Alterations In Protein Composition Research Articles

Cryopreservation of alfalfa (Medicago sativa L.) cells by encapsulation-dehydration

Our objective was to establish a cryopreservation protocol for alfalfa (Medicago sativa L.) cells and study the physiological changes occurring in cells during cryopreservation treatment. Cell cultures of Pioneer cvs. 5262 (fall-dormant, winter-hardy) and 5929 (non-dormant, non-hardy) plants initiated regrowth after cryopreservation by encapsulation-dehydration (ED). Pre-treatment of the encapsulated cells for 4 days in B5 medium containing 0.75 M sucrose and dehydration for 4 h in a laminar flow hood were necessary to achieve maximum cell viability after ED and cryopreservation in liquid N2 (EDN). Viability (measured as triphenyl tetrazolium chloride reduction) of the cv. 5262 cells after cryopreservation was two- to three-fold greater than that of the cv. 5929 cells. Cold acclimation of the cells (10 days at 2°C) improved viability after cryopreservation. The addition of 7.6 µM ABA to the B5 medium enhanced viability in ED but did not improve cell cryopreservability. Cold-acclimated cells had higher protein concentrations, but neither ABA nor cold acclimation influenced protein composition of cold-acclimated cells determined using SDS-PAGE. Encapsulated cells pre-treated for 4 days in B5 medium containing 0.75 M sucrose showed an increased concentration of cell protein and an altered protein composition. Suspension cultures were re-initiated from both ED and EDN treatments by transferring beads sequentially to B5 media containing 0.75, 0.5, 0.25 M sucrose and then to fresh B5 medium. The ED cells resumed rapid growth after two subcultures, whereas EDN cells needed four or five subcultures to resume rapid growth.

Male fertility defects in mice lacking the serine protease inhibitor protease nexin-1.

Understanding infertility and sterility requires knowledge of the molecular mechanisms underlying sexual reproduction. We have found that male mice deficient for the gene encoding the protease inhibitor protease nexin-1 (PN-1) show a marked impairment in fertility from the onset of sexual maturity. Absence of PN-1 results in altered semen protein composition, which leads to inadequate semen coagulation and deficient vaginal plug formation upon copulation. Progressive morphological changes of the seminal vesicles also are observed. Consistent with these findings, abnormal PN-1 expression was found in the semen of men displaying seminal dysfunction. The data demonstrate that the level of extracellular proteolytic activity is a critical element in controlling male fertility.

Kainate-induced seizures alter protein composition and N-methyl- d-aspartate receptor function of rat forebrain postsynaptic densities

The postsynaptic density is a highly dynamic structure, which is reorganized in an activity-dependent manner. An animal model for temporal lobe epilepsy, i.e. kainate-induced limbic seizures in rats, was used to study changes in postsynaptic density composition after extensive synaptic activity. Six hours after kainate injection, the protein content of the postsynaptic density fractions from rats that developed strong seizures was increased three-fold compared to saline-treated controls. Immunoblot analysis revealed that the relative amounts of metabotropic glutamate receptor 1α, N-ethylmaleimide-sensitive fusion protein, protein kinases C, Fyn and TrkB, as well as the neuronal nitric oxide synthase, were significantly higher in seizure-developing than in control rats. In contrast, the relative contents of the kainate receptor KA2 subunit, β-actin, α-adducin and the membrane-associated guanylate kinase homolog SAP90/PSD-95 were decreased. The relative amounts of additional postsynaptic density proteins, including α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate and N-methyl- d-aspartate receptor subunits, calcium/calmodulin-dependent kinase type II, casein kinase 2, tubulin, microtubule-associated protein 2B, the membrane-associated guanylate kinase homolog SAP102, and proline-rich synapse-associated protein 1/cortactin binding protein 1/Shank2 remained essentially unchanged. To assess possible changes in postsynaptic performance, postsynaptic densities were isolated from control and epileptic rats, incorporated into giant liposomes and N-methyl- d-aspartate receptor currents were recorded. A significant reduction in the mean conductance was observed in patches containing postsynaptic densities from animals with high seizure activity. This was due to the presence of reduced conductance levels in each membrane patch compared to control postsynaptic density preparations. From these data, we suggest that intense synaptic activity associated with seizures modifies the composition of postsynaptic densities and has profound consequences on the function of the N-methyl- d-aspartate receptors present in them. This rearrangement may accompany impairment of synaptic plasticity.

Free radicals alter maximal diaphragmatic mitochondrial oxygen consumption in endotoxin-induced sepsis

Recent studies indicate that sepsis is associated with enhanced generation of several free radical species (nitric oxide, superoxide, hydrogen peroxide) in skeletal muscle. While studies suggest that free radical generation causes uncoupling of oxidative phosphorylation in sepsis, no previous report has examined the role of free radicals in modulating skeletal muscle oxygen consumption during State 3 respiration or inhibiting the electron transport chain in sepsis. The purpose of the present study was to examine the effects of endotoxin-induced sepsis on State 3 diaphragm mitochondrial oxygen utilization and to determine if inhibitors/scavengers of various free radical species would protect against these effects. We also examined mitochondrial protein electrophoretic patterns to determine if observed endotoxin-related physiological derangements were accompanied by overt alterations in protein composition. Studies were performed on: (a) control animals, (b) endotoxin-treated animals, (c) animals given endotoxin plus PEG-SOD, a superoxide scavenger, (d) animals given endotoxin plus L-NAME, a nitric oxide synthase inhibitor, (e) animals given only PEG-SOD or L-NAME, (f) animals given endotoxin plus D-NAME, and (g) animals given endotoxin plus denatured PEG-SOD. We found: (a) no alteration in maximal State 3 mitochondrial oxygen consumption rate at 24 h after endotoxin administration, but (b) a significant reduction in oxygen consumption rate at 48 h after endotoxin, (c) no effect of endotoxin to induce uncoupling of oxidative phosphorylation, (d) either PEG-SOD or L-NAME (but neither denatured PEG-SOD nor D-NAME) prevented endotoxin-mediated reductions in State 3 respiration rates, (e) some mitochondrial proteins underwent tyrosine nitrosylation at 24 h after endotoxin administration, and (f) SDS-page electrophoresis of mitochondria from endotoxin-treated animals revealed a selective depletion of several proteins at 48 h after endotoxin administration (but not at 24 h); (g) administration of L-NAME or PEG-SOD prevented this protein depletion. These data provide the first evidence that endotoxin-induced reductions in State 3 mitochondrial oxygen consumption are free radical-mediated.

Alteration of protein composition in mouse thymocytes by signals through T-cell receptor.

To avoid destructive autoimmunity, T-cell precursors (thymocytes) expressing autoreactive T-cell receptor are deleted in the thymus via an apoptotic process by the signals from the T-cell receptor-CD3 complexes. In order to analyze the apoptotic mechanism, we established a cell-free system using the lysates from mouse thymocytes treated in vivo with anti-CD3 monoclonal antibody (mAb). The soluble cytosolic high molecular mass protein fraction from the anti-CD3-treated thymocytes revealed an activity that directly induces nuclear apoptotic morphological changes and DNA fragmentation. This fragmentation activity was not observed in the fraction from the thymocytes without anti-CD3 treatment. Proteins in both fractions were separated by two-dimensional electrophoresis. The silver-stained gels revealed differences in protein spots. These protein spots were identified by database searching of mass spectrometric (MS) and tandem mass spectrometric (MS/MS) data obtained from in-gel tryptic digests of the spots, using an integrated system of liquid chromatography/electrospray ionization/ion-trap mass spectrometry. In this study, the high mobility group protein HMG2 was identified as one of the cytosolic proteins that is increased by the signals from the T-cell receptor, and heterogeneous nuclear ribonucleoprotein A2/B1 and glyceraldehyde 3-phosphate dehydrogenase were found to be decreased by the signals.

Alteration of protein composition in mouse thymocytes by signals through T-cell receptor

Alteration of protein composition in mouse thymocytes by signals through T-cell receptor

Characterization of human lung cancer cells resistant to 4'-O-demethyl-4beta-(2"-nitro-4"-fluoroanilino)-4-desoxypodophyllotoxin , a unique compound in the epipodophyllotoxin antitumor class.

A new semi-synthetic podophyllotoxin derivative, 4'-O-demethyl-4beta-2"-nitro-4"-fluoroanilino)-4-desoxypo dophyllotoxin (compound 1), an analog of GL-331 (compound 2), is a potent and broad-spectrum inhibitor of cultured human cancer and drug-resistant cell growth. In general, 4'-demethylepipodophyllotoxin analogs, including 2, exert anti-tumor activity by targeting the nuclear enzyme DNA topoisomerase II, but 1 is not an enzyme inhibitor. Unlike the cytotoxic activity of compound 2, cell killing by 1 is dose-limiting and a significant fraction of cells (30-40%) survive treatment. As an approach to investigate mechanism of action, 1-resistant A549 (human lung cancer) sub-lines were selected and characterized. Results of the work show that 1-resistant cells: (i) are moderately cross-resistant (2- to 3-fold) to various cytotoxic drugs via a P-glycoprotein-independent mechanism, (ii) have an altered growth habit, (iii) are deficient in normal attachment on plastic and collagen substrata, and (iv) have an altered plasma membrane protein composition including several proteins in the 140->200 kDa molecular mass range and a doublet of phosphoserine-containing proteins of about 135 kDa. Since 1 treatment of cells affects neither cellular attachment or membrane-protein phosphorylation, the changes observed in 1-resistant cells are interpreted as a survival response to drug action.

A novel hnRNP protein (HAP/SAF-B) enters a subset of hnRNP complexes and relocates in nuclear granules in response to heat shock.

A two-hybrid screening in yeast for proteins interacting with the human hnRNP A1, yielded a nuclear protein of 917 amino acids that we termed hnRNP A1 associated protein (HAP). HAP contains an RNA binding domain (RBD) flanked by a negatively charged domain and by an S/K-R/E-rich region. In in vitro pull-down assays, HAP interacts with hnRNP A1, through its S/K-R/E-rich region, and with several other hnRNPs. HAP was found to be identical to the previously described Scaffold Attachment Factor B (SAF-B) and to HET, a transcriptional regulator of the Heat Shock Protein 27 gene. We show that HAP is a bona fide hnRNP protein, since anti-HAP antibodies immunoprecipitate from HeLa cell nucleoplasm the complete set of hnRNP proteins. Unlike most hnRNP proteins, the subnuclear distribution of HAP is profoundly modified in heat-shocked HeLa cells. Heat-shock treatment at 42 degrees C causes a transcription-dependent recruitment of HAP to a few large nuclear granules that exactly coincide with sites of accumulation of Heat Shock Factor 1 (HSF1). The recruitment of HAP to the granules is temporally delayed with respect to HSF1 and persists for a longer time during recovery at 37 degrees C. The hnRNP complexes immunoprecipitated from nucleoplasm of heat-shocked cells with anti-HAP antibodies have an altered protein composition with respect to canonical complexes. Altogether our results suggest an involvement of HAP in the cellular response to heat shock, possibly at the RNA metabolism level.

Two forms of sustained xanthophyll cycle‐dependent energy dissipation in overwintering Euonymus kiautschovicus

Seasonal differences in PSII efficiency (Fv/Fm), the conversion state of the xanthophyll cycle (Z + A)/ (V + A + Z), and leaf adenylate status were investigated in Euonymus kiautschovicus. On very cold days in winter, Fv/Fm assessed directly in the field remained low and Z + A high throughout day and night in both sun and shade leaves. Pre‐dawn transfer of leaves from subfreezing temperatures in the field to room temperature revealed that recovery (increases in Fv/Fm and conversion of Z + A to violaxanthin) consisted of one, rapid phase in shade leaves, whereas in sun leaves a rapid phase was followed by a slow phase requiring days. The pre‐dawn ATP/ADP ratio, as well as that determined at midday, was similar when comparing overwintering leaves with those sampled in the summer, although pre‐dawn levels of ATP + ADP were elevated in all leaves during winter relative to summer. After a natural transition to warmer days during the winter, pre‐dawn Fv/Fm and Z + A in shade leaves had returned to values typical for summer, whereas in sun leaves Fv/Fm and Z + A levels remained intermediate between the cold day in winter and the summer day. Thus two distinct forms of sustained (Z + A)‐dependent energy dissipation were identified based upon their differing characteristics. The form that was sustained on cold days but relaxed rapidly upon warming occurred in all leaves and may result from maintenance of a low lumenal pH responsible for the nocturnal engagement of (Z + A)‐dependent thermal dissipation exclusively on very cold days in the winter. The form that was sustained even upon warming and correlated with slow Z + A to violaxanthin conversion occurred only in sun leaves and may represent a sustained engagement of (Z + A)‐dependent energy dissipation associated with an altered PSII protein composition. In the latter, warm‐sustained form, uncoupler or cycloheximide infiltration had no effect on the slow phase of recovery, but lincomycin infiltration inhibited the slow increase in Fv/Fm and the conversion of Z + A to violaxanthin.

Regulation of surfactant proteins A and B by TNF-alpha and phorbol ester independent of NF-kappa B.

Acute lung inflammation is complicated by altered pulmonary surfactant phospholipid and protein composition. The proinflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) and the phorbol ester 12-O-tetradecanoyl phorbol-13-acetate (TPA) inhibit expression of surfactant-associated proteins A and B (SP-A and SP-B), both important for normal surfactant function. The transcription factor nuclear factor-kappa B (NF-kappa B) frequently mediates regulation of gene expression by TPA and TNF-alpha. In the present study, electrophoretic mobility shift assays (EM-SAs) and pyrrolidine dithiocarbamate (PDTC), an inhibitor of NF-kappa B activation, were utilized to determine the role of NF-kappa B activation in TPA and TNF-alpha inhibition of the surfactant proteins in NCI-H441 cells. Pentoxifylline (PTX), which inhibits TNF-alpha cellular effects without preventing NF-kappa B activation, was also tested. By EMSA, TPA and TNF-alpha increased nuclear NF-kappa B binding activity in temporally distinct patterns. PDTC decreased TPA- and TNF-alpha-induced NF-kappa B binding activity but did not limit their inhibition of SP-A and SP-B mRNAs. PDTC independently decreased both SP-A and SP-B mRNAs. PTX partially reversed TNF-alpha-but not TPA-mediated inhibition of SP-A and SP-B mRNAs without altering NF-kappa B binding. The effects of PDTC and PTX on NF-kappa B and the surfactant proteins suggest that NF-kappa B activation does not mediate TPA or TNF-alpha inhibition of SP-A and SP-B mRNA accumulation.

Apoptosis in the lacrimal gland and conjunctiva of dry eye dogs.

Keratoconjunctivitis sicca (KCS), or dry eye syndrome, is characterized by the development of ocular surface damage. KCS can occur as an individual phenomenon, or in association (as a secondary phenomenon) with various types of systemic autoimmune disorders. Sjogren’s syndrome (SS), with the signature symptoms of dry eye and dry mouth, has the strongest association with KCS. The most prominent characteristic of SS is a progressive, follicular, lymphoid infiltration found in biopsy specimens of the lacrimal and salivary glands. These infiltrates consist of primarily CD4+ T cells and B cells demonstrated by immunohistological staining. Functional studies have shown that this type of impairment of the lacrimal gland results in decreased tear secretion and altered tear protein composition.1

Altering protein composition by genetically removing phaseolin from common bean seeds containing arcelin or phytohemagglutinin

Arcelin seed proteins of common bean (Phaseolus vulgaris L.) confer resistance to bruchid pests, and in vitro results suggested that greater resistance could be achieved by increasing the concentration of arcelin. We created backcross lines having arcelin alleles (SMARC lines), or alleles for the related protein phytohemagglutinin (PHA) (SMPHA lines), and a null allele for phaseolin to determine if seeds lacking phaseolin would contain increased quantities of arcelin or PHA proteins. To test the affects of genetically removing phaseolin, SMARC and SMPHA lines were derived as pairs of phaseolin-containing and phaseolin-null lines. Parental, SMARC, and SMPHA lines were grown in a replicated greenhouse trial and measured for days-to-flower, days-to-maturity, seed weight, and for quantities of phaseolin, arcelin dimer, PHA, and total proteins. There were no differences between pairs of phaseolin and phaseolin-null lines for days-to-flower, seed weight or total protein, and inconsistent differences for days-to-maturity. Arcelin concentrations were significantly increased in two of four pairs of SMARC lines, and PHA concentration was significantly greater in four of five pairs of SMPHA lines. These or other changes in the seed protein composition in phaseolin null lines may improve resistance to bruchids.

Fructosamine measurement in ponies: validation and response following experimental cyathostome infection

Validation of an assay for measurement of fructosamine in equine serum and plasma utilised blood samples collected from 24 British native breed ponies. The results indicated that fructosamine can be measured easily using an assay which is precise and accurate. Paired plasma and serum fructosamine measurements were highly correlated, however, greater variations were observed within serum compared with those in plasma. A reference range for fructosamine in plasma was calculated to be 256·9±60·6 μmol litre −1 (mean±2 SD). In order to assess the fructosamine response following experimental cyathostome infection, nine British native breed ponies were allocated to one of three groups: Group 1 (3·9 million third-stage cyathostome larvae (L 3) over a nine-week-period), Group 2 (3·15 million L 3 over seven weeks) and Group 3 (uninfected controls). From four weeks prior to infection, blood was obtained, once weekly, from all animals for measurement of plasma fructosamine, plasma albumin and serum globulin concentrations. Plasma fructosamine concentrations decreased in all infected ponies post-infection due to enteric protein loss and/or altered protein composition and/or increased protein turnover.

Fibrin degradation products in post mortem brain tissue of schizophrenics: a possible marker for underlying inflammatory processes

Alterations in protein composition are often present in diseases of the central nervous system (CNS). Inflammatory processes causing these alterations have been suggested by many authors to underlie the cerebral disturbance in some cases of schizophrenia. In a previous study, two disease-associated additional polypeptides P1 and P2 could be detected in the cerebrospinal fluid (CSF) of 60 out of 123 (49%) schizophrenic patients. These polypeptides were identified as cleavage products of the β-chain of fibrin(ogen), an acute-phase protein which is increased in inflammatory processes. Because investigations of brain tissue might better reflect the disease process than cerebrospinal fluid, we examined 6 post mortem brains (3 schizophrenics, 3 normal controls) for the presence of fibrin(ogen) and its cleavage products by two dimensional gel electrophoresis followed by immunoblotting. We detected fibrin(ogen) and its degradation products P1 and P2 in all schizophrenic brain tissue samples, but not in that from controls. These results are consistent with previous CSF observations and suggest that a CNS inflammatory process may be occurring in a subgroup of schizophrenic patients.

Dormant/unculturable cells of the fish pathogen Aeromonas salmonicida

Viable cells of Aeromonas salmonicida remained in experimental marine systems after plate counts indicated an absence of culturable cells. These so-called viable but nonculturable (VBNC) cells were coccoid and smaller than their normal culturable counterparts. There was no reduction in lipopolysaccharide of the VBNC cells. There was an alteration in protein composition, however, with a decline in some (15, 70, 30, 22, and 17 kDa), but an increase in another protein (49 kDa). A significant loss of DNA occurred. The VBNC cells responded to fluorescent antibodies prepared against A. salmonicida by developing enlarged and bizarre shapes in the presence of yeast extract and nalidixic acid (the direct viable count technique), and they demonstrated respiratory activity. It was concluded that A. salmonicida survived in seawater, but major morphological changes occurred with cells retaining some viability but losing pathogenicity to Atlantic salmon (Salmo salar).

'Sheltered disruption' of Neurospora crassa MOM22, an essential component of the mitochondrial protein import complex.

MOM22 is a component of the protein import complex of the mitochondrial outer membrane of Neurospora crassa. Using the newly developed procedure of 'sheltered disruption', we created a heterokaryotic strain harboring two nuclei, one with a null allele of the mom-22 gene and the other with a wild-type allele. Homokaryons bearing the mom-22 disruption could not be isolated, suggesting that mom-22 is an essential gene. The mutant nucleus can be forced to predominate in the heterokaryon through the use of specific nutritional and inhibitor resistance markers. Cultivation of the heterokaryon under conditions favoring the mutant nucleus resulted in selective depletion of MOM22. MOM22-depleted cells did not grow and contained mitochondria with an altered morphology and protein composition. Protein import into isolated, MOM22-depleted mitochondria was abolished for most precursor proteins destined for all subcompartments. In contrast, precursors of MOM19, MOM22 and MOM72 became inserted normally into the outer membrane, defining a novel MOM22-independent import pathway which remained intact in mutant mitochondria. Furthermore, the specific binding of the ADP/ATP carrier to the outer membrane was unaffected, but subsequent transport across the outer membrane did not occur. Our data show that MOM22 is an essential component of Neurospora cells specifically required for the biogenesis of mitochondria.

Potential mechanisms of the age-related changes in the blood-brain barrier

A variety of age-related changes in the blood-brain barrier transport processes have been identified. These include reduced hexose and butyrate transport, reduced choline transport, reduced triiodothyronine transport without a change in the transport of neutral and basic amino acids. The potential mechanisms underlying these age-related changes include hemodynamic alterations in the cerebral circulation of aged rats, notably increased occurrence of arteriovenous shunting. Additional age-related changes in cerebral microvessels include alterations in protein composition, and increased accumulation of lipid peroxidation byproducts, along with changes in membrane fluidity of isolated cerebral microvessels. In addition, neurotransmitter activity notably beta adrenergic neurotransmission, is significantly reduced in cerebral microvessels of aged rats. These alterations taken together may account for some of the age-related changes in the blood-brain barrier.

Effects of chitosan andaspergillus flawson isozymes related to phenouc compound synthesis and protein profiles of peanut seeds

Abstract Changes in isozymes and peanut protein molecular weights were determined in mature peanuts treated with chitosan and/or Aspergillus flavus. Enzymes involved with the synthesis of phenolic compounds were analyzed. Cinnamyl alcohol dehydrogenase (CAD), glutamate dehydrogenase (GDH), and glucose‐6‐phosphate dehydrogenase (G6PDH) were resolved by native‐PAGE using gradient gels containing 8–25% polyacrylamide while polyphenoloxidases (PPO) were resolved with 10–15% polyacrylamide. Anodic peroxidase (PRX) and shikimate dehydrogenase (SKD) were detected by IEF‐PAGE (pi range of 4.0–6.5). Chitosan induced enzyme activities of PPO (Rf=0.34) and SKD (pl=5.22; pl=4.85) in viable seeds while A. flavus enhanced activities of G6PDH (Rf=0.44), GDH (Rf=0.14), PRX (pl=4.00), PPO (Rf=0.27), and SKD (pl=4.56; pl=4.27). Chitosan + A. flavus promoted GDH (Rf=0.14; Rf=0.20; Rf=0.25) and SKD (pl=4.56; pl=4.04) activities. SKD with five isozyme bands was a better marker for comparing the effects of elicitors. Chitosan and chitosan + A. flavus treated seeds did not have any protein molecular weight changes over 72 h. By 48 h after inoculation of mature seeds with A. flavus, an early change in protein composition of moderately susceptible cultivar of peanuts, Starr, was detected by non‐denaturing gel electrophoresis. Resistant cultivar J‐11 exhibited a delay in alteration of protein composition until 72 h. Manipulation of specific enzyme activities could help elucidate biochemical mechanisms in elicitor‐host interaction and control contamination by aflatoxin‐producing fungi.

Characterization of HIV replication complexes early after cell-to-cell infection.

In this study, we have characterized the HIV DNA-containing replication complexes present in cells early after cell-to-cell infection, using sucrose gradient sedimentation and immunoprecipitation. Six hours after cell-to-cell infection, a cytoplasmic HIV replication complex sedimented as a large structure (320S). This replication complex was precipitated by antisera to three virus-coded enzymes (reverse transcriptase, integrase, protease), to the matrix protein (p17), and to cellular histones but not to the major capsid protein (p24). This replication complex was not associated with cell membranes and could not be dissociated into smaller discrete subunits, using detergents. Nuclear extracts from the same cell-to-cell infection contained a smaller (80S) complex that lacked reverse transcriptase and matrix protein (p17). Cytoplasmic replication complexes from a cell-free virus infection sedimented as 160S structures under identical conditions, as previously reported. Our results indicate that, following cell-to-cell transmission of HIV, all the HIV pol gene products, the matrix protein p17, and cellular histones are present in cytoplasmic replication complexes that are taking part in or have completed reverse transcription. Transportation of the cytoplasmic replication complex to the nucleus is associated with structural changes, including a reduction in size and altered protein composition.

Sulphonate buffers affect the recovery of microtubule-associated proteins MAP1 and MAP2: evidence that MAP1A promotes microtubule assembly.

The influence of two commonly used sulphonate buffers, PIPES and MES, on the in vitro assembly of bovine brain microtubule protein was examined. Microtubule assembly was monitored by turbimetry and, after centrifugation, the polymerised protein was analysed by SDS-PAGE and western blotting. Assembly in MES when compared with PIPES resulted in a higher recovery of microtubule proteins at both pH 6.4 and pH 6.9 and in an altered protein composition. The buffer pH affected the total amount of protein polymerised but did not significantly affect the protein composition. At both pH conditions the recovery of HMW-MAPs was markedly increased in MES buffer and this increase was mostly due to an increase in the amount of MAP1.