Changes in potato tuber invertase and its endogenous inhibitor after slicing, including a study of assay methods

The increase in the invertase activity of extracts from freshly cut potato (Solanum tuberosum L.) by “foaming,” caused by selective denaturation of an endogenous invertase inhibitor, did not occur in extracts made from thin disks 2 days after slicing. Rather, foaming such extracts decreased invertase activity. Apparently, the inhibitor disappeared after slicing, and the enzyme became more labile to foaming. Such disappearance of inhibitor could account for up to 15% of the dramatic increase in total invertase activity that had occurred within 2 days after slicing. The difference between extracts from 0-day and 2-day slices was mainly in the first of two peaks of invertase activity eluted from diethylaminoethyl-cellulose columns. This peak was increased by foaming 0-day extracts, but even when foamed was much smaller than in 2-day extracts. The apparent loss in inhibitor was not caused by a decreasing susceptibility of the enzyme to the inhibitor. Both the increase in total invertase activity and the apparent loss of inhibitor after slicing were partially blocked by actinomycin D and completely blocked by cycloheximide.     

Partial purification of potato tuber invertase and its proteinaceous inhibitor

Improved purification of potato tuber invertase was achieved by utilizing a form of affinity chromatography between the enzyme and Concanavalin A (Con A) bound to Sepharose. Twenty-fold increases in specific activity were routinely obtained with this step and the enzyme was purified 190-fold over that found in the crude homogenate. The Con A-Sepharose chromatography step gave a greater purification than any other step in the invertase isolation procedure. There was up to 170% recovery of the activity loaded onto the column. α-Methyl-d-mannoside, sucrose, d-glucose and d-fructose eluted the enzyme from the Con A-Sepharose column with similar recoveries, although the volume of eluent required varied with the sugar. This unusually high recovery of invertase activity was obtained with some batches of tubers but not with others. There was evidence to suggest that the high recovery, or activation, may be due to the release of an inhibitor from the enzyme in the presence of Con A-Sepharose. Adsorption of invertase to Con A-Sepharose could be eliminated by incubation of the enzyme with α-mannosidase and β-glucosidase, indicating that potato tuber invertase is a glycoprotein. Proteinaceous inhibitor purification was improved by treatment of the tuber extract at low pH.     

Effect of temperature on invertase, invertase inhibitor, and sugars in potato tubers

The accumulation of reducing sugars in potato tubers exposed to low temperatures occurs with concomitant formation of the enzyme invertase. During the initial period of cold treatment when reducing sugars increase rapidly, invertase formation proceeds until the level of enzyme exceeds that of an endogenous macromolecular invertase inhibitor, resulting in a basal invertase activity. As the rate of sugar accumulation decreases and the sugar level becomes nearly constant, total invertase decreases, the basal activity disappears, and a low excess of inhibitor develops. On transfer of cold-stored tubers to warmer temperatures, sugars and invertase decrease sharply and a large excess of inhibitor develops. These changes in sugars, invertase and inhibitor occur reversibly when the tubers are subjected to alternating temperatures.     

Synthesis and apparent turnover of Acid invertase in relation to invertase inhibitor in wounded sweet potato root tissue

Previously we showed that acid invertase activity increased and then decreased rapidly in wounded sweet potato (Ipomoea batatas Liam.) root tissue, and that the tissue contained a heat-stable, proteinaceous inhibitor with a molecular weight of about 19,500 daltons.     

马铃薯转化酶抑制子St-inh cDNA克隆及在大肠杆菌中的表达和功能测定

用RT-PCR结合5’RACE方法从马铃薯(Solarium tuberosum L．)栽培种JH块茎中克隆了转化酶抑制子St-inh全长cDNA。序列分析表明,St-inh基因编码区全长663bp,编码221个氨基酸。将含St-inh基因cDNA的DNA片段克隆到pET28a( )上,转化大肠杆菌BL21(DE3)后成功实现了表达。基因表达产物与马铃薯栽培品种(系)E1、JH试管块茎以及番茄果实的转化酶提取物共孵育结果显示,转化酶活性分别下降了34．3％、21％和33．8％,说明St-inh的翻译产物具有转化酶抑制子功能。BLAST基因序列分析表明,St-inh与Kunitz-type C类基因序列同源性达95％以上,T-COFFEE氨基酸序列对比分析显示,该基因编码的蛋白质具有典型的Kunitz-type结构域[L,I,V,M]-X-D-X-[E,D,N,T,Y]-[D,G]-[R,K,H,D,E,N,Q]-X-[L,I,V,M]-X(5)-Y-X-[L,I,V,M],因此,St-inh基因可能为Kunitz-type家族成员。     

The behaviour of some naturally occurring strains of potato virus Y

Potato virus Y was obtained from field crops of potatoes in many strains which differed widely in virulence and caused diseases in the variety Majestic ranging from severe leaf-drop streak to mild mosaic. The symptoms caused by these strains in seven potato varieties and tobacco are described and compared with those caused by the serologically related potato virus C. No changes were noted in the behaviour of any of the strains over three years, during which they were transmitted to many different plants.
Potato virus C was not transmitted by Myzus persicae , the most efficient vector of other strains of virus Y. Nor was virus C transmitted by eleven other species of aphides, eight of which transmitted virus Y. The efficiency with which different species acted as vectors of virus Y varied greatly, and it is suggested that in some species only occasional individuals can transmit.
Possible mechanisms for the evolution of viruses C and Y are indicated, and the effects of changes in virus, vector, and host on the prevalence of insect-transmitted viruses are discussed.     

Gibberellin and growth inhibitor changes in potato tuber buds in response to cold treatment

Eye plugs removed from tubers of four potato cultivars previously stored for 14 days at 2 °C and then left at 15 °C for a further 14 days contained more gibberellin and less growth inhibitor activity than those kept at 15 °C for 28 days. Two gibberellin-active peaks were obtained when the extracts from cold-treated tubers were separated on paper chromatograms with an isopropanol/ammonia/water solvent. The main inhibitor did not appear to be abscisic acid.     

Antithrombotic and haemorrhagic effects of the naturally occurring thrombin inhibitor hirudin

Haemorrhagic effects of the naturally occurring thrombin inhibitor hirudin measured by the determination of bleeding time were compared with its antithrombotic actions in different models of experimental thrombosis. In mice and rats intravenous administration of hirudin caused a plasma concentration-dependent prolongation of bleeding time after transection of the tail tip and standardized incision of the tail, respectively. In rats intravenous infusion of hirudin prevented the formation of stasis-induced venous thrombosis of the jugular vein and the occurrence of thrombotic occlusion of the carotid artery after electrically induced damage of the vessel wall. Comparison of the haemorrhagic action of hirudin with its antithrombotic effectiveness showed that it caused haemorrhagic side effects only at plasma concentrations which are not required for the antithrombotic effects.     

Binding of mitochondrial ATPase from ox heart to its naturally occurring inhibitor protein: Localization by antibody binding

The naturally occurring ATPase inhibitor protein from ox heart mitochondria was cross-linked to its binding site on the mitochondrial ATPase using 1-ethyl-3-(dimethylamino)propyl carbodiimide. The cross-linked product, when transferred electrophoretically to a nitrocellulose sheet, reacted with antibodies directed against the inhibitor protein and the β-subunit of the ATPase. It was concluded that the binding site for the inhibitor protein lies on the β-subunit.     

Hydroxylation of dehydroepiandrosterone and its acetate with potato tuber slices

Evidence is presented for the view that the potato tuber tissue contains an unstable enzyme capable of hydroxylation of dehydroepiandrosterone and its acetate in low yield. The products of hydroxylation were isolated and identified. The possibility is discussed that auto-oxidation is a simultaneous reaction.     

The identification and properties of a naturally occurring inhibitor of malic enzyme

The inhibitor of malic enzyme present in potato tubers has been identified as oxalic acid. Oxalic acid proves to be a particularly potent inhibitor with a K_I = 50 μM. A kinetic analysis indicates that inhibition is not due to chelation of Mg²⁺ and suggests that oxalate binds tightly to malic enzyme after NADPH has been bound.     

Aspects of tuber resistance in hybrid potatoes to potato tuber worm

Tubers produced from crosses between the wild potato, Solanum berthaultii Hawkes (Solanaceae), and the cultivated species Solanum tuberosum L. (Solanaceae) are resistant to potato tuber worm (PTW), Phthorimaea operculella (Zeller) (Lepidoptera: Gelechiidae), infestation compared to those of the popular commercial North American cultivars Allegany, Atlantic, Chieftain, Katahdin, MaineChip, NorDonna, Norwis, Russet Norkotah, Snowden, and Yukon Gold. Given a choice between Atlantic and hybrid tubers, female PTW deposited ca. 50% fewer eggs on hybrid tubers than on those of Atlantic; larval survival and production of prepupae on hybrid tubers were reduced similarly. Time needed for neonates to penetrate eye buds was ca. 100 min greater on hybrid tubers compared to that on cv. Atlantic. Periderm of hybrid tubers is thicker than that of cv. Atlantic and may contribute to the delay in larval penetration of tubers and the success of initial establishment.     

Comparative compositional analysis of transgenic potato resistant to potato tuber moth (PTM) and its non-transformed counterpart

In this study, the compositions of transgenic potatoes (TPs) resistant to potato tuber moth (Phthorimaea operculella) were compared with those of its non-transgenic (NTP) counterparts. The light inducible promoter, phosphoenolpyruvate carboxylase led to the expression of Cry1Ab only in the leaves and light-treated tubers of the TPs. No significant differences were found in the moisture, ash, dry weight, total soluble protein, carbohydrate, starch, fiber, ascorbate, cations, anions, fatty acids, and glycoalkaloids contents of TP and NTP. Moreover, light treatment significantly affected the contents of ascorbate, acetate and nitrite anions, palmitic, stearic and linolenic fatty acids, α-haconine and α-solanine glycoalkaloids in TP and NTP tubers. While, significant differences were observed in the amino acid contents in light-treated tubers of TPs than the NTP ones. Although, light treatment in potato tubers resulted in marked metabolic changes, all the variations observed in the metabolites compositions were found to be within the desired reference ranges for potato plants. In conclusion, the results indicated that the TPs were substantially and nutritionally equivalent to the NTP counterparts.     

A thermodynamic analysis of the interaction between the mitochondrial coupling adenosine triphosphatase and its naturally occurring inhibitor protein

1. The naturally occurring ATPase (adenosine triphosphatase)-inhibitor protein, from bovine heart mitochondria, was obtained as a single pure protein. It was not identical with any of the five subunits (alpha-epsilon) of the isolated ATPase, and appeared to be a single polypeptide chain. 2. The inhibitor combined with the ATPase in a 1:1 molar ratio, producing a completely inhibited ATPase molecule. The affinity of the ATPase for its inhibitor is high; the K(d) is of the order of 10(-8)m. 3. The enthalpy of the ATPase-inhibitor complex-formation is positive, the value of K(d) decreasing as the temperature is raised. This suggests that the forces involved are largely hydrophobic in nature. 4. Hydrolysis of a nucleoside triphosphate promoted formation of the ATPase-inhibitor complex, although the equilibrium position was almost unaffected by the rate of hydrolysis. At low salt concentration, less than 200 turnovers of the ATPase suffice for the ATPase to combine with the inhibitor protein. At higher salt concentrations, a larger number of turnovers is required. It is suggested that the inhibitor binds to a form of the ATPase that is produced transiently during hydrolysis. 5. In the presence of 75mm-K(2)SO(4), the rates of association and dissociation are slow enough to allow their kinetics to be studied. Association is first-order in inhibitor concentration, but fractional order in ATPase concentration. Dissociation is first-order in ATPase-inhibitor complex concentration. The temperature coefficients of the ;on' and ;off' processes were also measured. 6. A simple kinetic model for the ATPase-inhibitor interaction is proposed that can be extended to take into account release of inhibitor protein under energized conditions on the membrane. 7. The isolated ATPase is inhibited by preincubation with Mg(2+), reversible by subsequent addition of EDTA, and by ADP, reversible by subsequent addition of ATP. These effects are not found on the membrane-bound ATPase. The mechanism of these effects is discussed.     

Post‐translational regulation of acid invertase activity by vacuolar invertase inhibitor affects resistance to cold‐induced sweetening of potato tubers

Cold‐induced sweetening (CIS) is a serious post‐harvest problem for potato tubers, which need to be stored cold to prevent sprouting and pathogenesis in order to maintain supply throughout the year. During storage at cold temperatures (below 10 °C), many cultivars accumulate free reducing sugars derived from a breakdown of starch to sucrose that is ultimately cleaved by acid invertase to produce glucose and fructose. When affected tubers are processed by frying or roasting, these reducing sugars react with free asparagine by the Maillard reaction, resulting in unacceptably dark‐coloured and bitter‐tasting product and generating the probable carcinogen acrylamide as a by‐product. We have previously identified a vacuolar invertase inhibitor (INH2) whose expression correlates both with low acid invertase activity and with resistance to CIS. Here we show that, during cold storage, overexpression of the INH2 vacuolar invertase inhibitor gene in CIS‐susceptible potato tubers reduced acid invertase activity, the accumulation of reducing sugars and the generation of acrylamide in subsequent fry tests. Conversely, suppression of vacuolar invertase inhibitor expression in a CIS‐resistant line increased susceptibility to CIS. The results show that post‐translational regulation of acid invertase by the vacuolar invertase inhibitor is an important component of resistance to CIS.