Purification, molecular cloning, and sequence analysis of sucrose-6F-phosphate phosphohydrolase from plants.

Sucrose-6(F)-phosphate phosphohydrolase (SPP; EC ) catalyzes the final step in the pathway of sucrose biosynthesis and is the only enzyme of photosynthetic carbon assimilation for which the gene has not been identified. The enzyme was purified to homogeneity from rice (Oryza sativa L.) leaves and partially sequenced. The rice leaf enzyme is a dimer with a native molecular mass of 100 kDa and a subunit molecular mass of 50 kDa. The enzyme is highly specific for sucrose 6(F)-phosphate with a K(m) of 65 microM and a specific activity of 1250 micromol min(-1) mg(-1) protein. The activity is dependent on Mg(2+) with a remarkably low K(a) of 8-9 microM and is weakly inhibited by sucrose. Three peptides from cleavage of the purified rice SPP with endoproteinase Lys-C showed similarity to the deduced amino acid sequences of three predicted open reading frames (ORF) in the Arabidopsis thaliana genome and one in the genome of the cyanobacterium Synechocystis sp. PCC6803, as well as cDNA clones from Arabidopsis, maize, and other species in the GenBank database of expressed sequence tags. The putative maize SPP cDNA clone contained an ORF encoding a 420-amino acid polypeptide. Heterologous expression in Escherichia coli showed that this cDNA clone encoded a functional SPP enzyme. The 260-amino acid N-terminal catalytic domain of the maize SPP is homologous to the C-terminal region of sucrose-phosphate synthase. A PSI-BLAST search of the GenBank database indicated that the maize SPP is a member of the haloacid dehalogenase hydrolase/phosphatase superfamily.

Cloning and expression of a prokaryotic sucrose-phosphate synthase gene from the cyanobacterium Synechocystis sp. PCC 6803.

Sucrose is one of several low-molecular-weight compounds that cyanobacteria accumulate in response to osmotic stress and which are believed to act as osmoprotectants. The genome of the cyanobacterium Synechocystis sp. PCC 6803 contains a 2163 bp open reading frame (ORF) that shows similarity to genes from higher plants encoding sucrose-phosphate synthase (SPS), the enzyme responsible for sucrose synthesis. The deduced amino acid sequence shows 35-39% identity with known higher-plant SPS sequences. The putative Synechocystis sps gene was cloned from genomic DNA by PCR amplification and expressed as a His6-tagged amino-terminal fusion protein in Escherichia coli. The expressed protein was purified and shown to be a functional SPS enzyme, confirming the identity of the ORF, which is the first sps gene to be cloned from a prokaryotic organism. The Synechocystis SPS has a molecular mass of 81.5 kDa, which is smaller than the typical higher-plant SPS subunit (117-119 kDa), and lacks the phosphorylation site motifs associated with light- and osmotic stress-induced regulation of SPS in higher plants. The enzyme has Km values for UDPG1c and Fru6P of 2.9 mM and 0.22 mM, respectively, with a Vmax of 17 micromol per minute per mg protein and a pH optimum of 8.5. Unlike the higher-plant enzyme, ADPG1c, CDPG1c and GDPG1c can substitute for UDPG1c as the glucosyl donor with Km values of 2.5, 7.2 and 1.8 mM, respectively. The enzyme is activated by Mg2+ but not by G1c6P, and is only weakly inhibited by inorganic phosphate. The purified protein was used to raise a high-titre antiserum, which recognises a low-abundance 81 kDa protein in Synechocystis sp. PCC 6803 extracts. There was no apparent increase in expression of the 81 kDa protein when the cells were exposed to moderate salt stress, and SPS activity was very low in extracts from both unstressed and salt-stressed cells. These results and the lack of evidence for sucrose accumulation in Synechocystis sp. PCC6803 lead to the conclusion that expression of the sps gene plays no obvious role in adaptation to osmotic stress in this species.

Carbohydrate Content and Enzyme Metabolism in Developing Canola Siliques.

Little biochemical information is available on carbohydrate metabolism in developing canola (Brassica napus L.) silique (pod) wall and seed tissues. This research examines the carbohydrate contents and sucrose (Suc) metabolic enzyme activities in different aged silique wall and seed tissues during oil filling. The silique wall partitioned photosynthate into Suc over starch and predominantly accumulated hexose. The silique wall hexose content and soluble acid invertase activity rapidly fell as embryos progressed from the early- to late-cotyledon developmental stages. A similar trend was not evident for alkaline invertase, Suc synthase (SuSy), and Suc-phosphate synthase. Silique wall SuSy activities were much higher than source leaves at all times and may serve to supply the substrate for secondary cell wall thickening. In young seeds starch was the predominant accumulated carbohydrate over the sampled developmental range. Seed hexose levels dropped as embryos developed from the early- to midcotyledon stage. Hexose and starch were localized to the testa or liquid endosperm, whereas Suc was evenly distributed among seed components. With the switch to oil accumulation, seed SuSy activity increased by 3.6-fold and soluble acid invertase activity decreased by 76%. These data provide valuable baseline knowledge for the genetic manipulation of canola seed carbon partitioning.

Localisation of sucrose-phosphate synthase and starch in leaves of C4 plants.

The activity and intercellular distribution of sucrose-phosphate synthase (SPS; EC 2.4.1.14) were determined in fully expanded leaves from a range of C4 plants. In Zea mays L. and Atriplex spongiosa F. Muell., SPS was located almost exclusively in the mesophyll cells. In other species, SPS was found in both cell types, with the activity in the bundle sheath cells ranging from 5% of the total leaf activity in Echinochloa crus-galli (L.) Beauv. to 35% in Sorghum bicolor Moench. At the end of the light period, starch was found only in the bundle sheath cells in all of the species examined. There appears to be little correlation between C4-acid decarboxylation type and the location of sucrose and starch synthesis in the leaves of C4 plants.

Whistle-blowing: a legal perspective.

Whistle-blowing needs to be explored from a legal perspective. Nurses who become aware of confidential information which they feel is a matter of public concern are faced with a legal as well as a moral dilemma as to when and how they may 'blow the whistle'.

Transport of inorganic pyrophosphate across the spinach chloroplast envelope.

Spinach-leaf chloroplasts take up PPi at a rate of 1.9 mumol/h per mg of chlorophyll (Chl) in the dark and 1.6 mumol/h per mg of Chl in the light. The Km for PPi transport is 32 microM in the dark and 6 microM in the light. Uptake is inhibited by pyridoxal phosphate, 4,4'-di-isothiocyanatostilbene-2,2'-disulphonic acid and imidodiphosphate, but not by NaF or EDTA. PPi does not appear to cross the chloroplast envelope in exchange for Pi, suggesting that it is not transported by the phosphate translocator. Exchange of PPi and adenine nucleotides across the chloroplast envelope is very slow and PPi does not competitively inhibit ATP uptake, suggesting that little, if any, PPi is transported by the adenine-nucleotide translocator. These results are consistent with the presence of a specific, high-affinity PPi translocator in the spinach chloroplast envelope. It is proposed that in vivo PPi is taken up into the chloroplast from the cytosol to replenish the Pi pool in the stroma.

Localization of ATP Sulfurylase and O-Acetylserine(thiol)lyase in Spinach Leaves.

The intracellular compartmentation of ATP sulfurylase and O-acetylserine(thiol)lyase in spinach (Spinacia oleracea L.) leaves has been investigated by isolation of organelles and fractionation of protoplasts. ATP sulfurylase is located predominantly in the chloroplasts, but is also present in the cytosol. No evidence was found for ATP sulfurylase activity in the mitochondria. Two forms of ATP sulfurylase were separated by anion-exchange chromatography. The more abundant form is present in the chloroplasts, the second is cytosolic. O-Acetylserine(thiol)lyase activity is located primarily in the chloroplasts and cytosol, but is also present in the mitochondria. Three forms of O-acetylserine(thiol)lyase were separated by anion-exchange chromatography, and each was found to be specific to one intracellular compartment. The cytosolic ATP sulfurylase may not be active in vivo due to the unfavorable equilibrium constant of the reaction, and the presence of micromolar concentrations of inorganic pyrophosphate in the cytosol, therefore its role remains unknown. It is suggested that the plant cell may be unable to transport cysteine between the different compartments, so that the cysteine required for protein synthesis must be synthesized in situ, hence the presence of O-acetylserine(thiol)lyase in the three compartments where proteins are synthesized.

Apparent equilibrium constant and mass-action ratio for sucrose-phosphate synthase in seeds of Pisum sativum.

The aim of this work was to use preparations from germinating seeds of Pisum sativum to determine the apparent equilibrium constant of the reaction catalysed by sucrose-phosphate synthase (EC 2.4.1.14) and to compare this with the mass-action ratio of the reaction in the seeds. The apparent equilibrium constant ranged from 5.3 at 0.25 mM-MgCl2, pH 7.0, to 62 at 10 mM-MgCl2, pH 7.5. The sucrose phosphate content of the seeds, 23 nmol/g fresh wt., was determined by separating sucrose phosphate from sucrose by ion-exchange chromatography and then measuring the sucrose released by alkaline phosphatase. Comparison of equilibrium constants and mass-action ratios in the cotyledons of 38 h-germinated seeds showed that the reactions catalysed by glucose-6-phosphate isomerase, phosphoglucomutase and UDP-glucose pyrophosphorylase are close to equilibrium, and those catalysed by sucrose-phosphate synthase and sucrose phosphatase are considerably displaced from equilibrium in vivo.

Attitudes to audit.

An exercise in audit was arranged jointly by the Local Medical Committee and the Royal College of General Practitioners in the Doncaster area. This was followed up by a questionnaire enquiring about attitudes to the audit.The proportion of doctors who did the audit was low (28 per cent overall and 7 per cent for doctors who had graduated abroad). The followup questionnaire was answered by 28 (78 per cent) of the doctors who had done the audit and by 25 (38 per cent) of those who had not. There was strong opposition to outside control of audit and some respondents even had doubts about control from within the profession.If the Doncaster findings are representative of other areas of the country, the future outlook for audit in general practice is bleak.

A four-year prospective study of the work of the practice nurse in the treatment room of a South Yorkshire practice.

During a four-year study period 43 985 patients were seen in the treatment room and 61 806 coded procedures carried out. Thirty per cent of these procedures were not part of usual nursing curricula and required initial supervision and assessment or training (or both). Nearly 15% of the patients seen were making a first visit and did not require referral to a doctor. A further 17% were also making a first visit but were referred to a doctor. The treatment room made an important contribution to the work of the practice, but this would not have been possible if the staff concerned had been attached nurses requiring area health authority authorisation for procedures carried out as opposed to practice nurses for whom procedures were authorised on a personal basis.

Undispensed prescriptions in a mining general practice.

Seven per cent of prescription forms issued in a mining practice were not presented at chemists for dispensing. The people least likely to present their prescription forms were men aged 25-34 years, particularly miners. To get sickness benefit these men have to consult a doctor, but the medical content of the consultation in these circumstances is often perceived by them as irrelevant and the medication rejected. Children and old people nearly always presented their prescriptions. The percentage of undispensed prescription forms for the trainee doctor was higher than for the trainer, but age may have been a factor: older patients tended to consult the established doctor, whom they knew. Drugs affecting the cardiovascular system, moderate or strong analgesics, hypnotics, sedatives, tranquillisers, and antidepressants were rarely rejected, but mild analgesics and drugs prescribed for symptomatic relief, such as those affecting the alimentary system, cough mixtures, and skin preparations, were more often rejected.

Organising a physiotherapy service in general practice.

This paper describes three years' experience of running a domiciliary physiotherapy service based on general practice and financed by limited voluntary funds.The need arose from the remoteness of, and lack of, open access to, hospital physiotherapy. This was particularly so for elderly patients who were often frail and mentally confused. In addition there were obvious advantages in properly instructing relatives in management and treatment, especially since many of the patients and their relatives expressed a desire for home treatment.There was also a desire on the part of the general practitioners, nurses, and ancillary workers to develop further the teamwork in the health services of the four villages involved. Details of the constitution of the voluntary service and its financial arrangements are given.The results of the service and the nature of its work are described. There were no difficulties experienced in selecting the correct patients for treatment and the type of equipment required was almost all normally available through the health authority nursing service. There was no great need for expensive or heavy equipment and no transport problems arose.It was found that one hour of physiotherapist's time per 1,000 patients per week was adequate to cover all patients requiring short-term intensive therapy and to allow a small amount of palliative therapy in addition, although this had not been the original intention of the service.The physiotherapist averaged about 40 hours work per month and under these conditions the travelling and costs averaged 1.54 miles and 83 pence per visit. With self-determined hours of work and flexible timing, these conditions proved ideal for a married physiotherapist with the responsibility of a young family. Expansion of the hours of work in this particular area would have led to wasteful visits devoted to palliative and placebo therapy; and extension of the service beyond the area defined, would have increased travelling time at the expense of working time. Thus there appear to be considerable advantages in keeping general-practice based domiciliary physiotherapy work on a part-time basis and looking for staff living close to the practice.