New trends in cryoenzymology: probing the functional role of protein dynamics by single-step kinetics

Cryoenzymology was initially used to slow down enzyme-catalyzed reactions so as to stabilize intermediates for further study. During the course of this early work, it became clear that cryoenzymology could be extended to other ends and some of these are described. First, the use of a cryosolvent on its own (or together with temperature) as a perturbant has allowed a resolution of the substrate binding steps of certain enzymes (myosin, D-amino acid oxidase, peroxidase and cytochrome P450). Second, by the use of cryosolvent and temperature, coupled with the classical physico-chemical perturbants, one can selectively modulate the various steps of an enzyme pathway. This approach can lead to an understanding of the mechanism of enzyme regulation. Finally, by carrying out experiments over a wide range of temperatures (-30 degrees C- +30 degrees C) and pressure (up to several kbars) in specially constructed fast reaction equipment, one can study the thermodynamic properties of the individual rate constants describing the interconversions of reaction intermediates. Experiments with creatine kinase, cytochrome P450 and peroxidase are described. The thermodynamic parameters delta H, delta G, delta S and delta V are thus measured and when this is done under different solvent conditions one can, at least within the theories available, attempt an approach to the problem of protein dynamics.     

Primary structure of a collagenic tail peptide of Torpedo acetylcholinesterase: co-expression with catalytic subunit induces the production of collagen-tailed forms in transfected cells.

The asymmetric forms of cholinesterases are synthesized only in differentiated muscular and neural cells of vertebrates. These complex oligomers are characterized by the presence of a collagen-like tail, associated with one, two or three tetramers of catalytic subunits. The collagenic tail is responsible for ionic interactions, explaining the insertion of these molecules in extracellular basal lamina, e.g. at neuromuscular endplates. We report the cloning of a collagenic subunit from Torpedo marmorata acetylcholinesterase (AChE). The predicted primary structure contains a putative signal peptide, a proline-rich domain, a collagenic domain, and a C-terminal domain composed of proline-rich and cysteine-rich regions. Several variants are generated by alternative splicing. Apart from the collagenic domain, the AChE tail subunit does not present any homology with previously known proteins. We show that co-expression of catalytic AChE subunits and collagenic subunits results in the production of asymmetric, collagen-tailed AChE forms in transfected COS cells. Thus, the assembly of these complex forms does not depend on a specific cellular processing, but rather on the expression of the collagenic subunits.     

Amphiphilic,glycophosphatidylinositol-specific phospholipase C (PI-PLC)-insensitive monomers and dimers of acetylcholinesterase

1. In a recent study, we distinguished two classes of amphiphilic AChE3 dimers in Torpedo tissues: class I corresponds to glycolipid-anchored dimers and class II molecules are characterized by their lack of sensitivity to PI-PLC and PI-PLD, relatively small shift in sedimentation with detergent, and absence of aggregation without detergent. 2. In the present report, we analyze the amphiphlic or nonamphiphilic properties of globular AChE forms in T28 murine neural cells, rabbit muscle, and chicken muscle. The molecular forms were identified by sucrose gradient sedimentation in the presence and absence of detergent and analyzed by nondenaturing charge-shift electrophoresis. Some amphiphilic forms showed an abnormal electrophoretic migration in the absence of detergent, because of the retention of detergent micelles. 3. We show that the amphiphilic monomers (G1a) from these tissues, as well as the amphiphilic dimers (G2a) from chicken muscle, resemble the class II dimers of Torpedo AChE. We cannot exclude that these molecules possess a glycolipidic anchor but suggest that their hydrophobic domain may be of a different nature. We discuss their relationship with other cholinesterase molecular forms.     

Synthesis in vitro of precursors of the catalytic subunits of acetylcholinesterase from Torpedo marmorata and Electrophorus electricus

We translated poly(A-rich messenger RNA prepared from the electric organs of Electrophorus electricus and Torpedo marmorata in a reticulocyte lysate system. In the case of Electrophorus, which appears to contain only one type of acetylcholinesterase catalytic subunit, an anti-(Electrophorus acetylcholinesterase) antiserum precipitated a single 65-kDa polypeptide from the products translation obtained in vitro. In the case of Torpedo, where a number of distinct catalytic subunits corresponding to different fractions of the enzyme have been described, an anti-(Torpedo acetylcholinesterase) antiserum precipitated two main polypeptides, 61 kDa and 65 kDa, both of which could be displaced by unlabelled purified Torpedo acetylcholinesterase. Synthesis in vitro thus appears to produce a single type of precursor of the acetylcholinesterase catalytic subunit for Electrophorus, and at least two distinct precursors for Torpedo, suggesting that several mRNAs code for the catalytic subunits in the latter species.     

Blood coagulation induced by the venom of Bothrops atrox. 2. Identification, purification, and properties of two factor X activators

We have characterized and purified the two components of the venom of Bothrops atrox that activate the coagulation factor X. Activator 1 and activator 2 were separated by ion-exchange chromatography but otherwise presented similar characteristics. They consist of a heavy polypeptide of Mr 59,000 and either one or two light chains forming a doublet of Mr 14,000-15,000. They are inactive on synthetic substrates and on prothrombin or fibrinogen and thus appear to act specifically on factor X. They are not sensitive to inhibitors of serine proteases or thiol esterases. The activation of factor X is activated by Ca2+ ions with a Hill coefficient of 2.4 and is inhibited by Hg2+, Ba2+, and Cd2+. Its pH dependency suggests that the activity depends on the ionization of a group with an apparent pK of 6.9. We studied the cleavage of purified bovine factor X by B. atrox activators and compared it to that obtained with the factor X activator from Vipera russelli venom. Like the physiological activators, the venom's activators cleave the heavy chain of factor X, producing the activated factor Xa alpha. They produce however two other cleavages: one near the N-terminal end of the heavy chain of factor X, generating factor Xmu, and a second one located at one extremity of the heavy chain of factor Xa alpha, generating factor Xav.     

Early and late passage C-6 glial cell growth: Similarities with primary glial cells in culture

Earlier studies in our laboratory have shown that C-6 glial cells in culture exhibit astrocytic properties with increasing cell passage. In this study, we tested the responsiveness of early and late passage C-6 glial cells to various cultures conditions: culture substrata (collagen, poly-L-lysine, plastic), or supplements for the culture medium, DMEM, [fetal calf, or heat inactivated (HI) serum, or media conditioned from mouse neuroblastoma cells (NBCM) or primary chick embryo cultured neurons (NCM)]. Glutamine synthetase (GS) and cyclic nucleotide phosphohydrolase (CNP), astrocytic and oligodendrocytic glial markers, were used. Cell numer and protein content increased exponentially with days in culture regardless of the type of the substratum or cell passage. Differences in cell morphology among the three types of substratum were also reflected on GS activity, which rose by three-fold on culture day 3 for cells grown on collagen; thereafter, GS profiles were similar for all substrata. This early rise in GS is interpreted to reflect differential cell adhesion processes on the substrata; specifically, cell adhesion on the collagen stimulated differentiation into astrocytic phenotype.Analogous to immature glia cells in primary cultures, early passage C-6 glial cells responded to neuronal factors supplied either from NCM or NBCM by expressing reduced GS activity, the astrocytic marker and enhanced CNP activity, the oligodendrocytic marker. Thus, early passage cells can be induced to express either astrocytic or oligodendrocytic phenotype. In accordance with our previous reports on primary glial cells, late passage C-6 cells exhibit their usual astrocytic behavior, responding to serum factors with GS activity. Moreover, whereas NCM or NBCM alone markedly lowered GS activity, a combination with serum restored activity. The present findings confirm our previous observations and further establish the C-6 glial cells as a reliable model to study immature glia.Special issue dedicated to Dr. Paola S. Timiras.     

Analysis of HIV-1 drug resistant mutations by line probe assay and direct sequencing in a cohort of therapy naive HIV-1 infected Italian patients

Background  

Recent studies of viral entry proteins from influenza, measles, human immunodeficiency virus, type 1 (HIV-1), and Ebola virus have shown, first with molecular modeling, and then X-ray crystallographic or other biophysical studies, that these disparate viruses share a coiled-coil type of entry protein.     

Subcellular distribution of acetylcholinesterase forms in chromaffin cells. Do chromaffin granules contain a specific secretory acetylcholinesterase?

The presence of acetylcholinesterase (AChE) in chromaffin granules has been controversial for a long time. We therefore undertook a study of AChE molecular forms in chromaffin cells and of their distribution during subcellular fractionation. We characterized four main AChE forms, three amphiphilic forms (Ga1, Ga2 and Ga4), and one non-amphiphilic form (Gna4). Each form shows the same molecular characteristics (sedimentation, electrophoretic migration, lectin interactions) in the different subcellular fractions. All forms are glycosylated and seem to possess both N-linked and O-linked carbohydrate chains. There are differences in the structure of the glycans carried by the different forms, as indicated by their interaction with some lectins. Glycophosphatidylinositol-specific phospholipases C converted the Ga2 form, but not the other amphiphilic forms, into non-amphiphilic derivatives. The distinct patterns of AChE molecular forms observed in various subcellular compartments indicate the existence of an active sorting process. Gna4 was concentrated in fractions of high density, containing chromaffin granules. We obtained evidence for the existence of a lighter fraction also containing chromogranin A, tetrabenazine-binding sites and Gna4 AChE, which may correspond to immature, incompletely loaded granules or to partially emptied granules. The distribution of Gna4 during subcellular fractionation suggested that this form is largely, but not exclusively, contained in chromaffin granules, the membranes of which may contain low levels of the three amphiphilic forms.     

Epigenetic chromatin modifiers in barley: IV. The study of barley Polycomb group (PcG) genes during seed development and in response to external ABA

Background  

Epigenetic phenomena have been associated with the regulation of active and silent chromatin states achieved by modifications of chromatin structure through DNA methylation, and histone post-translational modifications. The latter is accomplished, in part, through the action of PcG (Polycomb group) protein complexes which methylate nucleosomal histone tails at specific sites, ultimately leading to chromatin compaction and gene silencing. Different PcG complex variants operating during different developmental stages have been described in plants. In particular, the so-called FIE/MEA/FIS2 complex governs the expression of genes important in embryo and endosperm development in Arabidopsis. In our effort to understand the epigenetic mechanisms regulating seed development in barley (Hordeum vulgare), an agronomically important monocot plant cultivated for its endosperm, we set out to characterize the genes encoding barley PcG proteins.     

Image analysis of the heavy form of the acetylcholine receptor from Torpedo marmorata

The structure of the heavy (H) form of the acetylcholine receptor, which comprises two covalently linked 250,000 M_r oligomers, has been investigated by numerical analysis of electron microscope images. Na-cholate solubilized Torpedo marmorata H-form receptor was reintegrated into artificial lipid vesicles and negatively stained with uranyl acetate prior to imaging in a conventional transmission microscope. The reconstituted preparations exhibited the standard polypeptide composition of the purified receptor (α₂βγδ) and the same transmembrane arrangement as in the native subsynaptic membrane. Covalent disulfide linkage between the two oligomers took place exclusively through the δ chains.In agreement with previous work (Cartaud et al., 1980) the H-form appeared as “doublets” of two coplanar 9 nm rosettes at a center-to-center distance of 9.2 ± 1.1 nm. The relative angular orientation of the two rosettes in a doublet was examined by correlation analysis in the real space. It exhibited a marked variability, few of the doublets featuring any kind of symmetry, suggesting that the two oligomers of a doublet are connected via an extended and flexible chain or loop. The area of contact between the two rosettes of a doublet therefore does not necessarily represent a reliable clue as to the location of the δ chain within the structure.Averaged images obtained after reorientation and summation of up to 132 rosettes revealed the three major peaks and the two grooves already observed in previous studies. Two additional smaller peaks were identified.Tentative assignment of structural details to individual subunits was deduced from an examination of α-bungarotoxin-labeled doublets. The α subunits, which carry part or all of the acetylcholine binding sites, are probably located in nonadjacent positions in the vicinity of the newly found peaks. This assignment is consistent with the image analysis of receptor-toxin complexes recently reported by Zingsheim et al. (1982b).