Julio Pudles

State of actin during the cycle of cohesiveness of the cytoplasm in parthenogenetically activated sea urchin egg

Abstract By the DNase I inhibition assay it is shown that the cytoplasmic matrix isolated 60 min after procaine activation of Paracentrotus lividus eggs contains about 20% of the total egg actin, mostly in polymerized form (85%). Electron microscopy studies on this cytoplasmic structure after treatment with heavy meromyosin (HMM), reveal that the decorated actin filaments are organized in bundles which are distributed radially, with the arrowheads pointing towards the central region. In addition few microtubules and a network of non-decorated microfilaments of about 3 nm diameter are observed. From the cytoplasmic pH determination and the DNase I inhibition assay on homogenates of eggs which were taken at different times of activation, it cannot be inferred that a direct relationship between the increase in the cytoplasmic pH and the increase in the amount of polymerized actin or of cytoplasmic matrix exists. Activation experiments carried out in the presence of colchicine shows that, although the formation of the cytoplasmic matrix is inhibited, polymerization of actin still occurs. Moreover, from the inhibition effects of cytochalasin B (CB) added before the activator it is shown that polymerization of actin is a necessary step for the organization of the cytoplasmic matrix. However, the cycles of cohesiveness of the cytoplasm observed in the course of the activation process do not appear to depend on cycles of polymerization and depolymerization of actin.

The Mechanism of Association of Trypsin (or Chymotrypsin) with the Pancreatic Trypsin Inhibitors (Kunitz and Kazal) Kinetics and Thermodynamics of the Interaction

This paper is a survey of our recent work concerning the dynamics of the association of trypsin and chymotrypsin with the pancreatic trypsin inhibitors, that is, the Kunitz inhibitor (PTI)1 and the Kazal (secretory) inhibitor (PSTI).

Parthenogenetic activation of sea urchin egg induces a cyclical variation of the cytoplasmic resistance to hexylene glycol-triton X-100 treatment

Abstract During the time course of the activation of Paracentrotus lividus eggs with 2.5 mM procaine or 10 mM ammonium chloride, the amount of cytoplasmic material resistant to a detergent lysis treatment (1 M hexylene glycol/1% Triton X-100) was measured. Two peaks of resistance were obtained at 60 and 120 min after addition of activator. These two peaks were obtained whether or not the activated eggs were previously treated with dithiothreitol-pronase (pH 8.9). After this treatment the material taken from the two peaks appears to be the same membrane-less cytoplasmic matrix (70–80 μm), whereas without treatment, although the first peak is identical with the material described above, the second peak shows the presence of cytaster embedded in the central region of the cytoplasmic matrix. Electron microscopy studies on the cytoplasmic matrix (peak I) reveal that this material consists of a cortical region formed by a network of 7 nm microfilaments surrounding a more heterogeneous central region made up of numerous 7 nm microfilaments. In this region, microtubules can also be distinguished, although they are less numerous. Furthermore, the second peak isolated from activated eggs which were only dejellied, reveals also a cortical and a central region which are made essentially of radially distributed microtubules. Activation experiments carried out in the presence of either cytochalasin B (CB) or colchicine indicate that both actin filaments and microtubules are implicated in the formation of the cytoplasmic matrix and that the assembly of actin filaments in the early stage of the activation process might play some role in the ulterior organization of the cytaster fibrous system. These results suggest that parthenogenetic activators induce fluctuations in the cohesiveness of the sea urchin egg cytoplasm which are revealed by the isolation of a matrix constituted by a filamentous framework. This cytoplasmic organization appears to be an intermediate step in the development of the cytaster structure.

Isolation, characterization, and immunochemical properties of a giant protein from sea urchin egg cytomatrix.

A giant protein of apparent molecular weight (Mr) 2000 kDa, as determined by SDS-PAGE, was isolated and partially purified, under denaturing conditions, from the detergent-resistant cytomatrix of unfertilized sea urchin egg. Immunoblot analysis and indirect immunofluorescence microscopy observations indicated that this high-molecular-weight protein cross-reacted with the immunospecific serum raised against chicken breast muscle beta-connectin. However, rotary-shadowing electron microscopy images of the protein revealed short threadlike structures which appear morphologically different from beta-connectin structure. Indirect immunofluorescence localization of the protein with anti-beta-connectin serum showed a distribution throughout the whole unfertilized egg cytomatrix. This immunofluorescence pattern seems to change upon egg fertilization, since at metaphase the fluorescence stain appears to be excluded from the mitotic apparatus region as revealed by the double immunolabeling with anti-beta-connectin serum and monoclonal anti-alpha-tubulin antibody. Moreover, when egg cortical fragments were double-labeled with anti-beta-connectin serum and rhodamin-conjugated phalloidin, it was observed that the microfilaments assembled after fertilization seem to be in close association with the protein at the cleavage furrow and other locations. The possible significance of this sea urchin egg connectin(titin)-like protein is discussed.

Studies on proteolytic inhibitors: III. Stability of trypsin in trypsin-Inhibitor complexes to specific chemical inactivating agents

Abstract The addition of calcium ions during dissociation of trypsin-trypsin inhibitor complexes by 5% trichloroacetic acid (TCA) greatly improves the recovery of active trypsin. The complexes were formed by interaction of trypsin with chicken ovomucoid or Ascaris and pancreas (Kunitz) inhibitors. The dissociation procedure allows one to study the activity of trypsin after treatment of the complexes with specific chemical inactivators which react with the active serine and histidine residues of the enzyme. In addition to photoxidation, two reagents were used: diisopropylfluorophosphate (DFP), 1-chloro-3-tosylamido-7-amino-2-heptanone (TLCK). The results show that the enzyme is fully protected from inactivation when bound to the inhibitors. It was also demonstrated that the native enzyme, when inactivated by DFP does not interact with chicken ovomucoid, Ascaris , and pancreas inhibitors. In the case of TLCK and photoxidized trypsin the interaction with these inhibitors is almost negligible as measured by the competitive enzymatic assay. These data support recent suggestions that the binding between trypsin and natural inhibitors is of a covalent nature and that the “active serine and histidine residues” of the enzyme participate in this interaction.

Specific phosphorylation of yeast hexokinase induced by xylose and ATPMg: Properties of the phosphorylated form of the enzyme

Abstract The inactivation of yeast hexokinase A (ATP: d -hexose-6-phosphotransferase, EC 2.7.1.1), induced by d -xylose and ATPMg, is related to the phosphorylation of a serine residue. The phosphoenzyme can be specifically dephosphorylated and reactivated after treatment with alkaline phosphatase or when incubated with d -xylose or d -lyxose and ADPMg. The phosphorylation of hexokinase, as well as the dephosphorylation of the phosphoenzyme, are highly specific processes, depending on the formation of a quaternary complex (protein-pentose-nucleotide-Mg 2+ ). Glucose or β-γ-CH 2 -ATP can inhibit the reactivation of the phosphoenzyme induced by xylose and ADPMg. The phosphorylation of the yeast hexokinase does not seem to affect the protein structure as seen by circular dichroism or fluorescence studies significantly. Binding studies, based on a quenching effect of ATP and glucose on the intrinsic fluorescence of the protein, show that both substrates can bind to the phosphohexokinase, but with slightly higher dissociation constants than with the native enzyme. Our results suggest that the essential serine residue is located in the active center region and that the phosphorylation of this residue represents a dead-end pathway in the course of the activation of the ATPase activity induced by xylose or lyxose.

Tubulin dynamics during the cytoplasmic cohesiveness cycle in artificially activated sea urchin eggs

Sedimentation studies and [3H]colchicine-binding assays have demonstrated a relationship between the cytoplasmic cohesiveness cycles and the changes in tubulin organization in Paracentrotus lividus eggs activated by 2.5 mM procaine. The same amount of tubulin (20-25% of the total egg tubulin) is involved in these cyclic process and appears to undergo polymerization and depolymerization cycles. Electron microscopy studies reveal that the microtubules formed during these cytoplasmic cohesiveness cycles are under a particulate form which is sedimentable at low speed. Activation experiments carried out in the presence of cytochalasin B (CB) show that the increase in the cytoplasmic cohesiveness is highly reduced while tubulin polymerization and depolymerization cycles and pronuclear centration are not affected. Although tubulin or actin polymerization can be independently triggered in procaine-activated eggs, the increase in cytoplasmic cohesiveness requires the polymerization of both proteins. However, the cytoplasmic cohesiveness cycles appear to be regulated by tubulin polymerization and depolymerization cycles.

Effects of activators on chemically modified yeast hexokinase.

Enzymic studies performed with chemically modified yeast hexokinase (ATP : D-hexose-6-phosphotransferase) confirm previous results indicating that the sulfhydryl, imidazol and most of the reactive amino groups do not seem to be directly implicated in the enzyme active site. On the other hand the modification of these functional groups of the enzyme does not affect the transition between the acidic inactive form to an active enzyme form after deprotonation. The chemically modified forms of hexokinase and the native enzyme are affected in the same way by activators (citrate, D-malate, 3-phosphoglycerate and Pi) when the activity was measured at pH 6.6. Moreover the loss of enzyme activity observed in the course of the chemical modifications is accompanied by an increase of the activation effect. This increase must be related to some reorganization of the enzyme active site in presence of the effectors, since the same effect was observed when hexokinase was denatured with 3M urea at pH 7.5. However no increase in the activation effect was observed when the denaturation was carried out at pH 6.5 At this pH the loss in activity and the change of optical absorption at 286 nm were much slower than at pH 7.5, which indicates a great difference in the protein structure between these pHs.

A network of 2–4 nm filaments found in sea urchin smooth muscle: Protein constituents and in situ localization☆

In this report the coisolation of two proteins from sea urchin smooth muscle of apparent molecular weights (Mr) 54 and 56 kD respectively, as determined on SDS-PAGE, is described. Like the intermediate filament proteins, these two proteins are insoluble in high ionic strength buffer solution. On two-dimensional gel electrophoresis and by immunological methods it is shown that these proteins are not related (by these criteria) to rat smooth muscle desmin (54 kD) or vimentin (56 kD). Furthermore, in conditions where both desmin and vimentin assemble in vitro into 10 nm filaments, the sea urchin smooth muscle proteins do not assemble into filaments. Ultrastructural studies on the sea urchin smooth muscle cell show that the thin and thick filaments organization resembles that described in the vertebrate smooth muscle. However, instead of 10 nm filaments, a network of filaments, 2-4 nm in diameter, is revealed, upon removal of the thin and thick filaments by 0.6 M KCl treatment. By indirect immunofluorescence microscopy, and in particular by immunocytochemical electron microscopy studies on the sea urchin smooth muscle cell, it is shown that the antibodies raised against both 54 and 56 kD proteins appear to specifically label these 2-4 nm filaments. These findings indicate that both the 54 and 56 kD proteins might be constituents of this category of filaments. The possible significance of this new cytoskeletal element, that we have named echinonematin filaments, is discussed.

Dual effect of procaine in sea urchin eggs: Inducer and inhibitor of microtubule assembly☆

An increase in the amount of cytoplasmic filamentous structures (cytoplasmic matrix and aster) which were recovered after hexylene glycol/Triton X-100 treatment of sea urchin eggs (Paracentrotus lividus) activated by 0.2-2.5 mM procaine was observed. At higher activator concentrations, an opposite effect was observed and formation of these cytoplasmic structures was inhibited in the presence of 10 mM procaine. This inhibitory effect was reversed by diluting the drug in the incubation medium. DNase I inhibition assays on egg homogenates which were performed at different time points of the activation process, show that the same amount of actin was induced to polymerize in eggs activated either by 2.5 or 10 mM procaine. However, colchicine-binding assays on the 100 000 g particulate fractions of these homogenates show that in eggs activated by 10 mM procaine, in contrast to those activated by 2.5 mM, tubulin polymerization was inhibited and microtubules were disassembled. These results show that the dual effect of procaine in the organization of the egg cytoskeleton appears to be related to its effect on the state of tubulin.