Fruit Softening II. Precursor Incorporation into Pectin by Pear Tissue 6

The incorporation of radioactivity from precursors of methylester and galacturonosyl residues into pectin was investigatedusing tissue slices cut from ripening pear fruits. Incorporationfrom ¹⁴CH₃ methionine into methyl ester of water soluble pectinincreased 10 fold in 4 d at 18 °C and declined in laterstages of ripening. Activity from [³H]inositol could not bedetected in gaJacturonic acid released enzymically from solublepolysaccharides. When l³H]glucose was used as a precursor, activitycould be detected in galacturonic acid released from both thesoluble and insoluble polysaccharide fractions. Methionine wasa more efficient precursor of methyl ester groups than S-adenosylmethionine or S-methyl methionine; incorporation from all threeprecursors was inhibited under nitrogen. Radioactively labelledmethyl ester did not decline during a 225 min ‘chase’following a 15 min ‘pulse’ of [¹⁴CH₃]methionine;the total pectin content of slices increased by 20% during this4 h incubation.     

Cellulase, Fruit Softening and Abscission in Red RaspberryRubus idaeusL. cv Glen Clova

The ripening of raspberry fruit (Rubus ideausL. cv Glen Clova)is associated with a climacteric rise in ethylene production.As the fruit pigments change from green to red there is a progressivesoftening, loss of skin strength and a breakdown of cell wallsin the mesocarp. An increase in cellulase (endo-1,4-ß-D-glucanase)in both drupelets and receptacles accompanies these changes.The localization of cellulase in the regions of the fruit associatedwith abscission zones suggest the enzyme may be involved infruit separation as well as softening. Rubus idaeusL; raspberry; fruit ripening; ethylene; abscission; cell wall breakdown; cellulase; endo-1,4-ß-D-glucanase     

Fruit Softening I. Changes in Cell Wall Composition and endo-Polygalacturonase in Ripening Pears

Softening of pome fruits during ripening is characterized bythe solubilization of pectin. The activity of endo-polygalacturonase(endo-PG, EC 3.2.1.15 [EC] ) was determined in pears (Pyrus communisL.) ripened at 18 °C, after storage at –1°C. Theenzyme was assayed, using viscometry, in the presence of pectinesterase(EC 3.1.1.11 [EC] ) with citrus pectin as substrate. Endo-PG activitywas not detected in fruit assayed immediately from store at–1 °C but the enzyme was present after 2 d at 18 °Cwhen the fruit had started to soften and degradation of solublepectin was apparent.     

Effects of pH and Oxygen on Photosynthetic Reactions of Intact Chloroplasts

Oxygen inhibition of photosynthesis was studied with intact spinach (Spinacia oleracea L.) chloroplasts which exhibited very high rates of photosynthetic CO₂ reduction and were insensitive to additions of photosynthetic intermediates when CO₂ was available at saturating concentrations. Photosynthetic rates were measured polarographically as O₂ evolution, and the extent of the reduction of substrate was estimated from the amount of O₂ evolved. With CO₂ as substrate, inhibition of photosynthesis by O₂ was dependent on pH. At pH values above 8, rates of O₂ evolution were strongly inhibited by O₂ and only a fraction of the added bicarbonate was reduced before O₂ evolution ceased. The extent of O₂ evolution declined with increasing O₂ concentration and decreasing initial bicarbonate concentration. At pH 7.2, the initial photosynthetic rate was inhibited about 30% at high O₂ levels, but the extent of O₂ evolution was unaffected and most of the added bicarbonate was reduced. Photosynthetic O₂ evolution with 3-phosphoglycerate as substrate was similarly dependent on pH and O₂ concentration. In contrast, there was little effect of O₂ and pH on oxaloacetate-dependent oxygen evolution. Acid-base shift experiments with osmotically shocked chloroplasts showed that ATP formation was not affected by O₂. The results are discussed in terms of a balance between photosynthetic O₂ evolution and O₂ consumption by the ribulose diphosphate oxygenase reaction.     

Cleavage of Chlorophyll-Porphyrin (Requirement for Reduced Ferredoxin and Oxygen)

The chemical structures of some colorless catabolites that accumulate in senescent leaves have been established recently (B. Krautler, B. Jaun, W. Amrein, K. Bortlik, M. Schellenberg, P. Matile [1992] Plant Physiol Biochem 30: 333-346; W. Muhlecker, B. Krautler, S. Ginsburg, P. Matile [1993] Helv Chim Acta 76: 2976-2980). Such studies suggest that oxygenolytic cleavage of chlorophyll-porphyrin may occur by the action of a dioxygenase. We have attempted to demonstrate such an enzyme activity and to explore the requirements of the cleavage reaction in a reconstituted system of chloroplast (Chlpl) components prepared from senescent rape (Brassica napus L.) cotyledons. Intact senescent Chpls (also referred to as gerontoplasts) contain small amounts of two fluorescent chlorophyll catabolites, Bn-FCC-1 and Bn-FCC-2, probably representing primary cleavage products. Upon the incubation of Gpls in the presence of glucose-6-phosphate (Glc6P) or ATP, these catabolites (predominantly FCC-1) were produced in organello. In a reconstituted system of thylakoids and stroma fraction the FCCs (predominantly FCC-2) were produced in the presence of ferredoxin (Fd) and cofactors (NADPH, Glc6P) helping to keep Fd in the reduced state. Reduced Fd could not be replaced by other electron donors, suggesting that the putative dioxygenase requires Fd for the operation of its redox cycle. Production of FCC-2 did not occur in the absence of oxygen and it was inhibited by chelators of Fe2+. The contributions to the production of FCCs from both parts of the reconstituted system, thylakoids and stroma, are heat labile. The enzymic process in the thylakoids yields pheophorbide a, the presumptive precursor of FCCs. However, native senescent thylakoids could not be replaced as a "substrate" by free pheophorbide a. The stromal enzyme appears to have an affinity for senescent thylakoids; thus, "loaded" thylakoids capable of FCC production in the presence of Fd and cofactors were obtained upon homogenization of senescent cotyledons in a medium containing sorbitol and ascorbate. Such thylakoids were inactive if prepared from mature green cotyledons. As senescence was induced, the capacity to generate FCCs appeared and peaked when about half of the chlorophyll had disappeared from the cotyledons. The effectiveness of a relevant inhibitor showed that cytoplasmic protein synthesis was required for inducing the catabolic machinery in the loaded thylakoids. Thylakoids from mature Chlpls were ineffective as substrate of the stromal enzyme prepared from Gpls. However, senescent thylakoids yielded FCCs if challenged with stroma from either Chlpls or Gpls. Therefore, the stromal part of the system is likely to be a constitutive enzyme, and the pace-setting step of the pathway of chlorophyll breakdown seems to be located in the thylakoids.     

果实软化的胞壁物质和水解酶变化

果实软化通常被认为是由于胞壁水解酶如多聚半乳糖醛酸酶,果胶酯酶,纤维素酶降解胞壁物质导致。本文概述了这三种酶分子与果实软化关系的研究进展。反义基因证明,这三种酶基因的任一种表达被报制,果实能够正常软化,暗示果实的软化有其它因子的参与。其中由细胞内的淀粉酶和蔗糖酶引起的细胞膨压的变化及果胶的溶解可能是引起果肉软化的重要原因。     

溶氧及pH对产朊假丝酵母分批发酵生产谷胱甘肽的影响

在7 L发酵罐中研究了溶氧和pH对产朊假丝酵母分批发酵生产谷胱甘肽的影响。结果表明,当葡萄糖浓度为30 g/L且通气量控制在5 L/min时,搅拌转速达到300 r/min即可满足细胞生长和谷胱甘肽合成对溶解氧的需求。不同pH控制方式对谷胱甘肽分批发酵的影响有较大差异。不控制pH时,细胞干重和谷胱甘肽产量比控制pH为55的发酵分别低27%和95%,且有50%的谷胱甘肽向胞外渗漏。研究了将pH控制在4.0、4.5、5.0、5.5、6.0和6.5的谷胱甘肽分批发酵过程,发现在pH 5.5时谷胱甘肽总产量最高。用前期研究建立的动力学模型模拟了不同pH (4.0～6.5)下的分批发酵过程,并从动力学角度解释了pH对细胞生长和谷胱甘肽合成的影响。     

Oxygen binding constants and stepwise enthalpies for human and bovine hemoglobin at pH 7.6.

A high-precision thin-layer gas-solution microcalorimeter has been developed to study the binding reactions of gaseous ligands with ligand-binding macromolecules in a manner analogous to that of the Gill thin-layer optical apparatus [Doleman & Gill (1976) Anal. Biochem. 87, 127]. We have generated differential heat-binding curves of oxygen binding to human and bovine hemoglobin in phosphate buffer at pH 7.6, with the enzyme-reducing system of Hayashi et al. [(1973) Biochim. Biophys. Acta 310, 309]. Experiments were conducted at a number of different temperatures in order to expand the data field, allowing for separation of enthalpy and free energy parameters. This type of experimental analysis makes no assumptions of optical linearity between the various heme groups and reveals that the triply ligated species is measurably significant for both human and bovine hemoglobin. It was also determined that the concentration of doubly ligated species of bovine hemoglobin is relatively low. The experiments indicate that the reactions for both hemoglobins are enthalpy-driven for oxygen stepwise additions 1, 2, and 4 while being entropy-driven for step 3. Human hemoglobin oxygen-binding experiments were also performed with the Gill thin-layer optical apparatus under solution conditions identical to those used in the calorimeter. The experiments revealed that if optical linearity is assumed, the overall third equilibrium constant is negative or near zero. This indicated that either the optical cell's performance is much poorer than the thin-layer calorimeter or there is an appreciable nonlinear optical effect.     

The influence of pH on arsenazo III.

None of the methods already reported for elimination of pectins from rRNA extracts allowed the complete removal of methylated polysaccharides from methyl-labeled cytoplasmic 17 and 26 S rRNA preparations of sycamore (Acer pseudoplatanus L.) cells. An improved procedure for purifying large amounts of higher plant cytoplasmic rRNA labeled on the methyl groups was investigated. Bulk cellular RNA from sycamore cells incubated for 24 to 36 h with methyl-labeled methionine was extracted at 4°C by the phenol-extraction procedure. Most of the pectic compounds (that accounted for about 30% of the total label of RNA extracts) was selectively precipitated, before the 66% ethanol precipitation of nucleic acid, by bringing the deproteinized aqueous layer to 10% ethanol ?0.15 m sodium acetate. Cytoplasmic rRNA, 17 and 26 S, were isolated by repeated sucrose gradient sedimentations and further chromatographed on a methylated albumin kieselgurh (MAK) column. The old-fashioned MAK chromatography proved to be very useful for elimination of residual pectins, since these compounds eluted in the void volume of the column. This purification procedure gave in a reproducible way cytoplasmic 17 and 26 rRNA virtually free of any labeled DNA, mRNA, plastid rRNA, and pectic compounds.     

Cell Wall Metabolism in Ripening Fruit: IV. Characterization of the Pectic Polysaccharides Solubilized during Softening of ;Bartlett' Pear Fruit

Fractionation of pectic polysaccharides from the juice of ripening `Bartlett' pears (Pyrus communis) gave two general types of polyuronides. The major type was a homogalacturonan (HGA) whose molecular weight decreased upon ripening. The other type comprised heteropolymers composed of various amounts of arabinose, rhamnose, and galactose. Treatment of the major arabinose-containing heteropolymeric fraction of high molecular weight (400,000) with a pear exo-polygalacturonase to degrade contaminating HGA gave a polyuronide which was inert to tomato endopolygalacturonase. Glycosyl-linkage analysis of this arabinosyl-polyuronide gave results expected from a rhamnogalacturonan I-like polysaccharide with large, highly branched araban side chains (RG-I). A linkage between HGA and RG-I was not found. RG-I, in ripening pears, appeared to be degraded with the initial loss of much of its arabinose.     

柿果实采后软化过程中细胞壁组分代谢和超微结构的变化

柿果实采后果胶酯酶活性迅速上升,其活性与果实硬度的下降呈明显的负相关。多聚半乳糖醛酸酶活性增加缓慢,但其活性与果实硬度的下降无明显相关性。β-半乳糖苷酶活性迅速增加,其活性与果实硬度的下降呈明显的负相关。纤维素酶活性呈逐渐上升趋势,与果实硬度的下降也呈明显的负相关。伴随着细胞壁水解酶活性的增加,果实原果胶和纤维素含量迅速下降,而水溶性果胶含量则迅速上升。柿果刚采收时细胞壁结构完整,3d后细胞壁中胶层基本被溶解,甚至初生壁也局部发生降解。     

Requirement of multiple protein domains and residues for gating K(ATP) channels by intracellular pH.

ATP-sensitive K(+) channels (K(ATP)) are regulated by pH in addition to ATP, ADP, and phospholipids. In the study we found evidence for the molecular basis of gating the cloned K(ATP) by intracellular protons. Systematic constructions of chimerical Kir6.2-Kir1.1 channels indicated that full pH sensitivity required the N terminus, C terminus, and M2 region. Three amino acid residues were identified in these protein domains, which are Thr-71 in the N terminus, Cys-166 in the M2 region, and His-175 in the C terminus. Mutation of any of them to their counterpart residues in Kir1.1 was sufficient to completely eliminate the pH sensitivity. Creation of these residues rendered the mutant channels clear pH-dependent activation. Thus, critical players in gating K(ATP) by protons are demonstrated. The pH sensitivity enables the K(ATP) to regulate cell excitability in a number of physiological and pathophysiological conditions when pH is low but ATP concentration is normal.     

Requirement of a transmembrane pH gradient for the entry of diphtheria toxin into cells at low pH

The effects of acidification of the cytosol and of electrical depolarization on the entry of diphtheria toxin were studied. Entry of the toxin from the cell surface was induced by low pH, and the presence of the toxin in the cytosol was monitored as toxin-induced inhibition of protein synthesis. To reduce the membrane potential the cells were incubated in a buffer containing a high concentration of potassium. The cytosol was acidified either by incubating the cells with acetic acid, by incubating them with ammonium chloride which was subsequently removed in the presence of amiloride to prevent pH regulation by the Na+/H+ exchanger, or by incubating the cells in isotonic KCl in the presence of nigericin and valinomycin. The results showed that when the cytosol was acidified by either method toxin entry was inhibited, while a reduction in the membrane potential did not strongly interfere with the entry. A pH gradient across the membrane of at least 1 pH unit was required for entry. Possibly this gradient acts as a driving force for diphtheria toxin entry.     

Immobilized pH gradients for isoelectric focusing. III. Preparative separations in highly diluted gels

A further improvement on the preparative aspects of immobilized pH gradients (IPG) (J. Biochem. Biophys. Methods (1983) 8, 135–172) is described, based on the use of soft (highly diluted) polyacrylamide gels. While in conventional IPGs in 5%T gels an upper load limit of 40–45 mg protein/ml gel volume is found, in 2.5%T gels, containing the same amount of Immobiline, as much as 90 mg protein/ml gel can be applied, without overloading effects. This is an extraordinary amount of material to ba carried by a gel phase, and renders IPG by far the leading technique in any electrophoretic fractionation. A new, two-step casting technique, based on the formation of a %T step and a pH plateau around the application trench, is described. A new method for electrophoretic protein recovery from IPG gel strips, based on embedding on low-gelling agarose (37°C), is reported. The physico-chemical properties of highly diluted gels, in relation to their protein loading ability, are evaluated and discussed. It is recommended that diluted gels (e.g. 3.5%T) be used also in analytical runs, since sharper protein zones are obtained, due to the increased charge density on the polymer coil.