Differential expression and localization of brain-type and mitochondrial creatine kinase isoenzymes during development of the chicken retina: Mi-CK as a marker for differentiation of photoreceptor cells

The expression and the cellular- as well as subcellular-distribution of brain-type B-CK and mitochondrial Mi-CK during development of the chicken retina was studied by immunoblotting, immunofluorescence and immunogold methods. B-CK expression and accumulation in retina was high from early stages of embryonic development on, decreased slightly around hatching and remained high again during adulthood. At early stages of development (days 2-5), B-CK was more or less evenly distributed over the entire retina with the exception of ganglion cells, which were stained more strongly for B-CK than other retinal precursor cells. Then, at around day 10, the beginning of stratified immunostaining by anti-B-CK antibody was noted concomitant with progressing differentiation. Finally, a dramatic increase in staining of the differentiating photoreceptor cells was seen before hatching (day 18) with weaker staining of other cell types. At hatching, as in the adult state, most of the B-CK was localized within rods and cones. Thus, during retinal development marked changes in the immunostaining pattern for B-CK were evident. By contrast, Mi-CK expression was low during development in ovo and rose just before hatching with a predominant accumulation of this isoenzyme within the ellipsoid portion of the inner photoreceptor cell segments. Mi-CK accumulation in the retina coincided with functional maturation of photoreceptors and therefore represents a good marker for terminal differentiation of these cells. B-CK, present from early stages of retina development, seems to be relevant for the energetics of retinal cell proliferation, migration and differentiation, whereas the simultaneous expression of both B- and Mi-CK around the time of hatching indicates a coordinated function of the two CK isoforms as constituents of a PCr-circuit involved in the energetics of vision, which, in autophagous birds, has to be operational at this point in time.     

Site-directed mutagenesis of beta-galactosidase (E. coli) reveals that tyr-503 is essential for activity

By using the technique of site-directed mutagenesis we have succeeded in replacing tyr-503 of beta-galactosidase (E. coli) with a phe. A study of the kinetic and stability properties of this mutant enzyme (F-503 beta-galactosidase) showed that the loss in activity upon this change is due to the loss of a catalytic group (rather than a detrimental change in the enzyme's overall structure or a change in the enzyme's binding capacity). This confirms previous suggestions that this tyr residue is involved in catalysis.     

Identification of two histidines as copper ligands in Streptomyces glaucescens tyrosinase

The physiochemical properties of wild type and two mutants of Streptomyces glaucescens tyrosinase are reported. The native enzyme contains two coppers at the active site which are EPR nondetectable. The two coppers react stoichiometrically with one hydrogen peroxide molecule giving rise to oxytyrosinase. Its optical features are similar to those reported earlier for a molluscan hemocyanin. The two mutants in which histidine-62 and -189 were changed to asparagine by site-directed mutagenesis have lost their enzymatic activity and their ability to bind oxygen and contain only one copper ion which is fully EPR detectable. The EPR parameters indicate that the remaining copper is in a tetragonally distorted ligand environment. These data are in agreement with His-62 and His-189 serving as copper ligands in S. glaucescens tyrosinase.     

The use of limited proteolysis to probe interdomain and active site regions of beta-galactosidase (Escherichia coli)

Limited proteolysis by pancreatic elastase (EC 3.4.21.36) and chymotrypsin (EC 3.4.21.1) was used to study the domain structure and active site of beta-galactosidase (EC 3.2.1.23) (Escherichia coli). Treatment with elastase resulted in a rapid cleavage between residues Ala-732 and Ala-733. No inactivation accompanied this cleavage suggesting that this bond is in a hinge region of the protein. Some slow cleavages beyond the initial one were observed to occur and were accompanied by inactivation. Treatment of beta-galactosidase with chymotrypsin resulted in cleavages first between Trp-585 and Ser-586 and then between Phe-601 and Cys-602. The first of these cleavages resulted in total inactivation of beta-galactosidase. The presence of monovalent ions or isopropyl-beta-D-thiogalactopyranoside protected against the cleavages but when Mg2+ or Mn2+ was present in the reaction mixture, the bond between Trp-585 and Ser-586 was more susceptible to the action of chymotrypsin. These data demonstrate that the conformation of beta-galactosidase around Trp-585 and Ser-586 is dramatically affected by the binding of ions and isopropyl-beta-D-thiogalactopyranoside. The mutant M15 beta-galactosidase, which is missing residues 11 through 41 and is an inactive dimer rather than an active tetramer, was found to be much more labile to proteases than native beta-galactosidase, but the same initial cleavages were found to occur. In addition, trypsin cleaved the M15 protein between Arg-431 and Trp-432 while native beta-galactosidase was stable to trypsin.     

Reversion reactions of beta-galactosidase (Escherichia coli)

The reversion reactions of beta-galactosidase (Escherichia coli) produced beta-galactosyl-galactoses and beta-galactosyl-glucoses. About 10 beta-galactosyl-galactose and 10 beta-galactosyl-glucose gas-liquid chromatographic peaks were detected and it is thus very likely that every possible isomer of beta-galactosyl-galactose and beta-galactosyl-glucose was formed by the reversion reactions (taking into account both anomers for each isomer). The presence of lactose and allolactose among the beta-galactosyl-glucoses was confirmed with standards. An important finding relating to the role of allolactose as an inducer of the lac operon was that allolactose (beta-D-galactosyl-(1----6)-D-glucose) was the only disaccharide formed initially, and at equilibrium it was present in the largest amount (50%). Obviously the enzyme is specific in its ability to form allolactose, and allolactose is the most stable beta-galactosyl-glucose, both important inducer properties. The equilibrium constant (concentration of disaccharides divided by the concentration of reactants at equilibrium) of the reaction was about 9.5 mM-1. This is the first report of an equilibrium constant for the beta-galactosidase reaction. Of mechanistic significance is the fact that only three compounds were able to replace D-galactose as a reversion reactant. Two of these (L-arabinose and D-fucose) had alterations at carbon 6. The 6 position, therefore, is not essential for reactivity. The third compound was D-galactal. Any other sugars tested (even with very minor changes relative to D-galactose) did not react. Of special consequence is the 2 position. The results strongly suggest that there has to be either an equatorial hydroxyl at the 2 position of a sugar or a special reactivity (as with D-galactal) in order for the enzyme to catalyze the beta-galactosidase reaction.     

Crystal structure determination, refinement and the molecular model of the alpha-amylase inhibitor Hoe-467A

The crystal and molecular structure of the alpha-amylase inhibitor Hoe-467A has been determined and refined at high resolution. The polypeptide chain is folded in two triple-stranded sheets, which form a barrel. The topology of folding is as found in the immunoglobulin domains. The amino acid triplet Trp18-Arg19-Tyr20 has an exceptional conformation and position in the molecule and is possibly involved in inhibitory activity.     

Diurnal fluctuations in cotton leaf carbon export, carbohydrate content, and sucrose synthesizing enzymes

In fully expanded leaves of greenhouse-grown cotton (Gossypium hirsutum L., cv Coker 100) plants, carbon export, starch accumulation rate, and carbon exchange rate exhibited different behavior during the light period. Starch accumulation rates were relatively constant during the light period, whereas carbon export rate was greater in the afternoon than in the morning even though the carbon exchange rate peaked about noon. Sucrose levels increased throughout the light period and dropped sharply with the onset of darkness; hexose levels were relatively constant except for a slight peak in the early morning. Sucrose synthase, usually thought to be a degradative enzyme, was found in unusually high activities in cotton leaf. Both sucrose synthase and sucrose phosphate synthetase activities were found to fluctuate diurnally in cotton leaves but with different rhythms. Diurnal fluctuations in the rate of sucrose export were generally aligned with sucrose phosphate synthase activity during the light period but not with sucrose synthase activity; neither enzyme activity correlated with carbon export during the dark. Cotton leaf sucrose phosphate synthase activity was sufficient to account for the observed carbon export rates; there is no need to invoke sucrose synthase as a synthetic enzyme in mature cotton leaves. During the dark a significant correlation was found between starch degradation rate and leaf carbon export. These results indicate that carbon partitioning in cotton leaf is somewhat independent of the carbon exchange rate and that leaf carbon export rate may be linked to sucrose formation and content during the light period and to starch breakdown in the dark.     

m-Fluorotyrosine substitution in beta-galactosidase; evidence for the existence of a catalytically active tyrosine

The pH profiles of beta-galactosidase, having tyr replaced by m-fluorotyrosine, were compared to those of normal enzyme. The inflection point on the alkaline side was lowered about 1.5 pH units in the fluoro-enzyme, corresponding to the difference in the phenolic pKa values of m-fluorotyrosine and tyr. When glycosidic bond breakage was rate-limiting, the Vm at pH 7.0 was higher for the fluoro-enzyme. When hydrolysis was rate-limiting or when acceptors which made transgalactosylis rate-limiting were used, the Vm was lower for the fluoro-enzyme. This shows that a tyr in beta-galactosidase is a general-acid catalyst in the glycosidic bond breaking reaction and a tyr (probably the same one) is a general-base catalyst in the hydrolytic reaction.     

Kinetic studies of the reduction of yeast glutathione reductase by reduced nicotinamide hypoxanthine dinucleotide phosphate

The reduction of yeast glutathione reductase by reduced nicotinamide hypoxanthine dinucleotide phosphate (NHxDPH) has been examined by stopped-flow kinetic methods. Like reduced glutathione or NADPH, this pyridine nucleotide generates the catalytically active two-electron reduced form of the enzyme. This reductive half-reaction with NHxDPH has only one detectable kinetic step which shows saturation kinetics (Kd = 76 microM), and has a limiting rate constant of 56 s-1. Comparison of stopped-flow and steady-state turnover data indicates that the reductive half-reaction is rate-limiting in the overall catalytic reaction. No evidence was found for a preequilibrium charge-transfer complex between NHxDPH and the active site FAD, like that seen when NADPH is the electron donor.     

Endogenous Rhythms in Photosynthesis, Sucrose Phosphate Synthase Activity, and Stomatal Resistance in Leaves of Soybean (Glycine max [L.] Merr.)

Experiments were conducted with soybean (Glycine max [L.] Merr. cv `Ransom') plants to determine if diurnal rhythms in net carbon dioxide exchange rate (CER), stomatal resistance, and sucrose-phosphate synthase (SPS) activity persisted in constant environmental conditions (constant light, LL; constant dark DD) and to assess the importance of these rhythms to the production of nonstructural carbohydrates (starch, sucrose, and hexose). Rhythms in CER, stomatal resistance, and SPS activity were observed in constant environmental conditions but the rhythms differed in period length, amplitude, and phase. The results indicated that these photosynthetic parameters are not controlled in a coordinated manner. The activity of UDPG pyrophosphorylase, another enzyme involved in sucrose formation, did not fluctuate rhythmically in constant conditions but increased with time in plants in LL. In LL, the rhythm in CER was correlated positively with fluctuations in total chlorophyll (r = 0.810) and chlorophyll a (r = 0.791) concentrations which suggested that changes in pigment concentration were associated with, but not necessarily the underlying mechanism of, the rhythm in photosynthetic rate. Assimilate export rate, net starch accumulation rate, and leaf sucrose concentration also fluctuated in constant light. No single photosynthetic parameter was closely correlated with fluctuations in assimilate export during LL; thus, assimilate export may have been controlled by interactions among the endogenous rhythms in CER, SPS activity, or other metabolic factors which were not measured in the present study.