Aldolase exists in both the fluid and solid phases of cytoplasm.

We have prepared a functional fluorescent analogue of the glycolytic enzyme aldolase (rhodamine [Rh]-aldolase), using the succinimidyl ester of carboxytetramethyl-rhodamine. Fluorescence redistribution after photobleaching measurements of the diffusion coefficient of Rh-aldolase in aqueous solutions gave a value of 4.7 x 10(-7) cm2/S, and no immobile fraction. In the presence of filamentous actin, there was a 4.5-fold reduction in diffusion coefficient, as well as a 36% immobile fraction, demonstrating binding of Rh-aldolase to actin. However, in the presence of a 100-fold molar excess of its substrate, fructose 1,6-diphosphate, both the mobile fraction and diffusion coefficient of Rh-aldolase returned to control levels, indicating competition between substrate binding and actin cross-linking. When Rh-aldolase was microinjected into Swiss 3T3 cells, a relatively uniform intracellular distribution of fluorescence was observed. However, there were significant spatial differences in the in vivo diffusion coefficient and mobile fraction of Rh-aldolase measured with fluorescence redistribution after photobleaching. In the perinuclear region, we measured an apparent cytoplasmic diffusion coefficient of 1.1 x 10(-7) cm2/s with a 23% immobile fraction; while measurements in the cell periphery gave a value of 5.7 x 10(-8) cm2/s, with no immobile fraction. Ratio imaging of Rh-aldolase and FITC-dextran indicated that FITC-dextran was relatively excluded excluded from stress fiber domains. We interpret these data as evidence for the partitioning of aldolase between a soluble fraction in the fluid phase and a fraction associated with the solid phase of cytoplasm. The partitioning of aldolase and other glycolytic enzymes between the fluid and solid phases of cytoplasm could play a fundamental role in the control of glycolysis, the organization of cytoplasm, and cell motility. The concepts and experimental approaches described in this study can be applied to other cellular biochemical processes.

Muscle aldolase: the stress-dependent modification of catalytic and structural properties by rat muscle lysosomal cathepsin B.

Stress dependent variations in th properties of the rat muscle aldolase (D-fructose-1,6-bisphosphate D-glyceraldehyde-3-phosphate-lyase, EC 4.1.2.13) have been linked to the corresponding changes in the levels of proteolytic activities in rat muscle. Whole-body X-irradiation of rat was shown to result in loss of muscle aldolase activity towards fructose 1,6-bisphosphate by 50% while fructose 1-phosphate activity remained unchanged (Pote, M.S. and Altekar, W. (1980) Ind. J. Biochem, Biophys. 17, 255-262). Incubation of muscle extract of irradiated rat with that from control rat or rabbit muscle aldolase caused similar changes in aldolase activity. The changes are attributed to the action of catheptic enzymes possessing latency characteristics and capable of using aldolase as a substrate; the time course of their increase after irradiation corresponds to that of loss in muscle aldolase activities. Exposure of rats to stress resulted in an increase in the 'free' proteolytic activity, and the concomitant loss of 'bound' activity in muscle lysosomes indicates labilization of lysosomal membrane. The observed degradation of aldolase in vivo by muscle lysosomes is shown to be due to the action of cathepsin B (EC 3.4.22.1) present in the proteolytic enzymes released into cytosol under stress. Inactivation of rabbit muscle aldolase and rat muscle aldolase by rat muscle cathepsin B inhibited by leupeptin, antipain an iodoacetamide, but not be pepstatin. Inactivation is shown to be due to the release of C-terminal tyrosine if aldolase, required for its catalytic activity. Cathepsin B who acts as a rate-limiting enzyme in the degradation of aldolase. Such a proteolytic modification of aldolase in vivo could be relevant not only to the regulation of aldolase activity of glycolysis in muscle but also to the degradation of aldolase during stress conditions related to tissue damage and the maintenance of normal aldolase levels in the blood.

Sequential radiation damage in protein crystallography.

Radiation damage in protein crystals is described in terms of a sequential process of protein disordering. A new radiation-damage model has been tested against data from several protein crystals and can describe radiation damage corresponding to loss of the original intensity in excess of 80%. The model is an extension of previous models which characterize radiation damage in terms of successive conformational transitions of the protein from an undamaged to a spatially disordered to finally an amorphous state. The proposed model provides a more-general positional characterization of the disordered protein and includes, prior to the disordered state, a new dose-dependent state in which the protein conformation resembles the undamaged protein. Comparison of this model with the best previous model shows that the proposed model provides an improved fit to radiation-damage data.

[Changes in the kinetic properties of aldolase and lactate dehydrogenase in the brain cytoplasm of mice following chronic gamma irradiation at low doses]. / Izmenenie kineticheskikh svoistv al'dolazy i laktatdegidrogenazy tsitoplazmy mozga myshei posle khronicheskogo gamma-oblucheniia v malykh dozakh.

The effect of chronic low-dose (0.6 rad/day) 137Cs gamma-irradiation of mice was studied. It was shown that radiation causes changes in the kinetic parameters (Vmax, KM, Vmax/KM) and isoenzyme patterns of aldolase and lactate dehydrogenase of the brain cytoplasm of mice. The kinetic properties of both enzymes changes in 1, 2, 4 and 9 days after irradiation. The character of changes in Vmax of these enzymes depends on the original Vmax values. The level of the predominant LDH1 (H4) form increases while that of the specific tissular form (C4) decreases (acute on the 9th day). Different levels of sensitivity of aldolase, LDH, and their isoenzymes to low-doses of low-rate gamma-irradiation were observed.

[Effect of gamma radiation on fructose-1,6-diphosphate aldolase activity in the erythrocytes of healthy persons after submaximal physical exertion]. / Wpyw promieniowania gamma na aktywnosc aldolazy fruktozo-1,6-difosforanowej w krwinkach czerwonych ludzi zdrowych poddanych submaksymalnemu wysikowi fizycznemu.

The authors studied the effects of gamma radiation and submaximal physical exercise on aldolase activity in healthy men erythrocytes. Twelve men aged 20-22 were examined. They underwent physical exercise at doses of 2 W/kg body weight for 15 minutes. Erythrocytes were exposed to gamma radiation (500 Gy doses) from 60Co source. The activity of aldolase in erythrocytes was estimated by Fermognost test. The submaximal physical exercise was indicated to increase the aldolase activity. Gamma radiation at 500 Gy dose was found to increase aldolase enzymatic activity in erythrocytes both a rest and after submaximal physical exercise.

[Activity of several erythrocyte enzymes in chick embryos and chicks after gamma-irradiation]. / Aktivnost' nekotorykh fermentov éritrotsitov u kurinykh émbrinov i tsypliat posle gamma-oblucheniia.

After irradiation of chick embryos and chicks (1,000 rad), the activity of some erythrocyte enzymes undergoes significant changes. During the 1st day after irradiation of chick embryos, the activity of lactate dehydrogenase leucine aminopeptidase and glutamate pyruvate transaminase decreases. At the 3rd day, the decrease in the activity of glucose-6-phosphate dehydrogenase and acid phosphatase is also observed. In irradiated chicks, the activity of lactate dehydrogenase, leucine aminopeptidase and aldolase decreases within the 1st and the 3rd days, the decrease being most significant for the former two enzymes. At later period (10 and 15 days after irradiation), most significant decrease was found in the activity of glucose-6-phosphate dehydrogenase. The activity of the same enzymes in the blood plasma of irradiated embryos and chicks increases, the increase being most evident for glucose-6-phosphate dehydrogenase.

Light-induced gene expression of fructose 1,6-bisphosphate aldolase during heterotrophic growth in a cyanobacterium, Synechocystis sp. PCC 6803.

Synechocystis sp. PCC 6803 exhibits light-activated heterotrophic growth (LAHG) under dark conditions with glucose as a carbon source. The light activation is remarkable at a late period of photoautotrophic preculture, such as the late-linear and stationary growth phases. To understand the physiological effects of light irradiation and glucose under LAHG conditions, their effects on the expression of soluble proteins were analyzed by means of 2D-PAGE. Various soluble proteins, which were minimal under photoautotrophic preculture conditions, were observed clearly under LAHG conditions, suggesting that proteins were synthesized actively under these conditions. Fructose 1,6-bisphosphate aldolase, one of the glycolytic enzymes, was found to be induced under LAHG conditions on 2D-PAGE. The activity of fructose 1,6-bisphosphate aldolase, which had decreased during photoautotrophic preculture, also increased under LAHG conditions, similar to the mRNA level of the encoding gene, fbaA. In addition, we found that a deletion mutant of sll1330, a putative gene containing a helix-turn-helix DNA-binding motif, could not grow under LAHG conditions, whereas it could grow photoautotrophically. The increases in the protein level of FbaA and fbaA gene expression observed in wild-type cells under LAHG conditions were greatly inhibited in the deletion mutant. These results suggest that the regulation of fbaA gene expression by way of sll1330 is one of the important processes in Synechocystis sp. PCC 6803 under light pulse LAHG conditions.

Radiation inactivation of rabbit muscle aldolase.

Pulse radiolysis and steady-state X-radiolysis have been used to investigate the radiation inactivation of aldolase from rabbit muscle. Both eaq-and OH readily react with aldolase, and contribute to inactivation. The radical anions (CNS)2-and (Br)2-react with aldolase at neutral pH. The progressive addition of alkali results in an increase in the second-order rate constants, with an apparent pK approximately 10 +/- 0-3, and with the formation of an unstable intermediate, lambdamax approximately 400 nm resembling a phenoxyl radical. Steady-state radiolysis in the presence of (CNS)2-and (Br)2- at alkaline pH results in increased aldolase inactivation, with a pK of enzyme inactivation similar to that observed for reaction of the radical anions. We propose that a reaction of the radical anoins with tyrosine residues accounts for the resultant inactivation.

Photoregulation of some enzymes from Neurospora crassa.

Light-grown cultures of Neurospora crassa showed photoregulation of a number of enzymes. Proteases and cytosolic malate dehydrogenase showed an increase in activity. There was a decrease in the activity of mitochondrial malate dehydrogenase, isocitrate dehydrogenase and cytosolic glucose-6P-dehydrogenase, isocitrate dehydrogenase and isocitrate lyase.

[Possible effect of natural radioactivity on the induction of glucokinase synthesis in the liver of developing rats]. / K voprosu o vozmozhnom vliianii estestvennoi radioaktivnosti na induktsiiu sinteza gliukokinazy v pecheni razvivaiushchikhsia krys.

A decrease in the induced synthesis of glucokinase in the liver, at the time of spontaneous appearance of the enzyme, was observed in suckling rats kept for 10 days in a chamber with a decreased (by 10 times) natural radiation background. No changes were noted in the glucokinase synthesis induction after restoration of natural radioactivity by introducing of uranium salts.

[Fructose content and aldolase activity in the tissues of chickens under ultraviolet irradiation]. / Vmist fruktozy i al'dolazna aktyvnist' u tkanynakh kurei za ul'trafioletovoho oprominiuvannia.

The effect of UV-irradiation was studied as applied to the content of fructose and activity of aldolase in the liver tissue and muscles of chickens and embryos obtained from eggs eight months after the chicken irradiation. It is established that the content of fructose and activity of aldolase in the tissues of 19-day embryos are approximately the same as in 40-140 day chickens. Under the effect of UV-irradiation the content of fructose in the liver increases in 140-day chickens and in embryos of the group under experiment, and it decreases in the muscular tissue of 80-day chickens. In the liver of embryos obtained from the irradiated bird the activity of aldolase lowers and in young chickens no essential difference is observed between the groups. The value of the ratio of the aldolase activity for both substrates in the liver of embryos and chickens of the group under study at the age of 40 and 140 days is somewhat higher than in the control, and in the muscular tissue it is lower (in embryos and chickens at the age of 40- and 80-days).

Extreme X-ray sensitive modification of type I aldolases by blue dye ligand chromatography.

Aldolases purified by Blue dye ligand chromatography from a variety of vertebrate sources crystallize at room temperature in a habit similar to the monoclinic form of rabbit skeletal muscle aldolase. Crystals of aldolases thus purified including rabbit muscle aldolase are extremely sensitive to X-ray (Cu K alpha) radiation and shatter after short exposure to X-ray radiation (less than 5 min.). Crystals of aldolases purified by other techniques possess demonstrable diffraction patterns and are stable in the X-ray beam with lifetimes of the order of days. No clear distinction could be made on the basis of different biochemical assays between aldolases purified by Blue dye chromatography and those purified by other techniques.

The molecular chaperone alpha-crystallin inhibits UV-induced protein aggregation.

Solutions of gamma-crystallin, and various enzymes, at neutral pH and 24-26 degrees C, became turbid upon exposure to UV radiation at 295 or 308 nm. SDS-PAGE analysis revealed interchain cross-linking and aggregate formation compared to dark control solutions as reported previously. When alpha-crystallin was added to the protein solutions in stoichiometric amounts, UV irradiation resulted in significantly less turbidity than in the absence of alpha-crystallin. For example, addition of 0.5 mg of alpha-crystallin to 0.5 mg of gamma-crystallin in 1.0 ml solution yielded only 25% of the turbidity seen in the absence of alpha-crystallin. Addition of 2.0 mg of alpha-crystallin resulted in 20% of the turbidity. Given the molecular weights of alpha- and gamma-crystallin (about 800 kDa and 20 kDa, respectively), a gamma/alpha 1:1 weight ratio corresponds to a 40:1 molar ratio, and a gamma/alpha 1:4 weight ratio corresponds to a 10:1 molar ratio. Hence, the molar ratio of alpha-crystallin needed to effectively protect gamma-crystallin from photochemical opacification was gamma/alpha = n:1, where n was in the range 10-40. In terms of subunits, this ratio is gamma/alpha = 1:m, where m = 1-4. Thus, each gamma-crystallin molecule needs 1-4 alpha subunits for protection. Similar stoichiometries were observed for protection of the other proteins studied. The protection stems in part from screening of UV radiation by alpha-crystallin but more importantly from a chaperone effect analogous to that seen in thermal aggregation experiments.