight increase in basal plasma membrane protein kinase activity.     

High-resolution two-dimensional electrophoresis with isoelectric focusing in immobilized pH gradients

Due to the high reproducibility of pH gradient slope and width, immobilized pH gradients (IPG) have been used as the first dimension of two-dimensional techniques in order to generate maps of constant spot position in the $\text{p}\text{M}{}_{\mathrm{<mtext>r</mtext>}}$. However, when coupling IPG to SDS (sodium dodecyl sulphate) gels two problems were encountered: vertical streaking, due to incomplete zone solubilization in SDS, and horizontal streaking, due to spot fusion along the pH axis caused by the electroendosmosis of the charged Immobiline gels. Two methodical modifications are herewith described to overcome these drawbacks: (a) the SDS equilibrium time of the first-dimension gel has been prolonged to at least 30 min; (b) the SDS electrophoresis gel has been cast together with a starting gel, containing 2.5 mM of each Immobiline species used in the first dimension, which serves as a transition from the charged to the uncharged gel.     

Protoporphyrin-induced photodynamic effects on band 3 protein of human erythrocyte membranes

In previous studies it has been shown that protoporphyrin-induced photodynamic effects on red blood cells are caused by photooxidation of amino acid residues in membrane proteins and by the subsequent covalent cross-linking of these proteins. Band 3, the anion transport protein of the red blood cell membrane, has a relatively low sensitivity to photodynamic cross-linking. This cannot be attributed to sterical factors inherent in the specific localization of band 3 in the membrane structure. Solubilized band 3, for instance, showed a similar low sensitivity to cross-linking. By extracellular chymotrypsin cleavage of band 3 into fragments of 60 000 and 35 000 daltons it could be shown that both fragments were about equally sensitive to photodynamic cross-linking. The 17 000 dalton transmembrane segment, on the other hand, was completely insensitive. Inhibition of band 3-mediated sulfate transport proceeded much faster than band 3 interpeptide cross-linking, presumably indicating that the inhibition of transport is caused by photooxidation of essential amino acid residues or intrapeptide cross-linking. A close parallel was observed between photodynamic inhibition of anion transport and decreased binding of 4,4′-diisothiocyanodihydrostilbene-2,2′-disulfonate (H₂DIDS), suggesting that a photooxidation in the immediate vicinity of the H₂DIDS binding site may be responsible for transport inhibition.     

Purification and properties of two endonucleases specific for apurinic/apyrimidinic sites in DNA from Saccharomyces cerevisiae

Two distinct endonucleases from Saccharomyces cerevisiae, specific for apurinic/apyrimidinic sites (AP-endonucleases A and B), have been extensively purified and characterized. Both are free from unspecific and ultraviolet-specific endonucleases and exonucleases. The two enzymes are monomeric proteins of around 24 000 daltons. Both are sensitive to ionic strength and most active in the presence of 150 and 100 mM NaCl for AP-endonucleases A and B, respectively. They are not absolutely dependent on divalent cations, since they are insensitive to EDTA, although AP-endonuclease A is activated by Ca²⁺ or Mg²⁺ and AP-endonuclease B by Mg²⁺ only. ATP inhibits the enzymes. AP-endonuclease A reacts optimally between pH 6 and 8, and AP-endonuclease B at pH 8. AP-endonuclease A is more stable at 60°C (half-life of 17 min) than B (half-life of 4 min). AP-endonucleuase A is insensitive to N-ethylmaleimide or ρ-chloromercuribenzoate. AP-endonuclease B is also insensitive to N-ethylmaleimide, but ρ-chloromercuribenzoate inhibits its activity.     

The isolation of bovine-heart cytochrome c oxidase subunits Dependence on phospholipid and cholate content

The polypeptide chains of bovine-heart cytochrome c oxidase were preparatively isolated by a simple large-scale procedure based on gel permeation chromatography in the presence of sodium dodecyl sulphate.The resolution of the subunits as a function of the cholate and phospholipid content of the preparation was investigated.Cholate, and to a lesser extent, phospholipids interfere with the separation of the subunits; however, they do not prevent dissociation of the enzyme by SDS.Bovine-heart cytochrome c oxidase consists of six major subunits (estimated molecular weights in thousands: 40, 25, 20, 14, 12 and 10). In addition, the enzyme preparation contains at least five minor constituents, present in less than stoichiometric amounts.The first two of the three large subunits, all of which are hydrophobic, have amino-terminal N-formylmethionine. Subunit III, however, has a free methionine N-terminus.     

Stable transformation of Mesembryanthemum crystallinum (L.) with Agrobacterium rhizogenes harboring the green fluorescent protein targeted to the endoplasmic reticulum

Stable transformation of Mesembryanthemum crystallinum L. (common ice plant) with a green fluorescent protein (GFP) construct targeted to the endoplasmic reticulum was obtained. Seven and fourteen days after germination seedlings were infected with Agrobacterium rhizogenes strain ARqua1 either by direct coating of the cut radicles with bacteria growing on solid medium or by immersion of the cut surface in bacterial suspension at different optical densities. Both methods of infection resulted in production of GFP-positive roots with a frequency ranging from 6 to 20% according to the age of the explants and the application procedure. The green fluorescing roots displayed the typical hairy root phenotype and were easily maintained in liquid medium without growth regulators for over 2 years. Stable expression of the transgene in the roots was confirmed by polymerase chain reaction (PCR), immunoblotting and the capacity of roots to grow and produce callus on kanamycin-enriched medium. Nineteen endogenous cytokinins were determined in transgenic and non-transformed roots. The results revealed significantly lower levels of the free bases of isopentenyladenine, dihydrozeatin, cis- and trans-zeatin, as well as a conspicuous decline in concentrations of the corresponding nucleosides and most nucleotides in transgenic roots compared to the wild type. Comparison of the cytokinin profiles in transgenic and non-transformed roots suggested that transformation by A. rhizogenes disturbed cytokinin metabolism during the early steps of biosynthesis. Calli obtained from transformed roots were GFP-positive and remained non-regenerative or displayed high rhizogenic potential depending on the auxin/cytokinin ratio in the medium. Calli and callus-derived roots showed a strong GFP signal for over 2 years.     

Purification and Kinetic Characterisation of Lactate Dehydrogenase from Dioscorea cayenensis Tuber

Lactate dehydrogenase from yellow yam tuber (Dioscorea cayenensis Lam.) was isolated and purified using various chromatographic methods and electrophoresis. Only one form of the enzyme obtained, which obeyed Michaelis-Menten kinetics, was activated by Mg²⁺ and Ca²⁺ and inhibited by nucleotides and PEP. AMP, which activated the enzyme in the direction of pyruvate reduction, inhibited it in the direction of lactate oxidation. The enzyme is specific for pyruvate L-lactate and uses only NADH and NAD⁺ as the electron carriers. Polyacrylamide gel electrophoresis showed single band of lactate dehydrogenase activity. The average molecular mass obtained for the enzyme was 160 ± 1.2 kDa, while SDS gel electrophoresis indicated a dimer for the enzyme protein. The enzyme is very stable when frozen but its activity was hardly detectable when the tubers were stored in a well aerated place.     

Stoichiometric complexation of streptomyces subtilisin inhibitor and subtilisin

Subtilisin (Sbt) andStreptomyces subtilisin inhibitor (SSI) were analyzed either alone or together using sodium dodecylsulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). With all ratios of Sbt to SSI tested, the proteins formed a stoichiometric complex, and migrated abnormally at the top of the gel. Electroblotting and amino acid sequence analysis of the complex band showed both Sbt and SSI present at approximately equal molar ratios. When excess Sbt was present, it migrated as a free but still folded form slightly above the band corresponding to the complex. When excess SSI was present, it migrated as several species with molecular weights smaller than the intact form; in fact, the sequences of some of these species indicated that they lacked different amounts of N-terminal and possibly C-terminal residues.