Glyco-engineering of moss lacking plant-specific sugar residues.

The commercial production of complex pharmaceutical proteins from human origin in plants is currently limited through differences in protein N-glycosylation pattern between plants and humans. On the one hand, plant-specific alpha(1,3)-fucose and beta(1,2)-xylose residues were shown to bear strong immunogenic potential. On the other hand, terminal beta(1,4)-galactose, a sugar common on N-glycans of pharmaceutically relevant proteins, e.g., antibodies, is missing in plant N-glycan structures. For safe and flexible production of pharmaceutical proteins, the humanisation of plant protein N-glycosylation is essential. Here, we present an approach that combines avoidance of plant-specific and introduction of human glycan structures. Transgenic strains of the moss Physcomitrella patens were created in which the alpha(1,3)-fucosyltransferase and beta(1,2)-xylosyltransferase genes were knocked out by targeted insertion of the human beta(1,4)-galactosyltransferase coding sequence in both of the plant genes (knockin). The transgenics lacked alpha(1,3)-fucose and beta(1,2)-xylose residues, whereas beta(1,4)-galactose residues appeared on protein N-glycans. Despite these significant biochemical changes, the plants did not differ from wild type with regard to overall morphology under standard cultivation conditions. Furthermore, the glyco-engineered plants secreted a transiently expressed recombinant human protein, the vascular endothelial growth factor, in the same concentration as unmodified moss, indicating that the performed changes in glycosylation did not impair the secretory pathway of the moss. The combined knockout/knockin approach presented here, leads to a new generation of engineered moss and towards the safe and flexible production of correctly processed pharmaceutical proteins with humanised N-glycosylation profiles.

DNA-interactions and nuclear localisation of the chromosomal HMG domain protein SSRP1 from maize.

The structure-specific recognition protein 1 (SSRP1) is a member of the protein family containing a high mobility group (HMG) domain DNA-binding motif. We have functionally characterised the 71.4 kDa Zm-SSRP1 protein from maize. The chromatin-associated Zm-SSRP1 is detected by immunoblot analysis in maize leaves, kernels and suspension culture cells, but not in roots. Mediated by its HMG domain, recombinant Zm-SSRP1 interacts structure-specifically with supercoiled DNA and DNA minicircles when compared with linear DNA. In linear duplex DNA, the protein does not recognise a specific sequence, but it binds preferentially to sequences containing the deformable dinucleotide TG, as demonstrated by a random oligonucleotide selection experiment. Zm-SSRP1 modulates DNA structure by bending the target sequence, since it promotes the circularisation of short DNA fragments in the presence of DNA ligase. Moreover, Zm-SSRP1 facilitates the formation of nucleoprotein structures, as measured using the bacterial site-specific beta-mediated recombination reaction. Analysis of the subcellular localisation of various SSRP1-GFP fusions revealed that, in contrast to HMG domain transcription factors, the nuclear localisation sequence of Zm-SSRP1 is situated within a 20-amino acid residue region adjacent to the HMG domain rather than within the DNA-binding domain. The results are discussed in the context of the likely function of SSRP1 proteins in transcription and replication.

Occurrence of five different chromosomal HMG1 proteins in various maize tissues.

The nuclear HMG1 proteins of higher plants are small non-histone proteins that have DNA-bending activity and are considered architectural factors in chromatin. The occurrence of the chromosomal HMG1 proteins, HMGa, HMGc1/2 and HMGd, in various maize tissues was analyzed, and in the course of these studies a novel HMG1 protein, now termed HMGe, was identified. Purification and characterization of HMGe (M(r) 13,655) and cloning of the corresponding cDNA revealed that it displays only moderate similarity to other members of the plant HMG1 protein family. The five maize HMG1 proteins could be detected in kernels, leaves, roots and suspension culture cells, indicating that these proteins can be expressed simultaneously and occur relatively ubiquitously. However, the various HMG1 proteins are present in significantly different quantities with HMGa and HMGc1/2 being the most abundant HMG1 proteins in all tissues tested. Furthermore, the relative amounts of the various HMG1 proteins differ among the tissues examined. The HMG1 proteins were found to be relatively stable proteins in vivo, with HMGc1/2, HMGd and HMGe having a half-life of ca. 50 h in cultured cells, while the half-life of the HMGa protein is ca. 65 h. Collectively, these findings are compatible with the concept that the different plant HMG1 proteins might act as general architectural proteins in concert with site-specific factors in the assembly of certain nucleoprotein structures involved in various biological processes.

Structure of genes encoding chromosomal HMG1 proteins from maize.

The high mobility group (HMG) proteins of the HMG1 family are architectural proteins in chromatin that are considered to facilitate the formation of complex nucleoprotein structures in various biological processes such as transcription and recombination. Plants express a variety of these non-sequence-specific DNA-bending proteins. The sequences encoding the maize HMGa and HMGc1 proteins were isolated from a genomic DNA library. Determination of the nucleotide sequences of these genes revealed that the coding region of both genes has a similar genomic structure, comprising seven exons and six introns. The positioning of the introns is conserved between the two genes, whereas the number of introns and their positions are entirely different in the related animal genes. In the 5' flanking region of the hmgc1 gene, a copia-like retrotransposon was identified. In addition to the genes encoding HMGa and HMGc1, several genomic fragments (retropseudo gene, fragments of the genes) were isolated and characterised.

Protection against lymphocytic choriomeningitis virus infection induced by a reduced peptide bond analogue of the H-2Db-restricted CD8(+) T cell epitope GP33.

Recent investigations have suggested that pseudopeptides containing modified peptide bonds might advantageously replace natural peptides in therapeutic strategies. We have generated eight reduced peptide bond Psi(CH2-NH) analogues corresponding to the H-2Db-restricted CD8(+) T cell epitope (called GP33) of the glycoprotein of the lymphocytic choriomeningitis virus. One of these pseudopeptides, containing a reduced peptide bond between residues 6 and 7 (Psi(6-7)), displayed very similar properties of binding to major histocompatibility complex (MHC) and recognition by T cell receptor transgenic T cells specific for GP33 when compared with the parent peptide. We assessed in vitro and in vivo the proteolytic resistance of GP33 and Psi(6-7) and analyzed its contribution to the priming properties of these peptides. The Psi(6-7) analogue exhibited a dramatically increased proteolytic resistance when compared with GP33, and we show for the first time that MHC-peptide complexes formed in vivo with a pseudopeptide display a sustained half-life compared with the complexes formed with the natural peptide. Furthermore, in contrast to immunizations with GP33, three injections of Psi(6-7) in saline induced significant antiviral protection in mice. The enhanced ability of Psi(6-7) to induce antiviral protection may result from the higher stability of the analogue and/or of the MHC-analogue complexes.

Tumor-infiltrating lymphocytes exhibiting high ex vivo cytolytic activity fail to prevent murine melanoma tumor growth in vivo.

The identification of tumor-associated Ags recognized by CD8+ CTL and prevention of tumor outgrowth by adoptive transfer of these CTL demonstrates that CD8+ T cells play a major role in antitumor immunity. We have generated B16.F10 melanoma cells that express the glycoprotein epitope amino acid 33-41 (GP33) of the lymphocytic choriomeningitis virus (LCMV) to examine antitumor CD8+ T cell response in C57BL/6 mice immune to LCMV and in mice transgenic for the LCMV GP33-specific P14 TCR (P14 TCR mice). We find that B16.F10GP33 tumor cells grew in syngeneic C57BL/6 mice without inducing T cell tolerance. LCMV infection or adoptive transfer of LCMV-specific effector T cells delayed but did not prevent growth of preestablished tumors in these mice. However, B16.F10GP33 tumor cells were rejected in mice immune to LCMV and in mice treated with LCMV-specific effector T cells on the same day as the tumor. Surprisingly, B16.F10GP33 tumor cells grew in P14 TCR transgenic mice despite an abundance of tumor-associated Ag-specific CD8+ T cells. In these mice, freshly isolated tumor-infiltrating lymphocytes exhibited an activated phenotype and displayed high GP33-specific cytolytic activity when assessed ex vivo. Thus, B16.F10GP33 melanoma cells are able to initiate, but not to sustain, a GP33-specific CTL response sufficient to clear the tumor enduringly.

De novo minimal change disease.

Beyond the acute posttransplantation period, glomerular causes of proteinuria in the renal allograft include recurrent glomerulopathy, transplant-associated entities, and de novo disease. We present a case of de novo minimal change disease with reversible acute renal failure occurring 2.5 years posttransplantation in a 56-year-old man. The cause of end-stage renal disease in the native kidney was membranous glomerulopathy. De novo minimal change disease in the renal allograft is an extremely rare entity requiring stringent clinical-pathological criteria for diagnosis. Many of the cases previously reported as de novo minimal change disease fail to meet these criteria. We review the eight reported cases that appear to fulfill a strict definition of minimal change disease in the context of the current report.

Mapping of linear histone regions exposed at the surface of the nucleosome in solution.

Antibodies directed against defined regions of histone molecules represent one of the most specific probes for studying the topography and conformational changes of nucleosomes and chromatin. We have developed an assay involving a series of monoclonal and polyclonal antibody probes specifically reacting with a complete set of 40 overlapping synthetic peptides (6 to 28 residues long) covering the whole sequence of the four core histones H2A, H2B, H3 and H4. In this assay, mono-, di- and trinucleosomes, as well as a long chain of chromatin containing 20 to 35 nucleosomes, were used in solution as competitors of the antibody reaction. At least 11 surface-oriented linear regions were characterized on the mononucleosome; namely, the N-terminal domains of H2A (residues 1 to 20), H2B (residues 1 to 25) and H3 (residues 1 to 30), the C-terminal domains of H2A (residues 116 to 129) and H4 (residues 85 to 102), and six domains located in internal segments in the primary structures of core histones (33 to 49 H2A, 65 to 85 H2A, 60 to 78 H2B, 50 to 70 H3, 111 to 130 H3 and 42 to 59 H4). Only a few changes in the nucleosome topography were observed when free oligo- and polynucleosome structure were comparatively studied.

Induction of anti-polycation antibodies in H-2s mice by immunization with nuclear antigens.

Following administration of certain chemicals (heavy metals or lupus-inducing drugs), H-2s mice produce autoantibodies reacting with various nuclear antigens such as fibrillarin in the nucleolus and histones in chromatin. In the present study, we have immunized A.SW (H-2s) mice and their congenic counterparts A.BY (H-2b) mice with bovine thymus nuclei in Freund's adjuvant. As was previously observed with lupus-prone mice, such active immunization did not elicit antinuclear antibodies in any of the experimental groups. Surprisingly, the A.SW immunized with nuclei in adjuvant developed high titers of IgG antibodies that reacted exclusively with synthetic polycations. We obtained several monoclonal IgG antibodies from these mice and verified that these polycation-reactive antibodies were not directed against a specific nuclear antigen. The genetic analysis of the monoclonal antibodies further confirmed their clonal diversity. The mechanisms leading to the appearance of antibodies reactive with highly basic molecules in A.SW mice may be related to their predisposition to produce autoantibodies to cationic nuclear antigens (fibrillarin, histones) during chemically-induced autoimmunity.

Variability in Arabidopsis thaliana chromosomal high-mobility-group-1-like proteins.

The vertebrate high-mobility-group (HMG) protein HMG1 is an abundant non-histone protein which is considered as an architectural element in chromatin. In the monocotyledonous plant maize, four different HMG1-like proteins (HMGa, HMGc1/2, HMGd) have been identified, whereas other eukaryotes usually express only two different proteins of this type. We have examined here the HMG1-like proteins of the dicotyledonous plant Arabidopsis thaliana. The isolation and analysis of cDNAs encoding five different so far uncharacterised HMG1-like proteins (now termed HMG alpha, HMG beta1/2, HMG gamma, HMG delta) from Arabidopsis indicates that the expression of multiple HMG1-like proteins is a general feature of (higher) plants. The Arabidopsis HMG1-like proteins contain an HMG domain as a common feature, but outside this conserved DNA-binding motif the amino acid sequences are significantly different indicating that this protein family displays a greater structural variability in plants than in other eukaryotes. The five HMG1-like proteins were expressed in Escherichia coli and purified. They bind with somewhat different affinity to linear double-stranded DNA. The recognition of DNA structure is evident from their preferential interaction with DNA minicircles relative to linear DNA. Reverse-transcribed PCR suggested that the five HMG1-like genes are simultaneously expressed in Arabidopsis leaves and suspension culture cells.

Specificity of monoclonal anti-nucleosome auto-antibodies derived from lupus mice.

Recently, anti-nucleosome antibodies, which do not bind to DNA or to individual histones, have been identified in longitudinal studies in lupus mice. These anti-nucleosome antibodies occur early in spontaneous SLE and are formed prior to other anti-nuclear specificities. However, nucleosomal epitopes are yet to be fully characterized. We selected a panel of six monoclonal anti-nucleosome antibodies (mAbs) (#2, #32, #34, PL2-6, LG8-1 and LG10-1) derived from lupus mice. These mAbs were tested in ELISA on subnucleosome structures and on a panel of 53 histone peptides, covering the entire sequence of the five histones. Two mAbs reacted with one of these peptides, but the reactivity hardly exceeded the background reactivity. Based on the nucleosome and subnucleosome ELISA we identified different recognition patterns. Three mAbs showed the highest reactivity towards the intact nucleosome. For two of them (#32 and LG8-1) the nucleosomal epitope was primarily located on H2A-H2B/DNA, whereas for mAb #34 this primary epitope was located on H3/H4/DNA. Two mAbs (#2 and PL2-6) showed the highest reactivity with H2A-H2B/DNA and one mAb (LG10-1) recognized H3-H4/DNA. In the subnucleosome ELISA all but one (mAb #32) recognized more than one epitope, including DNA complexed to a variety of cationic molecules. Comparing these reactivities we identified for all mAbs one specific nucleosomal epitope, whereas reactivity with other subnucleosomes was comparable to the reactivity towards DNA complexed with cationic molecules. In inhibition experiments both in ELISA and in immunofluorescence it was found that only one of the mAbs (i.e. PL2-6), recognizing an epitope on H2A-H2B/DNA as primary epitope, could be inhibited by H2A-H2B/DNA in fluid phase. The two mAbs recognizing an epitope on H3-H4/DNA as primary epitope could be inhibited by H3-H4/DNA in fluid phase. From these analyses, we conclude first that for these nucleosome specific mAbs linear histone peptides are not very important. Second, that these mAbs all recognize different epitopes on both H2A/H2B-DNA and H3/H4-DNA and third that some solid phase H2A/H2B-DNA epitopes are not expressed on fluid phase H2A/H2B-DNA. Our findings suggest that in SLE the nucleosome can act as auto-antigen and that there is no immunodominant beta cell epitope within the nucleosome.

Dual reactivity of several monoclonal anti-nucleosome autoantibodies for double-stranded DNA and a short segment of histone H3.

We have shown previously that four IgG monoclonal autoantibodies (mAbs) reacted in ELISA with both double-stranded (ds) DNA and peptide 83-100 of histone H3. The peptide 83-100 contains a cysteine residue at position 96 and readily dimerizes at pH 7-8. We describe here that only the 83-100 dimers, and not the 83-100 monomers, are recognized by the four antibodies and inhibit in ELISA the binding of mAbs to dsDNA. The equilibrium affinity constants (Ka) and kinetic rate constants of two of these mAbs were measured in a biosensor system. Ka values were significantly higher when these mAbs were tested with dsDNA as compared with the 83-100 dimer. Further higher Ka values were measured with mononucleosomes containing DNA and histones. It is proposed that these four mAbs are directed against a topographic determinant formed by DNA and the region 83-100 of H3 present as a dimer at the surface of nucleosome, and that they react, although significantly less well, with DNA and peptide dimer tested separately. This study provides a quantitative and kinetic basis to interaction between several antibodies and distinct antigenic structures and allows us to better understand the structural basis of apparent autoantibody cross-reactivity.

Mapping of B-cell epitopes recognized by antibodies to histones in subsets of juvenile chronic arthritis.

The sera of 138 patients with juvenile chronic arthritis (JCA) were tested in ELISA with the five individual histones, 34 histone peptides covering the full length of the four core histones, and two peptides corresponding to the N- and C-terminal domains of H1. The occurrence of IgG antibodies was examined regarding the different subsets of JCA (pauciarticular, polyarticular, and systemic onset) and regarding clinical features (chronic anterior uveitis, CAU) and other serological features (antinuclear antibodies, ANA). Seventy-two percent of the 138 sera reacted with at least 1 histone peptide. The peptides 204-218 of H1, 1-25 of H2B, and 111-130 of H3 were recognized by 22-28% of JCA sera, and 42% of JCA sera reacted with one or both peptides 1-25 of H2B and 111-130 of H3. The frequency of occurrence of anti-histone antibodies (AHA) regarding the type of histone fraction (H1, H2A, H2B, H3 and H4) or the regions of histones was not significantly different in the three subsets of JCA. No obvious association was found between IgG antibodies to histone peptides and uveitis. In the subset of pauciarticular JCA, 13/31 patients (41.9%) with CAU against 14/41 patients (34.1%) without CAU possessed IgG antibodies reacting with peptides of the C-terminal domain 83-135 of H3. This difference is not statistically significant. Finally, the presence of antibodies to histones and histone peptides cannot completely explain ANA reactivity found in patients' sera. Although antibodies to histone peptides occur frequently in the serum of children with JCA, the antibody profiles seem to be highly individual and do not correlate with disease subtype or activity. Identification of AHA present not only in the circulation but also in tissue deposits may provide better insight into the identification of pathogenic antibodies.

Mapping of linear epitopes of human histone H1 recognized by rabbit anti-H1/H5 antisera and antibodies from autoimmune patients.

Seventeen synthetic peptides of 15-16 residues, covering the complete sequence of the major human H1b variant, were tested for their capacity to bind serum IgG antibodies from 128 patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and Sjögren's syndrome (pSS). One peptide (residues 111-127) of the human H1 degree variant and six synthetic and natural fragments of H5 were also tested. Results were compared to those obtained with antibodies from 11 rabbits immunized against chicken H1 and H5, and calf H1. The activity of peptides was tested in direct ELISA and in inhibition assays with free peptides in solution. A major epitope recognized by antibodies from SLE, RA and pSS patients as well as by rabbit antibodies was identified in the C-terminus of H1b (residues 204-218). Other peptides in the globular (residues 79-94) and C-terminal domains of H1b and peptide 111-127 of H1 degree were also recognized, albeit at a lower level and frequency, and some of them contain sequence homologies with peptide 204-218. Patients' antibodies and rabbit antisera were tested with complete H1 proteins from HeLa cells, calf thymus and chicken erythrocytes and with chicken H5. Less than 25% of autoimmune sera contained IgG antibodies reacting with H1/H5 in a direct ELISA. In dot-immunoassay, antigenic activity with intact H1/H5 proteins was detected in a larger number of sera. Using antibodies raised in rabbits against peptides 1-16 and 204-218 of H1b, we found no reaction with H1 immobilized on a solid-phase. In contrast, peptides 144-159, 170-185 and 204-218, which contain identical structural domains, compete with H1 in solution indicating that any of these three regions are accessible at the surface of free H1 and may be involved in the induction of specific antibodies in autoimmune patients.

Renal biopsy in patients 65 years of age or older. An analysis of the results of 334 biopsies.

We studied the clinical and pathological data for 334 patients age 65 or more who underwent renal biopsy for acute renal failure (ARF, n = 55), subacute renal failure (SRF, n = 72), chronic renal failure (CRF, n = 57), proteinuria (n = 137), and hematuria (n = 13). Tissue diagnoses were glomerulopathy (n = 252, 75.4%), acute tubular lesions (n = 18), interstitial nephritis (n = 23), vascular diseases (n = 36, including 14 with cholesterol emboli), and five miscellaneous diagnoses. Of the 55 patients with ARF, 23 had a glomerular lesion, 15 had acute tubular necrosis, and 8 had acute interstitial nephritis. Of 72 patients with SRF, 49 had a glomerulopathy, 12 had a vascular disorder, and six had acute interstitial nephritis. Hence, patients with ARF or SRF exhibited a high potential for reversible lesions. Only 11.3% of patients with CRF had potentially reversible causes. The most common causes of proteinuria were membranous glomerulopathy (34.3%), minimal change disease (14.6%), focal segmental sclerosis (11.7%), and amyloidosis (8.8%). Of the 25 patients with advanced nephrosclerosis, 24 had renal failure, 20 were hypertensive, and 13 had cholesterol emboli. Of 33 patients with diabetes mellitus, 66.7% were found to have lesions not related to diabetes. We conclude that renal biopsy is most useful in older patients with ARF or SRF because of potentially reversible renal disease. Old age alone is not a contraindication to performing a renal biopsy.

Comparison of the effects of sodium bicarbonate versus sodium citrate on renal acid excretion.

Citrate is used commonly as an alkalinizing agent and in the management of nephrolithiasis, but its quantitative effect on acid-base homeostasis, as judged by changes in renal net acid excretion, has not been delineated. We therefore administered 61 mEq of sodium citrate/day for 4 days to 10 normal volunteers and compared the results to those obtained in 10 normal subjects (4 of whom also participated in the citrate protocol) given 60 mEq/day for 4 days of sodium bicarbonate, the prototypical alkalinizing agent. We found that the sodium citrate group experienced an average reduction in net acid excretion (45.5 +/- 7.2 mEq/day) that was very similar to that (42.0 +/- 7.2 mEq/day) induced by the same amount of sodium bicarbonate. In both groups, the reduction in net acid excretion was equivalent to approximately 70% of the alkali administered. The latter appeared to relate to an average negative hydrogen ion balance of approximately 15 mEq/day, since there was an increase in blood [HCO3] in each group of about 2.5 mEq/l. We conclude that the findings demonstrate that the short-term effects of sodium citrate on acid-base homeostasis in normal subjects are indistinguishable from those of sodium bicarbonate.

Impairment of beta 2-adrenoceptor-stimulated potassium uptake in end-stage renal disease.

An abnormality of extrarenal mechanisms is believed to contribute importantly to the impaired potassium homeostasis in chronic renal failure. We evaluated the plasma potassium response to inhalation of albuterol, a beta 2 agonist, in eight patients who had end-stage renal disease and who were undergoing chronic hemodialysis and in eight control subjects. The purpose was to assess if an abnormality of the beta 2 adrenoceptor mechanism is present in uremia. The maximal decrement in plasma potassium concentration in the patients (0.12 +/- 0.04 mEq/L) was significantly less than that of the control subjects (0.30 +/- 0.05). Furthermore, the final plasma potassium concentration slightly exceeded baseline in the patients but was significantly reduced in controls, leading to the conclusion that an abnormal responsiveness of the beta 2 adrenoceptor may contribute to the impaired potassium tolerance found in patients who have end-stage renal disease.

Effect of routine doses of antacid on renal acidification.

Ten healthy volunteers took a magnesium and aluminium hydroxide antacid for 4 days, and their urinary acid excretion was measured. During antacid ingestion, blood bicarbonate levels did not change significantly, but there were highly significant rises in urine pH and bicarbonate excretion and falls in the 24 h excretion of titratable acid, ammonium, and net acid; the average change in net acid excretion was 41 +/- 4 mmol (72 +/- 9%) per 24 h. This large reduction in net acid excretion appears to result from neutralisation of more hydrochloric acid than sodium bicarbonate in the gastrointestinal tract rather than from absorption of exogenous alkali. Although metabolic alkalosis does not occur with their use in normal individuals, these antacids should not be termed "non-systemic". They might cause important changes in renal drug handling, solubility of excreted substances, or acid-base status in patients at risk.

Hepatic microsomal glucose-6-phosphatase of normal and alloxan-diabetic rats. Thermotropic effects on kinetics and interaction with deoxycholate and 1-anilino-8-naphthalene sulfonate.

Thermotropic effects on the kinetics of glucose-6-phosphatase (D-glucose-6-phosphate phosphohydrolase, EC 3.1.3.9) activity of hepatic microsomes from normal and alloxan-diabetic rat liver were investigated by determining V, Km and Ki (substrate inhibition) values. Influence of deoxycholate (0.1%) and 1-anilino-8-naphthalene sulfonate (2.5 mM) on the kinetics was also evaluated. 1. Substrate inhibition occurred at 0.06 M for the enzyme from normal rats and at 0.0-0.025 M for the enzyme from diabetic rats. 2. The enzyme from diabetic rats showed a transition that extended between 22.7 and 27 degrees C in the Arrhenius plot (log V vs. T-1) instead of at 19.5 degrees C. 3. Deoxycholate increased the V value of both enzymes without affecting substrate inhibition at all the temperatures but did not completely abolish the transition in the Arrhenius plot of the enzyme from diabetic rats. 4. 1-Anilino-8-naphthalene sulfonate eliminated substrate inhibition and activated the enzyme of normal rats above 27.5 degrees C by increasing both V and Km values. Below this temperature, the enzyme showed biphasic or allosteric kinetics. At low substrate concentrations it was activated as both V and Km values were increased. The enzyme from diabetic rats, on the other hand, was activated at all the temperatures and exhibited linear kinetics. 5. Binding of 1-anilino-8-naphthalene sulfonate to the microsomal fraction increased with decreasing temperature as revealed by the increase of relative fluorescence. The microsomal fraction of diabetic rats showed a more anomalous fluorescence response between 13-18 degrees C. 6. Enthalpy changes for glucose 6-phosphate binding to the inhibition site were slightly larger than binding to the active site. Calculated entropies of activation for transition state complex of glucose-6-phosphatase reaction were fairly large and negative. The free energy of activation (28-30 kcal/mol) was independent of temperature and experimental conditions. 7. In the microsomal fraction (total as well as rough), phospholipid content and fatty acid unsaturation index of phospholipids were decreased after diabetes. The level of free cholesterol remained unchanged but the molar ratio of cholesterol to phospholipid increased. The different thermal response and 1-anilino-8-naphthalene sulfonate interaction to the enzyme from diabetic rat and liver could be ascribed to the altered lipid environment of the enzyme on the endoplasmic reticulum membrane.