Biomedical subjects

J Preiss

Publications and source records attributed to J Preiss.

At least 73 records · Page 4Linked to original sources

PubMed1994

Characterization of the kinetic, regulatory, and structural properties of ADP-glucose pyrophosphorylase from Chlamydomonas reinhardtii.

ADP-glucose pyrophosphorylase (ADP-Glc PPase) from Chlamydomonas reinhardtii cells was purified over 2000-fold to a specific activity of 81 units/mg protein, and its kinetic and regulatory properties were characterized. Inorganic orthophosphate and 3-phosphoglycerate were the most potent inhibitor and activator, respectively. Rabbit antiserum raised against the spinach leaf ADP-Glc PPase (but not the one raised against the enzyme from Escherichia coli) inhibited the activity of the purified algal enzyme, which migrated as a single protein band in native polyacrylamide gel electrophoresis. Two-dimensional and sodium dodecyl sulfate-polyacrylamide gel electrophoresis indicate that the enzyme from C. reinhardtii is composed of two subunits with molecular masses of 50 and 53 kD, respectively. The molecular mass of the native enzyme is estimated to be 210 kD. Antisera raised against the spinach leaf holoenzyme and against the 51-kD spinach subunit cross-reacted with both subunits of the algal ADP-Glc PPase in immunoblot hybridization, but the cross-reaction was stronger for the 50-kD algal subunit than for the 53-kD subunit. No cross-reaction was observed when antiserum raised against the spinach leaf pyrophosphorylase 54-kD subunit was used. These results suggest that the ADP-Glc PPase from C. reinhardtii is a heterotetrameric protein, since the enzyme from higher plants and its two subunits are structurally more related to the small subunit of the spinach leaf enzyme than to its large subunit. This information is discussed in the context of the possible evolutionary changes leading from the bacterial ADP-Glc PPase to the cyanobacterial and higher plant enzymes.

Animals↗

PubMed1994

Expression of branching enzyme I of maize endosperm in Escherichia coli.

The gene encoding for mature branching enzyme (BE) I (BEI) of maize (Zea mays L.) endosperm has been expressed in Escherichia coli using the T7 promoter. The expressed BEI was purified to near homogeneity so that amylolytic activity and bacterial BE could be completely eliminated from the BE preparation. The recombinant enzyme showed properties very similar to those of BEI purified from developing maize endosperm with respect to branching amylose and amylopectin. This result confirmed our earlier report that maize endosperm BEI had a higher rate of branching amylose and a much lower rate (less than 10% of that of branching amylose) of branching amylopectin. This study also showed a great advantage in purifying BEI from the bacterial expression system rather than from developing maize endosperm. Most important, this study has established the system with which to study the structure-function relationships of the maize BEI using site-directed mutagenesis.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1994

Expression of branching enzyme II of maize endosperm in Escherichia coli.

A cDNA clone encoding maize branching enzyme II (BEII) has been independently isolated from a maize endosperm cDNA library. The deduced protein sequence of maize BEII was compared with that of BE from diverse sources. The gene encoding mature BEII of maize endosperm has been expressed in E. coli using the T7 promoter. The expressed BEII was purified to near homogeneity so that amylolytic activity and bacterial BE could be completely eliminated from the BE preparation. The expressed enzyme showed very similar properties to those of BEII purified from developing maize endosperm. This result confirmed our earlier report that BEII had a lower rate of branching amylose and the rate of branching amylopectin was twice that of branching amylose. This study also showed a greater advantage of purifying BEII from the bacterial expression system than from developing maize endosperm. Most importantly, this study has established a useful tool to study the structure-function relationships of the maize BE using site-directed mutagenesis.

1,4-alpha-Glucan Branching Enzyme↗

PubMed1994

[Comparison of 3 forms of psychosocial rehabilitation in patients with epilepsy].

The submitted paper is based on experience 1. with enlisting epileptic patients in programmes for neurotic patients during hospitalization (1968 - 1983, a total of 20 patients), 2. an ambulatory psychotherapeutic group for epileptic patients only (period 1983 - 1989, a total of 41 subjects, i.e. 39 patients and two mothers of patients) and 3. a club for epileptic patients (1990 - 1993, a total of 56 subjects, incl. 44 patients and relatives, partners and friends). The majority of patients are not only treated at the neurological department but are also psychiatric patients. The author mentions also excursions made with patients who attend the club and on two arts exhibitions of work made by the patients. He presents also results and discusses positive and negative of these tested forms of psychosocial work. The advantage of the programme for neuroses is the relatively intensive action. The advantage of the ambulatory group is the possibility to discuss in detail specific problems of patients with epilepsy. The club is acceptable for a much wider circle of patients than the other two forms. The author recommends a combination of the described three procedure and the application of the widest possible range of other rehabilitation methods.

Epilepsy↗

PubMed1993

Kinetic mechanism and regulation of ADP-glucose pyrophosphorylase from barley (Hordeum vulgare) leaves.

Barley (Hordeum vulgare, cv. Bomi) leaf ADP-glucose pyrophosphorylase (AGP) was purified to near-homogeneity, using ammonium sulfate fractionation and heat treatment as well as ion exchange, hydrophobic, and dye-ligand chromatography. The enzyme was found to be composed of two subunit types of 51 and 54 kDa, which were recognized by the respective rabbit antibodies against the small and/or large subunit of spinach leaf AGP. Substrate kinetics and product inhibition studies, carried out in the direction of ADP-glucose synthesis, suggested a sequential Iso Ordered Bi Bi kinetic mechanism for the enzyme. In addition, inhibition patterns with CrATP, a dead-end inhibitor of ATP-utilizing enzymes, were consistent with ATP (synthesis reaction) and ADP-glucose (pyrophosphorolysis reaction) binding first to different free forms of the enzyme. The AGP was potently activated by 3-phosphoglycerate (PGA) (K alpha of 5 microM) and inhibited by orthophosphate (P(i)), with the ratio of the two effectors playing a major role in modulating AGP activity. At low [PGA]/[P(i)] ratios, P(i) could reverse the activating effect of PGA. However, at the [PGA]/[Pi] ratios of 1.4, or higher, some activating effect of P(i) in the presence of PGA was observed. PGA decreased the Km values for all substrates of AGP (in both the synthesis and pyrophosphorolysis directions). Properties of the barley leaf AGP are compared to those of AGP from barley seed endosperm, which is insensitive to PGA/P(i) regulation.

Adenosine Triphosphate↗

PubMed1993

Expression of the potato tuber ADP-glucose pyrophosphorylase in Escherichia coli.

cDNA clones encoding the putative mature forms of the large and small subunits of the potato tuber ADP-glucose pyrophosphorylase have been expressed separately and together in an Escherichia coli B mutant deficient in ADP-glucose pyrophosphorylase activity. Expression of both subunits from compatible vectors resulted in restoration of ADP-glucose pyrophosphorylase activity. Maximal enzyme activity required both subunits. The expressed ADP-glucose pyrophosphorylase was purified and characterized. The recombinant enzyme exhibited catalytic and allosteric kinetic properties very similar to the enzyme purified from potato tuber. The expressed enzyme activity was neutralized by incubation with antibodies raised against potato tuber and spinach leaf ADP-glucose pyrophosphorylases but not with anti-Escherichia coli enzyme serum. 3-Phosphoglycerate was the most efficient activator and its effect was increased by dithiothreitol. In the ADP-glucose synthesis direction, 3-phosphoglycerate activated the recombinant enzyme nearly 100-fold in the presence of dithiothreitol, with an A0.5 value of 57 microM. The recombinant ADP-glucose pyrophosphorylase was less sensitive to P(i) inhibition and more sensitive to heat denaturation than the potato tuber enzyme. Results suggest that bacterial expression of potato tuber cDNAs could be used to study the role and interaction of the subunits of the native ADP-glucose pyrophosphorylase.

Allosteric Regulation↗

PubMed1993

Cloning, expression, and sequence of an allosteric mutant ADPglucose pyrophosphorylase from Escherichia coli B.

Escherichia coli B mutant strain SG14 accumulates glycogen at 28% of the rate observed for the parent strain. This is due to the presence of an ADPglucose pyrophosphorylase with altered allosteric properties including lower apparent affinities for substrates, the activator, fructose-1,6-bisphosphate, and the inhibitor, AMP. The mutant enzyme also is completely insensitive to activation by NADPH. To clone this mutant, an SG14 library was constructed by insertion of the chromosomal DNA into the PstI site of pBR322. Screening of the library via colony hybridization with a wild-type gene (glgC) probe resulted in the successful isolation of a recombinant plasmid, designated pPJ2, which contained the mutant glgC gene. The enzyme expressed from pPJ2 was partially purified and found to be very similar in kinetic and allosteric properties to the enzyme isolated from the SG14 strain. The mutant glgC gene, a HincII fragment from pPJ2, was then subcloned into pUC118/119 for dideoxy sequencing of both strands. One amino acid change was found in a region that is highly conserved in all known sequences: a single point mutation at the deduced amino acid residue 44 resulted in a change of alanine to threonine. The properties of this mutant are discussed in comparison to other known allosteric mutants.

Adenosine Monophosphate↗

PubMed1993

Insensitivity of barley endosperm ADP-glucose pyrophosphorylase to 3-phosphoglycerate and orthophosphate regulation.

Crude extracts of starchy endosperm from barley (Hordeum vulgare cv Bomi) contained high pyrophosphorolytic activity (up to 0.5 mumol of glucose-1-P formed min-1 mg-1 of protein) of ADP-glucose pyrophosphorylase (AGP) when assayed in the absence of 3-phosphoglycerate (3-PGA). This high activity was observed regardless of whether AGP had been extracted in the presence or absence of various protease inhibitors or other protectants. Western blot analysis using antibodies specific for either the small or large subunit of the enzyme demonstrated that the large, 60-kD subunit was prone to proteolysis in crude extracts, with a half-time of degradation at 4 degrees C (from 60 to 53 to 51 kD) on the order of minutes. The presence of high concentrations of protease inhibitors decreased, but did not prevent this proteolysis. The small, 51-kD subunit of barley endosperm AGP was relatively resistant to proteolysis, both in the presence or absence of protease inhibitors. For the crude, nonproteolyzed enzyme, 3-PGA acted as a weak activator of the ADP-glucose synthetic reaction (about 25% activation), whereas in the reverse reaction (pyrophosphorolysis) it served as an inhibitor rather than an activator. For both the synthetic and pyrophosphorolytic reactions, inorganic phosphate (Pi) acted as a weak competitive or mixed inhibitor of AGP. The relative insensitivity to 3-PGA/Pi regulation has been observed with both the nonproteolyzed crude enzyme and partially purified (over 60-fold) AGP, the latter characterized by two bands for the large subunit (molecular masses of 53 and 51 kD) and one band for the small subunit (51 kD). Addition of 3-PGA to assays of the partially purified, proteolyzed enzyme had little or no effect on the Km values of all substrates of AGP, but it reduced the Hill coefficient for ATP (from 2.1 to 1.0). These findings are discussed with respect to previous reports on the structure and regulation of higher plant AGP.

Adenosine Diphosphate Glucose↗

PubMed1993

Neurohumoral relations in neurological and psychiatric patients.

Sixteen patients-epileptics, alcoholics, psychotics and post-contusion cases-were subjected to neurological, psychiatric, psychological, morphological (X-ray, CT, NMR), physiological (EEG, polysomnography, evoked potentials), immunological and SPECT examination. Most had CT and SPECT signs of focal brain damage. The results were but exceptionally due to injury, most were of unknown etiology. This tallied with EEG focal abnormalities, mainly sleep disorders, sometimes subjective ones, but invariably detectable objectively. In each case there was REM, in some also NONREM sleep deficiency. Half of the cases were tested immunologically, each time with positive results: increased levels of acute-phase protein and stress-reaction proteins. As copious literature and our experience show, the above pathological condition, rather than being accidental, constitute regardless of etiology, an integrated entity which could be called "programme cerebropathy". The primary role is played there by the epileptic focus and its properties, in particular, the speed of discharge and localization and brain stem humoroergic systems, i.e., their performance and mutual balance. There is a program sequence of events, first, and epileptic focus acting on the neighborhood, the rise of secondary and tertiary foci, and influence over the thalamocortical system. Soon afterwards, epileptic activity begins to act on brain-stem structures. This results in changes in sleep, mood, psychic output, immunity, endosecretion, and in paroxysms.

Adult↗

PubMed1993

[The value of spiral-CT in primary and secondary liver neoplasms].

Sixty-one patients in whom primary or secondary malignomas of the liver were suspected on the basis of ultrasound examination were examined with spiral CT and sequential CT during i.v. administration of contrast medium. In four further cases spiral CT-angiography (spiral-CTA) was performed. Advantages and disadvantages of spiral volume CT are described.

Adult↗

PubMed1993

[Neuropsychological tests and smooth pursuit eye movements in schizophrenic twins].

The authors compares results of neuropsychological tests/Halstead-Reitan Neuropsychological Battery, Stroop test, Wechsler Memory Scale, Seashore Tonal Memory Test/with results of smooth pursuit eye movements in nine pairs of twins/5 monozygotic and 4 dizygotic pairs/. In all instances at least one twin suffered from schizophrenia. The majority of neuropsychological tests differentiated clearly schizophrenic and healthy twins. Of seven parameters of smooth pursuit eye movements big saccades differentiated significantly. The authors found also correlations between neuropsychological tests and smooth pursuit eye movements. Significant correlations pertained to motor velocity (correlation of the Oscillation test with big saccades and with small saccades), complex psychomotor abilities (correlation of the Tactual Performance Test-Localization with big saccades), general indicators of cerebral activity or neuropsychological performance (correlation of the Tonal Memory test with big saccades and correlation of the Rhythm test with the amplitude of the curve), memory (correlation of the memory quotient MQ and the Logical Memory sub-test with small saccades) and work performance during a perception or psychic load (correlation of Stroop sub-test with big saccades). The method of smooth pursuit eye movements is not time consuming and does not involve major demands on the patient's cooperation. On the other hand, neuropsychological tests have the advantage that they reveal the impact of possible pathological conditions of the brain on behaviour. On the whole the results support the hypothesis that in schizophrenic patients a disease with structural or functional abnormalities of the brain is involved.

Adult↗

PubMed1993

[Detection of cerebropathy in neurologic and psychiatric diseases].

The authors made a comprehensive examination of 16 patients--epileptics, alcoholics, psychotic subjects and patients after cerebral contusion. The patients were subjected to a neurological, psychiatric, psychological examination as well as to a morphological examination (X-ray, CT, NMR), physiological examination (EEG, polysomnography, evoked potentials), immunological examination and SPECT. The majority of patients had positive CT and SPECT findings suggesting focal brain damage. These results could be explained only in exceptional cases by injury, the majority was of unknown aetiology. With this corresponded focal EEG abnormalities and in particular sleep disorders, sometimes subjective but always detectable by objective methods. There was always a shortage of REM, sometimes also NONREM sleep. Half the patients were subjected to an immunological examination which was always positive and comprised elevated acute stage proteins and proteins associated with the stress reaction. Numerous data in the literature and the authors' experience indicate that the mentioned pathological findings are not incidental and form, independently on the aetiology, an integrated unit for which the term cerebropathy can be used. A primary role is played by the epileptic focus and its quality, i.e. above all the rate of discharge and site and humoroergic systems of the brain stem, in particular their efficiency and mutual balance. The events have a programmed sequence. At the beginning an epileptic focus develops which influences the surrounding area, secondary and tertiary foci are formed and the thalamo-cortical system is affected. Soon this is followed by an apparent influence of the epileptic activity on structures of the brain-stem. The consequence are changes affecting sleep, mood, mental performance, immunity, endosecretion and paroxysms. Subsequently individual symptoms are already prepared but have a different latency of manifestation and the latter depends also on external provoking influences. The thalamo-cortical reaction is characterized by the manifestation of epileptic paroxysms and sets in after a different interindividual incubation following injury. The same applies to the hippocampal reaction manifested by the organic psychosyndrome. Some symptoms such as changes of immunity, sleep or endosecretory function are not necessarily manifested if the influence of the focus on structures of the brain-stem is not sufficiently intense. Conversely if the effect on the brain-stem and limbic structures is greater and the effect on the thalamo-cortical system smaller, psychotiform behaviour develops. Then there are marked changes of phoria, dynamogeny, rate, affectivity, sleep and hormonal secretion and its equilibrium.

Adult↗

PubMed1993

[Epilepsy and intelligence. II. Correlation among 13 variables].

In a group of 215 ambulatory adult patients the authors assessed links with full-scale IQ, verbal IQ, performance IQ and different Wechsler-Bellevue sub-tests. They revealed marked correlations in particular with the frequency of grand mal seizures, invalidity and the onset of the disease. More important than the close correlation of IQ and education is the finding that an earlier onset of the disease markedly reduces the opportunities of good education. Less marked but still significant are correlations with the known aetiology of the disease, the duration of the disease, the frequency of other seizures than grand mal and the course of the disease. Dramatically increased correlations with the frequency of other seizures than grand mal were recorded in a sub-group of 77 men whose data on the disease can be considered valid (in WPSI, the Washington Psychosocial Seizure Inventory, values of scale L within the range of 0-3). No marked correlations were found with seizures in relatives. The position of bachelors is often adverse also as far as intellectual performance is concerned. Patients with a history of contact with psychiatry have lower intellectual performance. Patients who admit subjective needs of psychiatric or psychological assistance have a markedly better performance. It is probable that rehabilitation procedures which improve the collaboration and adherence to the therapeutic regime, in particular as regards taking prescribed drugs may promote also better cognitive performance.

Adult↗

PubMed1992

Molecular cloning and expression of the gene encoding ADP-glucose pyrophosphorylase from the cyanobacterium Anabaena sp. strain PCC 7120.

Previous studies have indicated that ADP-glucose pyrophosphorylase (ADPGlc PPase) from the cyanobacterium Anabaena sp. strain PCC 7120 is more similar to higher-plant than to enteric bacterial enzymes in antigenicity and allosteric properties. In this paper, we report the isolation of the Anabaena ADPGlc PPase gene and its expression in Escherichia coli. The gene we isolated from a genomic library utilizes GTG as the start codon and codes for a protein of 48,347 Da which is in agreement with the molecular mass determined by SDS-PAGE for the Anabaena enzyme. The deduced amino acid sequence is 63, 54, and 33% identical to the rice endosperm small subunit, maize endosperm large subunit, and the E. coli sequences, respectively. Southern analysis indicated that there is only one copy of this gene in the Anabaena genome. The cloned gene encodes an active ADPGlc PPase when expressed in an E. coli mutant strain AC70R1-504 which lacks endogenous activity of the enzyme. The recombinant enzyme is activated and inhibited primarily by 3-phosphoglycerate and Pi, respectively, as is the native Anabaena ADPGlc PPase. Immunological and other biochemical studies further confirmed the recombinant enzyme to be the Anabaena enzyme.

Amino Acid Sequence↗

PubMed1992

Comparison of proteins of ADP-glucose pyrophosphorylase from diverse sources.

The primary structures of 11 proteins of ADP-glucose pyrophosphorylase are aligned and compared for relationships among them. These comparisons indicate that many domains are retained in the proteins from both the enteric bacteria and the proteins from angiosperm plants. The proteins from angiosperm plants show two main groups, with one of the main groups demonstrating two subgroups. The two main groups of angiosperm plant proteins are based upon the two subunits of the enzyme, whereas the subgroups of the large subunit group are based upon the tissue in which the particular gene had been expressed. Additionally, the small subunit group shows a slight but distinct division into a grouping based upon whether the protein is from a monocot or dicot source. Previous structure-function studies with the Escherichia coli enzyme have identified regions of the primary structure associated with the substrate binding site, the allosteric activator binding site, and the allosteric inhibitor binding site. There is conservation of the primary structure of the polypeptides for the substrate binding site and the allosteric activator binding site. The nucleotide sequences of the coding regions of the genes of 11 of these proteins are compared for relationships among them. This analysis indicates that the protein for the small subunit has been subject to greater selective pressure to retain a particular primary structure. Also, the coding region of the precursor gene for the small subunit diverged from the coding region of the precursor gene for the large subunits slightly prior to the divergence of the two coding regions of the genes for the two tissue-specific large subunit genes.

Allosteric Site↗

PubMed1992

Evidence for an arginine residue at the allosteric sites of spinach leaf ADPglucose pyrophosphorylase.

The covalent modification of spinach leaf ADPglucose pyrophosphorylase leads to inactivation of both activator-stimulated and -unstimulated activity. Inactivation can be prevented if either the activator 3PGA or the inhibitor Pi are present during the modification. Pi proved to be more effective at protecting the enzyme from inactivation as it afforded 50% protection at 51 microM compared to 50% protection by 405 microM 3PGA. Partial modification of the enzyme using [14C]-phenylglyoxal leads to a decrease in both Vmax, A0.5 and a decrease in the ability of the 3PGA to stimulate the enzyme's activity. Modification increased the enzyme's susceptibility to inhibition by Pi and completely abolished the cooperative binding of Pi seen in the unmodified enzyme in the presence of 3PGA. Thus, phenylglyoxal appears to interfere, with the normal allosteric regulation of ADPglucose pyrophosphorylase from spinach leaf. Greater than 90% of the enzyme's activity is lost when 7.2 mol [14C]-phenylglyoxal are bound per mole of tetramer and this label is present in both the larger and small subunits. In addition, inactivation appears to involve two different arginine residues having different rates of modification.

Allosteric Site↗

PubMed1992

Involvement of arginine residues in the allosteric activation and inhibition of Synechocystis PCC 6803 ADPglucose pyrophosphorylase.

ADPglucose pyrophosphorylase (EC 2.7.7.27) from the cyanobacterium Synechocystis PCC 6803 was desensitized to the effects of allosteric ligands by treatment with the arginine reagent, phenylglyoxal. Enzyme modification by phenylglyoxal resulted in inactivation when the enzyme was assayed under 3P-glycerate-activated conditions. There was little loss of the catalytic activity assayed in the absence of activator. Pi, 3P-glycerate, and pyridoxal-P were able to protect the enzyme from inactivation, whereas substrates gave minimal protection. The protective effect exhibited by Pi and 3P-glycerate was dependent on effector concentration. MgCl2 enhanced the protection afforded by 3P-glycerate. The enzyme partially modified by phenylglyoxal was more resistant to 3P-glycerate activation and Pi inhibition than the unmodified form. Vmax at saturating 3P-glycerate concentrations and the apparent affinity of the enzyme toward Pi were decreased upon phenylglyoxal modification. Incorporation of labeled phenylglyoxal into the enzyme was proportional to the loss of activity. Pi and 3P-glycerate nearly completely prevented incorporation of the reagent to the protein. Results suggest that one arginine residue per mol of enzyme subunit is involved in the binding of allosteric effector in the cyanobacterial ADPglucose pyrophosphorylase.

Amino Acid Sequence↗

PubMed1992

Cloning, expression, and nucleotide sequence of glgC gene from an allosteric mutant of Escherichia coli B.

The Escherichia coli B mutant strain CL1136 accumulates glycogen at a 3.4- to 4-fold greater rate than the parent E. coli B strain and contains an ADPglucose synthetase with altered kinetic and allosteric properties. The enzyme from CL1136 is less dependent on the allosteric activator, fructose 1,6-bisphosphate, for activity and less sensitive to inhibition by AMP than the parent strain enzyme. The structural gene, glgC, for the allosteric mutant enzyme was selected by colony hybridization and cloned into the bacterial plasmid pBR322 by insertion of the chromosomal DNA at the PstI site. One recombinant plasmid, designated pKG3, was isolated from the genomic library of CL1136 containing glgC. The cloned ADPglucose synthetase from the mutant CL1136 was expressed and characterized with respect to kinetic and allosteric properties and found to be identical to the enzyme purified from the CL1136 strain. The mutant glgC was then subcloned into pUC118/119 for dideoxy sequencing of both strands. The mutant glgC sequence was found to differ from the wild-type at the deduced amino acid residue 67 where a single point mutation resulted in a change from arginine to cysteine.

Allosteric Regulation↗