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The purpose of this study was to examine the interaction between fatty acids and plasma membranes from liver cells. We were unable to reproduce the reported effect of heating on the capacity of these membranes to bind [3H]oleate (Stremmel et al. 1985 Proc. Natl. Acad. Sci. USA. 82: 4-8). In fact, the distribution of [3H]oleate between plasma membranes and unilamellar vesicles of lipids extracted from these membranes was in favor of the lipids, indicating the absence of a detectable amount of binding to a putative fatty acid binding protein in plasma membranes. Radius of curvature of vesicles (125 A vs 475 A) had no effect on the partitioning of fatty acid. In addition, the distribution of [3H]oleate between plasma membranes and other phases had the properties of a partition coefficient over a 200-fold range of [3H]oleate. There was no evidence in this experiment for a binding isotherm, i.e., binding of [3H]oleate at a specific site, superimposed on the nonspecific partitioning of [3H]oleate into the lipids of the plasma membrane. There was no competition between [14C]oleate and [3H]palmitate for entry into plasma membranes. Finally, rates of uptake of [14C]oleate and [3H]palmitate by perfused rat liver were not affected by the presence of the other fatty acid in perfusates. These data indicate that the avidity of hepatocyte plasma membranes for [3H]oleate is a simple consequence of the physical chemical properties of oleate, lipids, and water. The data exclude the idea that the uptake of fatty acids into cells is the result of binding proteins and/or catalyzed reactions at the water-membrane interface of the cell or within the plane of the plasma membrane.

Background and Objective: Chest pain is one of the most common complaints in emergency departments (EDs). Self-reported computerized history taking (CHT) programmes can be used for interpretation of the clinical significance of medical information coming directly from patients. The adoption of CHT in clinical practice depends on reactions and attitudes to the technology from patients and their belief that the technology will have benefits for their medical care. The study objective was to explore the user experience of the self-reported CHT programme Clinical Expert Operating System (CLEOS) in the setting of patients visiting an ED for acute chest pain., Methods: This qualitative interview study is part of the ongoing CLEOS-Chest Pain Danderyd Study. A subset (n = 84) of the larger sample who had taken part in self-reported history taking during waiting times at the ED were contacted by telephone and n = 54 (64%) accepted participation. An interview guide with open-ended questions was used and the text was analysed using directed content analysis., Results: The patients' experiences of the CLEOS programme were overall positive although some perceived it as extensive. The programme was well accepted and despite the busy environment, patients were highly motivated and deemed it helpful to make a diagnosis. Six categories of user experience emerged: The clinical context, The individual context, Time aspect, Acceptability of the programme, Usability of the programme and Perceptions of usefulness in a clinical setting., Conclusions: The programme was well accepted by most patients in the stressful environment at ED although some found it difficult to answer all the questions. Adjustments to the extent of an interview to better suit the context of the clinical use should be a future development of the programme. The findings suggest that CHT programmes can be integrated as a standard process for collecting self-reported medical history data in the ED setting., (© 2022 The Authors. Health Expectations published by John Wiley & Sons Ltd.)

Background: Chest pain is one of the most common chief complaints in emergency departments (EDs). Collecting an adequate medical history is challenging but essential in order to use recommended risk scores such as the HEART score (based on history, electrocardiogram, age, risk factors, and troponin). Self-reported computerized history taking (CHT) is a novel method to collect structured medical history data directly from the patient through a digital device. CHT is rarely used in clinical practice, and there is a lack of evidence for utility in an acute setting., Objective: This substudy of the Clinical Expert Operating System Chest Pain Danderyd Study (CLEOS-CPDS) aimed to evaluate whether patients with acute chest pain can interact effectively with CHT in the ED., Methods: Prospective cohort study on self-reported medical histories collected from acute chest pain patients using a CHT program on a tablet. Clinically stable patients aged 18 years and older with a chief complaint of chest pain, fluency in Swedish, and a nondiagnostic electrocardiogram or serum markers for acute coronary syndrome were eligible for inclusion. Patients unable to carry out an interview with CHT (eg, inadequate eyesight, confusion or agitation) were excluded. Effectiveness was assessed as the proportion of patients completing the interview and the time required in order to collect a medical history sufficient for cardiovascular risk stratification according to HEART score., Results: During 2017-2018, 500 participants were consecutively enrolled. The age and sex distribution (mean 54.3, SD 17.0 years; 213/500, 42.6% women) was similar to that of the general chest pain population (mean 57.5, SD 19.2 years; 49.6% women). Common reasons for noninclusion were language issues (182/1000, 18.2%), fatigue (158/1000, 15.8%), and inability to use a tablet (152/1000, 15.2%). Sufficient data to calculate HEART score were collected in 70.4% (352/500) of the patients. Key modules for chief complaint, cardiovascular history, and respiratory history were completed by 408 (81.6%), 339 (67.8%), and 291 (58.2%) of the 500 participants, respectively, while 148 (29.6%) completed the entire interview (in all 14 modules). Factors associated with completeness were age 18-69 years (all key modules: Ps<.001), male sex (cardiovascular: P=.04), active workers (all key modules: Ps<.005), not arriving by ambulance (chief complaint: P=.03; cardiovascular: P=.045), and ongoing chest pain (complete interview: P=.002). The median time to collect HEART score data was 23 (IQR 18-31) minutes and to complete an interview was 64 (IQR 53-77) minutes. The main reasons for discontinuing the interview prior to completion (n=352) were discharge from the ED (101, 28.7%) and tiredness (95, 27.0%)., Conclusions: A majority of patients with acute chest pain can interact effectively with CHT on a tablet in the ED to provide sufficient data for risk stratification with a well-established risk score. The utility was somewhat lower in patients 70 years and older, in patients arriving by ambulance, and in patients without ongoing chest pain. Further studies are warranted to assess whether CHT can contribute to improved management and prognosis in this large patient group., Trial Registration: ClinicalTrials.gov NCT03439449; https://clinicaltrials.gov/ct2/show/NCT03439449., International Registered Report Identifier (irrid): RR2-10.1136/bmjopen-2019-031871., (©Helge Brandberg, Carl Johan Sundberg, Jonas Spaak, Sabine Koch, David Zakim, Thomas Kahan. Originally published in the Journal of Medical Internet Research (https://www.jmir.org), 27.04.2021.)

Introduction: Management of acute chest pain focuses on diagnosis or safe rule-out of an acute coronary syndrome (ACS). We aim to determine the additional value of self-reported computerised history taking (CHT)., Methods and Analysis: Prospective cohort study design with self-reported, medical histories collected by a CHT programme (Clinical Expert Operating System, CLEOS) using a tablet. Women and men presenting with acute chest pain to the emergency department at Danderyd University Hospital (Stockholm, Sweden) are eligible. CHT will be compared with standard history taking for completeness of data required to calculate ACS risk scores such as History, ECG, Age, Risk factors and Troponin (HEART), Global Registry of Acute Coronary Events (GRACE), and Thrombolysis in Myocardial Infarction (TIMI). Clinical outcomes will be extracted from hospital electronic health records and national registries. The CLEOS-Chest Pain Danderyd Study project includes (1) a feasibility study of CHT, (2) a validation study of CHT as compared with standard history taking, (3) a paired diagnostic accuracy study using data from CHT and established risk scores, (4) a clinical utility study to evaluate the impact of CHT on the management of chest pain and the use of resources, and (5) data mining, aiming to generate an improved risk score for ACS. Primary outcomes will be analysed after 1000 patients, but to allow for subgroup analysis, the study intends to recruit 2000 or more patients. This ongoing project may lead to new and more effective ways for collecting thorough, accurate medical histories with important implications for clinical practice., Ethics and Dissemination: This study has been reviewed and approved by the Stockholm Regional Ethical Committee (now Swedish Ethical Review Authority). Results will be published, regardless of the outcome, in peer-reviewed international scientific journals., Trial Registration Number: This study is registered at https://www.clinicaltrials.gov (unique identifier: NCT03439449)., Competing Interests: Competing interests: DZ is the inventor on US patents for technology related to the CLEOS program. All patent rights and copyrights to technology, language, images and knowledge content are assigned without royalty rights by DZ to Karolinska Institutet, Stockholm, Sweden, which is a public university. Apart from Karolinska Institutet and its subsidiaries, no individuals or companies may be owners or receive royalties or other revenue from use of CLEOS technology, language, images, knowledge content or from clinical insights and/or computer algorithms generated from analysis of data acquired by the program. All CLEOS-CPDS steering group members (see above) will have full access to the final trial data set., (© Author(s) (or their employer(s)) 2020. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Basic life science research holds the promise of personalizing medical care. However, translation steps from the laboratory to the bedside are not trivial. Results from clinical research are difficult to replicate in part because study cohorts are poorly defined phenotypically. Here, we discuss how computer technology can improve the collection of clinical data to enable translation of insights from basic science to validated clinical guidelines., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Puumala virus (PUUV) is the most common species of hantavirus in Central Europe. Nephropathia epidemica (NE), caused by PUUV, is characterized by acute kidney injury (AKI) and thrombocytopenia. The major goals of this study were to provide a clear clinical phenotyping of AKI in patients with NE and to develop an easy prediction rule to identify patients, who are at lower risk to develop severe AKI., Methods: A cross-sectional prospective survey of 456 adult patients with serologically confirmed NE was performed. Data were collected from medical records and prospectively at follow-up visit. Severe AKI was defined by standard criteria according to the RIFLE (Risk, Injury, Failure, Loss, End-stage kidney disease) classification. Fuller statistical models were developed and validated to estimate the probability for severe AKI., Results: During acute NE, 88% of the patients had AKI according to the RILFE criteria during acute NE. A risk index score for severe AKI was derived by using three independent risk factors in patients with normal kidney function at time of diagnosis: thrombocytopenia [two points; odds ratios (OR): 3.77; 95% confidence intervals (CI): 1.82, 8.03], elevated C-reactive protein levels (one point; OR: 3.02; 95% CI: 1.42, 6.58) and proteinuria (one point; OR: 3.92; 95% CI: 1.33, 13.35). On the basis of a point score of one or two, the probability of severe AKI was 0.18 and 0.28 with an area under the curve of 0.71., Conclusion: This clinical prediction rule provides a novel and diagnostically accurate strategy for the potential prevention and improved management of kidney complications in patients with NE and, ultimately, for a possible decrease in unnecessary hospitalization in a high number of patients., (© The Author 2014. Published by Oxford University Press on behalf of ERA-EDTA. All rights reserved.)

Human infection with Puumala virus (PUUV), the most common hantavirus in Central Europe, causes nephropathia epidemica (NE), a disease characterized by acute kidney injury and thrombocytopenia. To determine the clinical phenotype of hantavirus-infected patients and their long-term outcome and humoral immunity to PUUV, we conducted a cross-sectional prospective survey of 456 patients in Germany with clinically and serologically confirmed hantavirus-associated NE during 2001-2012. Prominent clinical findings during acute NE were fever and back/limb pain, and 88% of the patients had acute kidney injury. At follow-up (7-35 mo), all patients had detectable hantavirus-specific IgG; 8.5% had persistent IgM; 25% had hematuria; 23% had hypertension (new diagnosis for 67%); and 7% had proteinuria. NE-associated hypertension and proteinuria do not appear to have long-term consequences, but NE-associated hematuria may. All patients in this study had hantavirus-specific IgG up to years after the infection.

Background: Validated guidelines to manage low-density lipoprotein (LDL)-cholesterol are utilized inconsistently or not at all even though their application lowers the incidence of coronary events. New approaches are needed, therefore, to implement these guidelines in everyday practice., Methods and Results: We compared an automated method for applying The National Cholesterol Education Panel (NCEP) guidelines with results from routine care for managing LDL-cholesterol. The automated method comprised computerized history-taking and analysis of historical data without physician input. Results from routine care were determined for 213 unselected patients and compared with results from interviews of the same 213 patients by a computerized history-taking program. Data extracted from hospital charts showed that routine care typically did not collect sufficient information to stratify risk and assign treatment targets for LDL-cholesterol and that there were inconsistencies in identifying patients with normal or elevated levels of LDL-cholesterol in relation to risk. The computerized interview program outperformed routine care in collecting historical data relevant to stratifying risk, assigning treatment targets, and clarifying the presence of hypercholesterolemia relative to risk., Conclusions: Computerized history-taking coupled with automated analysis of the clinical data can outperform routine medical care in applying NCEP guidelines for stratifying risk and identifying patients with hypercholesterolemia in relation to risk.

Objective: Giant cell arteritis (GCA) and polymyalgia rheumatica (PMR) generally respond well to treatment with glucocorticoids (GC). We sought to determine the value of clinical, histopathologic, immunohistochemical, and genetic findings and the expression of the glucocorticoid receptor (GR) for discriminating between patients who achieve complete remission, or partial remission, or who do not improve with glucocorticoid treatment., Methods: We examined biopsies of the temporal artery from 60 patients, of whom 27 had GCA, 13 PMR, and 20 arteriosclerosis., Results: Of the clinical variables evaluated, jaw claudication was correlated with the histologic classification of the biopsies (p < 0.0001). Erythrocyte sedimentation rate was significantly higher in patients with PMR and GCA than in patients with arteriosclerosis (p < 0.0001). There were significant differences between patients with GCA versus PMR in the numbers of CD3-, CD8-, and CD4-positive T cells, in CD68-positive monocytes (p < 0.0001), and antigen-presenting cells (p < 0.0001). CD138-positive and CD20-positive cells were absent in patients with PMR but present in patients with GCA (p < 0.0001). In GCA and chronic inflammation most monocytes and lymphocytes expressed GR (88.9%). The number of CD68-positive cells and the extent of GR-staining in chronic inflammation reflected the success of treatment in logistic regression analysis (p < 0.05). GR polymorphism showed that more than 90% of patients had the wild-type (homozygote) of the R23K or N363S polymorphism. There was no evidence that this polymorphism influenced response to treatment with GC (Fisher's exact test 1.0)., Conclusion: Expression of GR and the presence of CD20-, CD3-, CD4-, CD8-, CD68-, CD138-positive cells and antigen-presenting cells differ between GCA and PMR. The presence of CD68-positive cells and the extent of GR-staining in chronic inflammation are suitable to predict complete remission in GCA.

Background: The medical history is acknowledged as the sine qua non for quality medical care because recognizing problems is pre-requisite for managing them. Medical histories typically are incomplete and inaccurate, however. We show here that computers are a solution to this issue of information gathering about patients. Computers can be programmed to acquire more complete medical histories with greater detail across a range of acute and chronic issues than physician histories., Methods: Histories were acquired by physicians in the usual way and by a computer program interacting directly with patients. Decision-making of what medical issues were queried by computer were made internally by the software, including determination of the chief complaint. The selection of patients was from admissions to the Robert-Bosch-Hospital, Stuttgart, Germany by convenience sampling. Physician-acquired and computer-acquired histories were compared on a patient-by-patient basis for 45 patients., Results: The computer histories reported 160 problems not recorded in physician histories or slightly more than 3.5 problems per patient. However, physicians but not the computer reported 13 problems. The data show that computer histories reported problems across a range of organ systems, that the problems detected by computer but not physician histories were both acute and chronic and that the computer histories detected a significant number of issues important for preventing further morbidity., Conclusion: A combination of physician and computer-acquired histories, in non-emergent situations, with the latter available to the physician at the time he or she sees the patient, is a far superior method for collecting historical data than the physician interview alone.

Sphingomyelinase (SMase), a water-soluble enzyme from Bacillus cereus, is shown to bind with high affinity to vesicles of sphingomyelin (SM) but not to vesicles of phosphatidylcholine (PC). The reaction progress by SMase bound to SM vesicles occurs in the scooting mode with virtually infinite processivity of the successive interfacial turnover cycles. Three conditions for the microscopic steady state during the reaction progress at the interface are satisfied: the bound SMase does not leave the interface even after all the SM in the outer layer is converted to ceramide; the SMase-treated vesicles remain intact; and the ceramide product does not exchange with SM present in excess vesicles or in the inner layer of the hydrolyzed vesicle. Within these constraints, on accessibility and replenishment of the substrate, the extent of hydrolysis in the scooting mode reaction progress is a measure of the number of vesicles containing enzyme. The slope of the Poisson distribution plot, for the enzyme per vesicle versus the logarithm of the fraction of the total accessible substrate remaining unhydrolyzed in excess vesicles, shows that a single 32 kDa subunit of SMase is fully catalytically active. The maximum initial rate of hydrolysis, at the limit of the maximum possible substrate mol fraction, X(S)*=1, is 400 s(-1) in H(2)O and 220 s(-1) in D(2)O, which is consistent with the rate-limiting chemical step. The integrated reaction progress suggests that the ceramide product does not codisperse ideally on the hydrolyzed vesicles. Furthermore, complex reaction progress seen with covesicles of SM+PC are attributed to slow secondary changes in the partially hydrolyzed SM vesicles.

There is continuing controversy about the mechanism for transfer of fatty acids (FA) between plasma and the interior of cells and vice versa. One view is that this is a spontaneous process. The generally accepted view is that each step of the process is facilitated by a specialized protein. Whether uptake is spontaneous or facilitated, the components of the uptake system, e.g., albumin, water, FA, plasma membrane, and putative transport proteins of the plasma membrane, must behave according to the rules of the physical chemistry of the system. We review these features to illustrate the constraints they impose on the design of experiments to adduce the mechanism of uptake. Analysis of the literature in the context of the physical chemistry of the uptake system indicates that arguments for a facilitated mechanism of uptake for FA are not supported by any data extant. By contrast, comparison of the rates for individual steps of the pathway traversed by FA moving from albumin to the inside of a cell (or vesicles of a model system) with rates of uptake of FA of tissues in the steady state shows that the rates of the former are sufficient to account for the rate of the latter.

Two events in the last decade have set the stage for the large-scale clinical testing of chemopreventive agents for colorectal cancer in people at low to moderate risk for this disease. One of these is the discovery of a cause-effect relationship between the activities of cyclooxygenases (COX) and carcinogenesis in the colon, which can be interdicted by inhibitors of the enzymes. The other is the development of selective inhibitors of COX-2. These agents, when used in animals, also inhibit carcinogenesis in the colon. Additionally, they appear to be safe enough in humans to allow large-scale testing in healthy people. We review the key data implicating a causal relationship between the activity of COX and carcinogenesis and its possible mechanisms of action. We also emphasize work that points to other molecular targets for chemoprevention of colorectal cancer, which is emerging from studies of the link between COX and carcinogenesis.

Infrared absorption spectra of formalin-fixed, paraffin-embedded human cervical tissue are reported for normal, dysplastic and neoplastic samples. The spectral differences found in this study between these states of the tissues are far less than those observed for single cells by us and others. Nevertheless, we find a direct correspondence between spectral data from tissue sections, obtained from biopsies, and individual exfoliated cells, typically obtained during a pap procedure. We also find that spectra due to dysplastic samples fall about halfway between the spectral features of normal and cancerous samples.

Infrared spectral results for the different epithelial layers of human cervical squamous tissue are reported. The layers, representing different cellular maturation stages, exhibit quite different spectral patterns. Thus, infrared spectroscopy presents a powerful tool to monitor cell maturation and differentiation. Furthermore, a detailed understanding of the spectra of the individual layers of tissue permit a proper interpretation of the state of health of cells exfoliated from such tissue. Part II of this series describes the use of the spectral information presented here to interpret the infrared spectra of exfoliated cells.

A comparison of infrared absorption spectra obtained from the different layers of squamous epithelium from the human cervix, and infrared spectra obtained from exfoliated cervical cells, is presented. Infrared spectroscopy has been shown (in part I of this series) to be a sensitive tool to monitor maturation and differentiation of human cervical cells; therefore, this spectroscopic technique provides new insights into the composition and state of health of exfoliated cells.

We investigated the potential of dietary saturated fatty acids to decrease endotoxemia and suppress expression of cyclooxygenase 2 (Cox-2) and tumor necrosis factor alpha (TNF-alpha) in established alcohol-induced liver injury. Six groups (five rats/group) of male Wistar rats were studied. Rats in group 1 were fed a fish oil-ethanol diet for 6 weeks. Rats in groups 2, 3, and 4 were fed fish oil and ethanol for 6 weeks. Ethanol administration was stopped at this time, and the rats were switched to isocaloric diets containing dextrose with fish oil (group 2), palm oil (group 3), or medium-chain triglycerides (group 4) as the source of fat for an additional 2 weeks. Rats in groups 5 and 6 were fed fish oil-ethanol and fish oil-dextrose, respectively, for 8 weeks. Liver samples were analyzed for histopathology, lipid peroxidation, and levels of messenger RNA (mRNA) for Cox-2 and TNF-alpha. Concentrations of endotoxin were determined in plasma. The most severe inflammation and fibrosis were detected in groups 1 and 5, as were the highest levels of endotoxin, lipid peroxidation, and mRNA for Cox-2 and TNF-alpha. After ethanol was discontinued, there was minimal histological improvement in group 2 but near normalization of the histology, including regression of fibrosis, in groups 3 and 4. Histological improvement was associated with decreased levels of endotoxin, lipid peroxidation, and reduced expression of Cox-2 and TNF-alpha. The data indicate that a diet enriched in saturated fatty acids (groups 3 and 4) effectively reverses alcohol-induced liver injury, including fibrosis. The therapeutic effects of saturated fatty acids may be explained, at least in part, by reduced endotoxemia and lipid peroxidation, which in turn result in decreased levels of TNF-alpha and Cox-2.

An expanding body of evidence indicates that downregulation of the cyclooxygenases (Cox-1 and Cox-2) will be an important strategy for preventing cancer because cyclooxygenases catalyze the formation of prostaglandins (PGs), and PGs have multiple effects that favor tumorigenesis. PGs also are more abundant in cancers than in the normal tissues from which cancers arise. Overexpression of Cox-2 in epithelial cells inhibits apoptosis and increases the invasiveness of tumor cells; inhibitors of Cox (e.g., NSAIDS) are chemopreventive; and tumorigenesis is inhibited in Cox-2 knockout mice. We focus in this review on strategies to selectively inhibit and downregulate the Cox-2 isoform. This is important because simultaneous inhibition of Cox-1 (constitutively expressed) and Cox-2 (inducible isoform), which is achieved with classical NSAIDs, interferes with the housekeeping functions of Cox-1 and thereby causes serious side effects, such as peptic ulcer disease. Simultaneous inhibition of Cox-1 and Cox-2 hence is not a realistic approach for chemoprevention in individuals at low to moderate risk for cancer. On the other hand, it appears possible to avoid many NSAID-dependent side effects by selective inhibition of Cox-2, which is also the isoform that is upregulated in benign and malignant tumors. Through understanding the biochemistry of these enzymes and the regulation of Cox-1 and Cox-2 gene expression, we review how Cox-2 can be regulated selectively as a target for chemopreventive therapy. We also discuss the potential importance and advantages of a multifaceted approach to diminishing the function of Cox-2 (i.e., combining inhibitors of enzyme function with inhibitors of gene expression).

The rate of movement of fatty acids (FA) across phospholipid bilayers is an important consideration for their mechanism of transport across cell membranes but has not yet been measured. When FA move undirectionally across phospholipid bilayers, the rapid movement of un-ionized FA compared to ionized FA results in transport of protons. We have previously used this property to show that FA move spontaneously ("flip-flop") across the bilayer of small unilamellar vesicles within approximately 1 s (Kamp & Hamilton, 1992, 1993). This work extends the time resolution of this assay into the millisecond time range by use of stopped flow fluorometry. In small unilamellar vesicles (diameter, approximately 25 nm) at neutral pH, flip-flop of all fatty acids studied (lauric, myristic, palmitic, oleic, and stearic) was > or = 80% complete within 5-10 ms. In large unilamellar vesicles (diameter, approximately 100 nm), the same fatty acids exhibited fast flip-flop but with a measureable rate (t 1/2 = 23 +/- 12 ms). The calculated pseudounimolecular rate constant of the un-ionized FA (kFAH) approximately 15 s-1. There was no dependence of the flip-flop rate on the fatty acid chain length or structure. We also monitored the rate of desorption and transbilayer movement of (anthroyloxy)stearic acid in small unilamellar vesicles. Whereas previous studies suggested slow flip-flop of this FA analogue, the present studies suggest that (anthroyloxy)stearic acid flip-flops rapidly and that earlier studies did not truly measure the transbilayer movement step. These findings further support the view that proteins are not required for translocation of FA across cell membranes.

Addition of oleoyl-CoA to microsomes inhibited UDP-glucuronosyltransferase (assayed with 1-naphthol or p-nitrophenol) at concentrations within the physiologic range of total long-chain acyl-CoAs in liver. Inhibition of activity was associated with changes in the regulatory properties of the enzyme indicating that oleoyl-CoA altered the functional state of UDP-glucuronosyltransferase. The effect of oleoyl-CoA on the state of UDP-glucuronosyltransferase depended on the concentration of oleoyl-CoA, whether oleoyl-CoA was added in the presence or absence of substrates, the duration of treatment with oleoyl-CoA, and the aglycone with which activity was assayed. When oleoyl-CoA was added to microsomes in the presence of aglycones or UDP-glucuronic acid, inhibition by oleoyl-CoA was reversed by albumin, which by itself had no effect on activity. But UDP-glucuronosyltransferase, assayed with either aglycone, did not revert to the native state on removing oleoyl-CoA. Instead sequential treatment with oleoyl-CoA and albumin, in the presence of at least one substrate, produced a form of UDP-glucuronosyltransferase that was more active than the native state. When oleoyl-CoA was added to microsomes in the absence of aglycones or UDP-glucuronic acid, the activity of enzymes assayed with 1-naphthol decayed irreversibly to zero. Similar treatment followed by assay with p-nitrophenol as aglycone led to an active form of the enzyme that was inhibited further by albumin. The data are compatible with the idea that long-chain acyl-CoAs could regulate the functional state of UDP-glucuronosyltransferase.

The thermotropic properties of multilamellar vesicles of dimyristoylphosphatidylcholine (DMPC), dipalmitoylphosphatidylcholine (DPPC), and distearoylphosphatidylcholine (DSPC), as a function of the concentration of bilirubin in the range of 0.1 to 1 mol%, were measured. The exact effects of bilirubin depended on the chain length of the polymethylene chains. But the general effects of bilirubin were the same in all systems. At the lowest concentrations tested (0.1 mol bilirubin/100 mol phospholipid (0.1 mol%)), bilirubin broadened and shifted to higher temperatures the main phase transitions of all bilayers. For DPPC and DSPC, but not DMPC, this concentration of bilirubin was associated with a new transition at 25 degrees C (DPPC) or 34 degrees C (DSPC). Bilirubin at 0.2 mol% was required for the detection of a similar transition (at 13.7 degrees C) in DMPC. Higher concentrations of bilirubin (> 0.2 mol%) suppressed completely the main phase transitions in all bilayers but increased the enthalpy of the new transition. Maximal values of delta H for these transitions were reached at 0.5, 0.25, and 0.2 mol% bilirubin in DMPC, DPPC, and DSPC, respectively. Values of delta H and delta S for these transitions were far larger than for the corresponding gel-to-liquid crystal transitions in pure lipid bilayers but were equal to those expected for a transition between crystalline and liquid crystalline phases.

We have examined the idea that membrane enzymes are regulated by the viscosity of surrounding lipids using data compiled from the literature for the effect of the change in membrane viscosity ([symbol: see text]) at the gel- to liquid-crystal-phase transition on the activities of several enzymes. The analysis was not extended explicitly to the problem of viscosity-dependent regulation of membrane enzymes in liquid-crystalline lipids because of the absence of exact data for values of [symbol: see text] in liquid-crystalline phases of variable composition. For most membrane enzymes studied, energies of activation are discontinuous, while kcat is continuous, at the main-phase transition. We consider that the energy of activation contains terms related to the height of the chemical barrier to reaction and terms due to the mechanical properties of the bilayer, such as the work of expansion during the catalytic cycle and the temperature dependence of [symbol: see text]. We find that the differences in energies of activation, above and below the break points in Arrhenius plots, are orders of magnitude larger than can be accounted for by the above mechanical factors. Thus, discontinuities in energies of activation at the phase transition appear to reflect changes in the chemical barrier to reaction, which is independent of [symbol: see text]. The theorectical analysis indicates too that values of [symbol: see text] for bilayers in the liquid-crystalline phase would have to be several orders of magnitude larger than those for gel phases in order to provide a basis for viscosity-dependent regulation of membrane enzymes in liquid-crystalline phases.(ABSTRACT TRUNCATED AT 250 WORDS)

In contact with mother liquor, crystalline beta-cyclodextrin (beta-CD) hydrate has composition approximately beta-CD.12H2O. If crystals are dried at ambient conditions (18 degrees C, approximately 50% humidity), the unit cell volume diminishes approximately 30 to 50 A3. X-ray structure analysis of a dry crystal (0.89 A resolution, 4617 data, R = 0.059) showed the composition beta-CD.10.5 H2O, with approximately 5.5 water molecules in the beta-CD cavity (7 partially and 2 fully occupied sites) and approximately 5.0 between the beta-CD molecules. The positions of the beta-CD host and of most of the hydration waters are conserved during dehydration, but the occupancies of the waters in the beta-CD cavity diminish. Dry crystals put into solvent re-hydrate to the original form. The mechanism of de- and re-hydration is not evident.

The effect of a fat-free diet on the amount and functional state of UDP-glucuronosyltransferase was studied in rat liver microsomes. Measurements of enzyme activity showed that activity was approximately 30% lower in untreated microsomes in response to the fat-free diet as compared with the control diet. Immunoblotting with anti-UDP-glucuronosyltransferase showed approximately 200% less enzyme in rats fed the fat-free diet. A kinetic method for measuring total UDP-glucuronosyltransferase confirmed the result of the immunoblot. Thus, the total amount of enzyme declined to a greater extent than enzyme activity. Responses of the enzyme to activation by palmitoyl-lysophosphatidylcholine or UDP-N-acetyl-glucosamine suggested that rats fed the fat-free diet had a greater activity per molecule of UDP-glucuronosyltransferase than did rats fed the control diet. This result explained the relatively small decline in enzyme activity as compared with enzyme concentration in microsomes prepared from animals fed the fat-free diet. Fatty acid analysis of microsomal lipids demonstrated that the fat-free diet was associated with lower levels of arachidonic and linoleic acids and greater amounts of palmitoleic, oleic and cis-vaccenic acids.

Studies of the mechanism of lipid-induced regulation of the microsomal enzyme UDP-glucuronosyltransferase have been extended by examining the influence of charge within the polar region on the ability of lipids to activate delipidated pure enzyme. The effects of net negative charge, of charge separation in phosphocholine, and of the distribution of charge in the polar region of lipids were studied using the GT2p isoform isolated from pig liver. Prior experiments have shown that lipids with net negative charge inhibit the enzyme (Zakim, D., Cantor, M., and Eibl, H. (1988) J. Biol. Chem. 263, 5164-5169). The current experiments show that the extent of inhibition on a molar basis increases as the net negative charge increases from -1 to -2. The inhibitory effect of negatively charged lipids is on the functional state of the enzyme and is not due to electrostatic repulsion of negatively charged substrates of the enzyme. Although the inhibitory effect of net negative charge is removed when negative charge is balanced by a positive charge due to a quaternary nitrogen, neutrality of the polar region is not a sufficient condition for activation of the enzyme. In addition to a balance of charge between Pi and the quaternary nitrogen, the distance between the negative and positive charges and the orientation of the dipole created by them are critical for activation of GT2p. The negative and positive charges must be separated by the equivalent of three -CH2- groups for optimal activation by a lipid. Shortening this distance by one -CH2- unit leads to a lipid that is ineffective in activating the enzyme. Reversal of the orientation of the dipole in which the negative charge is on the polymethylene side of the lipid-water interface and the positive charge extends into water also produces a lipid that is not effective for activating GT2p. On the other hand, lipids with phosphoserine as the polar region, which has the "normal" P-N distance but carries a net negative charge, do not inhibit GT2p. This result again illustrates the importance of the dipole of phosphocholine for modulating the functional state of GT2p.

Rate constants for the hydration of bilirubin bound to unilamellar bilayers of dioleoylphosphatidylcholine and albumin were measured by stopped-flow methods. Rate constants for association of bilirubin with these vesicles and albumin were calculated from measured rate constants for dissociation and the equilibrium binding constants of bilirubin and lipids or albumin. Rate constants for hydration (dissociation) for bilirubin bound to dioleoylphosphatidylcholine and albumin were 71 s-1 and 1.8 s-1 respectively. Rate constants for association were 4.0 10(7) s-1 and 1.1 10(9) M-1 s-1, respectively. Both rates for interactions of bilirubin with bilayers were essentially independent of temperature in the range 0-40 degrees C, indicating that barriers to entry and exit of bilirubin from bilayers were entropic. Rates of transbilayer movement of bilirubin in dioleoylphosphatidylcholine were too fast to resolve by measuring rates of hydration of bilirubin. Rate constants for hydration of bilirubin bound to bilayers with less avidity for bilirubin as compared with dioleoylphosphatidylcholine also were too fast to measure with stopped-flow methods. In addition to providing details of the energetic basis for interactions between bilirubin and membranes, the data allow for calculating the maximal rates at which bilirubin could transfer spontaneously from sites on albumin in blood to the interior of cells. The data show, in this regard, that this rate is 10-50 fold faster than measured rates of uptake of bilirubin by intact liver.

The effects of high pressure on the kinetic properties of microsomal UDP-glucuronosyltransferase (assayed with 1-naphthol as aglycon) were studied in the range of 0.001-2.2 kbar to clarify further the basis for regulating this enzyme in untreated microsomes. Activity changed in a discontinuous manner as a function of pressure. Activation occurred at pressure as low as 0.1 kbar, reaching one of two maxima at 0.2 kbar. As pressure was increased above 0.2 kbar, activity decreased, reaching a minimum at about 1.4 kbar followed by a second activation. The pathway for activation at pressure greater than 1.4 kbar was complex. The immediate effect of 2.2 kbar was nearly complete inhibition of activity. The inhibited state relaxed, however, over about 10 min (at 10 degrees C), to a state that was activated as compared with enzyme at 0.001 kbar or enzyme at pressures between 1.4 and 2.2 kbar, which was the highest pressure we could test. Examination of the detailed kinetic properties of UDP-glucuronosyltransferase indicated that the effects of pressure were due to selective stabilization of unique functional states of the enzyme at 0.2 and 2.2 kbar. Activation at 0.2 kbar was reversible when pressure was released. This was true as well as for activation at pressure greater than 1.4 kbar, but after prolonged treatment at 2.2 kbar, UDP-glucuronosyltransferase became activated irreversibly on release of pressure. The process by which prolonged treatment at 2.2 kbar led to permanent activation of UDP-glucuronosyltransferase after release of pressure was not reflected, however, by time-dependent changes in the functional state of UDP-glucuronosyltransferase at this pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of cholesterol on the activity and thermal properties of a pure, delipidated isoform of UDP-glucuronosyltransferase were examined after incorporation of enzyme into unilamellar bilayers of distearoylphosphatidylcholine (DSPC) or dioleoylphosphatidylcholine (DOPC). Cholesterol, in bilayers of DSPC, decreased enzyme activity and lowered the temperature (from 37 to 30 degrees C) for a reversible transition from the active form of the enzyme to a less active form. These effects could be separated from each other in that the effect on reversible inactivation of the enzyme occurred at lower concentrations of cholesterol than the effect on activity of the active form of the enzyme. In addition, cholesterol in bilayers of DSPC stabilized UDP-glucuronosyltransferase against irreversible thermal inactivation. The extent of stabilization increased with increasing concentration of cholesterol in the bilayers. The effects of cholesterol on UDP-glucuronosyltransferase depended, however, on the nature of the bilayer containing cholesterol. Cholesterol had small effects, if any, on the properties of UDP-glucuronosyltransferase in bilayers of DOPC.

Diclofenac sodium, a phenylacetic acid-derived nonsteroidal anti-inflammatory drug (NSAID) recently released in the United States, was associated with the development of significant hepatitis in seven patients, with one associated death. Signs and symptoms developed within several weeks of initiation of drug use and generally resolved 4 to 6 weeks following discontinuation of use of the drug. The only patient rechallenged with the drug developed a recurrence of her hepatic abnormalities. In one patient, fatal, fulminant hepatitis developed despite early withdrawal of the drug. Review of the European literature disclosed three additional fatalities associated with diclofenac therapy. It is unclear whether the incidence of hepatotoxicity is higher with this drug compared with other nonsteroidal anti-inflammatory drugs. Careful patient monitoring is advised, and prompt discontinuation of the drug is suggested when signs or symptoms of liver disease develop.

Because of their high cost, lysolecithins are generally not considered useful detergents for the purification of membrane-bound enzymes. Therefore, we have synthesized a structural analog of lysolecithin with similar physical properties for which synthesis is straightforward. This analog is 1-palmitoylpropanediol-3-phosphocholine. To compare the efficacy of the two detergents for the purification of a membrane-bound enzyme, we have purified UDP-glucuronosyltransferase from pig liver microsomes using lysophosphatidylcholine or the synthetic analog. The catalytic properties of UDP-glucuronosyltransferase purified with 1-palmitoylpropanediol-3-phosphocholine or lysolecithin were identical. Sodium dodecyl sulfate-gel electrophoresis indicated that the purity of the UDP-glucuronosyltransferase preparation was the same whether lysophosphatidylcholine or its synthetic analog was used. The advantage of using 1-palmitoylpropanediol-3-phosphocholine in preference to lysophosphatidylcholine is that the former can be synthesized for about 1% the cost of the latter. In addition, the method for synthesis of 1-palmitoylpropanediol-3-phosphocholine is general in that the structural features of the polymethylene chain can be varied, allowing for the inexpensive synthesis of a series of detergents.

Treatment of microsomes at pressures as high as 2.25 kbar led to an apparent irreversible activation of UDP-glucuronylsyltransferase when pressure was released. The response of the enzyme to pressure, as reflected by activity measured after release of pressure, appeared to be discontinuous in that no activation was seen for any preparation at pressures less than 1.2 kbar. In addition, activation was temperature dependent. Maximum activation at 2.25 kbar occurred at about 12 degrees C; the extent of activation in 10 min was less for either higher or lower temperatures. Activation was also time dependent. Maximum activation at 2.25 kbar and 9 degrees C required 90 min of pressure treatment. Activation appeared to occur more slowly at lower pressure. Pressure-induced activation was associated with a loss of sensitivity of the enzyme to allosteric activation by UDP-N-Ac-Glc and a conversion of the kinetic pattern from non-Michaelis-Menten to Michaelis-Menten. Pressure did not activate enzyme that had previously been activated maximally by adding detergent to microsomes. Pressure also did not activate pure UDP-glucuronosyltransferase reconstituted into unilamellar vesicles of dioleoylphosphatidylcholine. Pressure treatment did not release UDP-glucuronosyltransferase from microsomes into water. Pressure had a continuous effect on the polarization and excimer/monomer formation of fluorescent probes incorporated into microsomes, and the properties returned essentially to their values at 1 atm when pressure was released. Measurements of activity at 2.2 kbar showed that pressure-induced activation of UDP-glucuronosyltransferase in microsomes occurred via two intermediates that were inactive and that the activated state of the enzyme was generated during/after release of pressure.(ABSTRACT TRUNCATED AT 250 WORDS)

The kinetic properties of UDP-glucuronosyltransferase were measured after brief treatment of liver microsomes at alkaline pH, followed by assay with p-nitro-phenol as aglycone, at pH 7.5. Enzyme activity increased in a graded fashion as the pH of pretreatment was increased above 8.0, with apparent maximal activation of eight-fold for a pretreatment pH of 11.1. The pH for half maximal activation was 10.6. Brief treatment at alkaline pH prior to assay at pH 7.5 was associated too with a graded conversion of the kinetics of the enzyme from non-Michaelis-Menten to Michaelis-Menten at pH 11.7. Sensitivity to the allosteric modulator, UDP-N-acetylglucosamine decreased as the pH increased. A fifty percent loss of sensitivity to UDP-N-acetylglucosamine-induced activation occurred at pH 10.6. Thus, pretreatment at alkaline pH had irreversible effects on the properties of UDP-glucuronosyltransferase in microsomes. In order to establish the cause for the irreversibility of the changes induced by alkaline pH, microsomes were treated at pH 11.6 prior to purifying UDP-glucuronosyltransferase. Enzyme purified from alkali-treated and untreated microsomes had approximately the same specific activity. More importantly, responses to activation by lipids, and regeneration of allosteric properties were the same for both purified enzymes (from alkali-treated and control microsomes). Pure enzyme was not activated by pretreatment at alkaline pH. We interpret these data to mean that the irreversible effects of alkaline pH on the properties of UDP-glucuronosyltransferase in microsomes were not due to direct effects on the enzyme, but to how the enzyme interacted normally with molecules within the plane of the membrane.

The location of bilirubin IXa in lipid bilayers of dimyristoylphosphatidylcholine or dioleoylphosphatidylcholine was studied by determining the effects of bilirubin on the infrared spectra of the lipids as a function of pressure. It was found for both bilayers that bilirubin intercalated into the polymethylene chain region of the bilayer, being located between the carbonyl region and the methylene group two carbons from the methyl terminus. Small amounts of bilirubin interacted with the choline region of dioleoylphosphatidylcholine. Lesser amounts interacted with the choline region of dimyristoylphosphatidylcholine. This difference between the two types of bilayers was attributed to the degradation of small amounts of bilirubin IXa to more polar isomers in the presence of dioleoylphosphatidylcholine. In dioleoyl- but not dimyristoylphosphatidylcholine, bilirubin interacted with the C = O region, probably indicating that bilirubin in the latter type of bilayer was intercalated into the polymethylene chains above and below the double bond. Bilirubin decreased the pressure required for the liquid-crystal to gel-phase transition in both bilayers at 28 degrees C. Bilirubin was not forced out of either bilayer at pressures as high as 20 kbar.

The thermal stability of glucose-6-phosphatase in rat liver microsomes was examined in untreated and cholate-treated microsomes. Activity of the enzyme was measured with both glucose-6-P and mannose-6-P as substrates. Heat treatment did not cause glucose-6-phosphatase activity to decline to zero with a single rate constant in untreated microsomes. Instead, heat treatment produced an enzyme with a small residual activity that was stable. The residual level of activity was not stimulated by addition of detergent. In untreated microsomes the energies of activation for the processes of decay were different for glucose-6-phosphatase and mannose-6-phosphatase activities, suggesting that the rate-limiting steps for the hydrolysis of these compounds were different. Treatment of microsomes with detergent increased the rate constants for the thermal decay of glucose-6-phosphatase by about 150 times, and, in contrast to untreated microsomes, glucose-6-phosphatase and mannose-6-phosphatase decayed to zero with a single rate constant in cholate-treated microsomes. Also, rate constants for thermal inactivation of glucose-6-phosphatase and mannose-6-phosphatase were the same in cholate-treated microsomes. Removal of cholate increased the stability of glucose-6-phosphatase but did not regenerate the form of the enzyme present in untreated microsomes. The data for the stability of glucose-6-phosphatase under different conditions provide evidence that the enzyme can exist in at least five different stable states that are enzymatically active.

The effects of bile acids on the detoxification of compounds by glutathione conjugation have been investigated. Bile acids were found to inhibit the total soluble-fraction glutathione S-transferase activity from rat liver, as assayed with four different acceptor substrates. Dihydroxy bile acids were more inhibitory than trihydroxy bile acids, and conjugated bile acids were generally less inhibitory than the parent bile acid. At physiological concentrations of bile acid, the glutathione S-transferase activity in the soluble fraction was inhibited by nearly 50%. This indicates that the size of the hepatic pool of bile acids can influence the ability of the liver to detoxify electrophilic compounds. The A, B and C isoenzymes of glutathione S-transferase were isolated separately. Each was found to be inhibited by bile acids. Kinetic analysis of the inhibition revealed that the bile acids were not competitive inhibitors of either glutathione or acceptor substrate binding. The microsomal glutathione S-transferase from guinea-pig liver was also shown to be inhibited by bile acids. This inhibition, however, showed characteristics of a non-specific detergent-type inhibition.

The substrate specificity of fatty-acyl-CoA ligase in liver microsomes has been studied in a system in which fatty acids are present initially as complexes with unilamellar vesicles of phosphatidylcholine. The latter were prepared by cosonication of phospholipids and different fatty acids. As compared with previous studies of the enzyme the activity of acyl-CoA ligase is several-fold higher for assays carried out with fatty acid substrates added as components of a bilayer. This was true for all fatty acids studied. Also as compared with data reported previously in the literature there was a systematic relationship between the structure of fatty acids, activity at Vmax for synthesis of acyl-CoA and avidity of binding to the ligase. Activity at Vmax was greatest for lauric acid and decreased with increasing chain length. The apparent avidity of enzyme for fatty acids was greatest for octanoic acid and decreased as chain length increased.

Glutathione S-transferase is present in rat liver microsomal fraction, but its activity is low relative to the transferase activity present in the soluble fraction of the hepatocyte. We have found, however, that the activity of microsomal glutathione S-transferase is increased 5-fold after treatment with small unilamellar vesicles made from phosphatidylcholine. The increase in activity is due to the removal of an inhibitor of the enzyme from the microsomal membrane. The inhibitor is present in the organic layer of a washed Folch extract of the microsomal fraction. When this fraction of the microsomal extract is reconstituted in the form of small unilamellar vesicles, it inhibits microsomal glutathione S-transferase that had been activated by prior treatment with small unilamellar vesicles of pure phosphatidylcholine, but does not affect the activity of unactivated microsomal glutathione S-transferase. The inhibitor did not seem to be formed during the isolation of the microsomal fraction, and hence may be a physiological regulator of microsomal glutathione S-transferase. In this regard, both free fatty acid (palmitate) and lysophosphatidylcholine were shown to inhibit the enzyme reversibly. The results indicate that the activity of microsomal glutathione S-transferase is far greater than appreciated until now, and that this form of the enzyme may be an important factor in the hepatic metabolism of toxic electrophiles.

Synthesis of glyco- or tauro-cholate from choloyl-CoA and the respective amino acid is shown to be catalysed by the soluble fraction of liver cells. Kinetic evidence supports the conclusion that there are separate enzymes for the synthesis of glycocholate and taurocholate. The kinetic parameters of these enzymes were determined.

UDP-glucuronyltransferase (EC 2.4.1.17) in intact, untreated microsomes from pig liver is activated by relatively low concentrations of UDP-N-acetylglucosamine. This property is absent after treatment of microsomes with detergents or phospholipases, and also is not a characteristic of pure, delipidated enzyme. Sensitivity to activation by UDP-N-acetylglucosamine was reconstituted, however, by incorporation of pure, delipidated enzyme into unilamellar bilayers of phosphatidylcholine that were in a gel phase. Warming of these bilayers to just above the temperature for the gel to liquid crystalline phase transition led to an abrupt loss of sensitivity of UDP-glucuronyltransferase to activation by UDP-N-acetylglucosamine. These experiments establish that sensitivity to activation by UDP-N-acetylglucosamine is an inherent property of UDP-glucuronyltransferase. The data also suggest that the lipid environment of UDP-glucuronyltransferase in intact, untreated microsomes can modulate the sensitivity of this enzyme to allosteric activation by UDP-N-acetylglucosamine, and that this lipid environment is in a gel phase in intact microsomes at 37 degrees C.

The affinity of delipidated microsomal UDP-glucuronyltransferase (EC 2.3.1.17) for UDP is greater than that for UDP-glucuronic acid. Measurement of KIglucuronic acid reveals that glucuronic acid binds to the enzyme. Hence, the difference in affinity of the enzyme for UDP versus UDP-glucuronic acid indicates that inherent binding energy for interactions between enzyme and this substrate is used for purposes other than enhancing binding. A reasonable interpretation of these data is that the binding of UDP-glucuronic acid to enzyme requires distortion of the substrate and/or the enzyme. Inherent binding energy due to interactions between enzyme and UDP and glucuronic acid is utilized to effect such distortions. This type of mechanism can cause significant rate enhancement. Phospholipid activators of UDP-glucuronyltransferase activate by amplifying this basic mechanism. Thus, addition of various species of lysophosphatidylcholine to the delipidated enzyme increase the activity at Vmax and enhance the affinity for UDP, glucuronic acid, and UDP-glucuronic acid. However, activators enhance the affinity of the enzyme for UDP-glucuronic acid to a significantly smaller extent than they enhance affinity for the UDP and glucuronic acid portions of the substrate. Calculations of the amount of binding energy for interactions between enzyme and UDP-glucuronic acid that can be used for stimulating activities at Vmax yield values in agreement with the observed enhancement of activities at Vmax for enzyme reconstituted with various types of lysophosphatidylcholine.

The activation of delipidated microsomal UDP-glucuronosyltransferase from pig liver (GT2P type of enzyme) was studied as a function of several structural modifications of 1-palmitoyl-sn-glycero-3-phosphocholine, which is known to be a good activator of the enzyme. The following types of compounds were tested: substitution of H for OH at position 2; substitution of an ether for an acyl link at position 1; variation of the phosphorus-nitrogen or acyl ester-phosphate ester distances; removal of the glycerol backbone; optical isomers; and substitution of phosphoethanolamine for phosphocholine. Although there were variations in the extent to which these compounds activated delipidated enzyme, all the above types of lipids were effective in this regard. By contrast, lipids with a net negative charge did not activate the enzyme. They inhibited it reversibly. Positively charged lipids, even those lacking a phosphate group, were effective activators. These results indicate that GT2P is unlikely to interact with specific chemical groups of its phospholipid milieu. Effective activation appears instead to depend on the physical properties of the lipid environment.

The temperature dependence of two kinetic properties of the GT2P isoform of microsomal UDP-glucuronosyltransferase was studied for enzyme in intact microsomes and for pure enzyme reconstituted into different types of lipid bilayers. The properties studied were the non-Michaelis-Menten kinetics of the enzyme and activity at Vmax(app). For enzyme in intact microsomes, the pattern of non-Michaelis-Menten kinetics was seen at all temperatures in the range tested (23 to 48 degrees C), and the slopes of the Hill plots of the data were constant across this range of temperatures. Although non-Michaelis-Menten kinetics were present for pure enzyme in bilayers of 1,2-dimyristoylphosphatidylcholine or 1,2-dipalmitoylphosphatidylcholine only in the gel phase (Hockman, Y., Kelley, M., and Zakim, D. (1983) J. Biol. Chem. 258, 6509-6519), it was not possible to reconstitute this pattern of kinetics for enzyme at T greater than 40 degrees C. For example, GT2P displayed Michaelis-Menten kinetics in bilayers of 1,2-distearoylphosphatidylcholine at 44 degrees C. For enzyme in microsomes, activities at Vmax(app) increased with increasing temperature in the range 23 to 48 degrees C, with a discontinuity in the slope of the Arrhenius plot at 34 degrees C. This thermotropic property also could not be reconstituted with pure GT2P. Instead, activities at Vmax(app) for GT2P reconstituted in 1,2-dioleoylphosphatidylcholine, 1,2-distearoylphosphatidylcholine, or 1,2-stearoyl oleoylphosphatidylcholine increased in the range 23 to 37 degrees C, but then decreased at T greater than 37 degrees C. The fall in activity at T greater than 37 degrees C was reversible, indicating that GT2P undergoes a reversible change at 37 degrees C to a less active form of the enzyme. The differences between pure, reconstituted GT2P and microsomal GT2P indicate that the thermotropic properties of the microsomal enzyme are not properties of the enzyme per se but depend on interactions between it and other components in the microsome. The data suggest, therefore, that the properties of GT2P in microsomes results in part from an organization of components in the microsomal membrane.

A comparison has been made of the enzymes catalyzing the transfer of mannose, glucose and N-acetylglucosamine from, respectively, GDPmannose, UDP-glucose and UDP-N-acetylglucosamine to endogenous dolichol phosphate (Dol-P) in liver Golgi membranes. Evidence is presented with suggests that all three reactions utilize the same pool of Dol-P. The transfer of mannose from GDP-Man to Dol-P is not inhibited by 0.1 mM UDP or UMP; 0.1 mM GDP did block the accumulation of mannose in Dol-P-Man. The net transfer of glucose and N-acetylglucosamine to Dol-P is prevented by 0.1 mM UDP but not 0.1 mM GDP. UDPglucose inhibits the reverse of the glucose transfer reaction but not the reverse of the N-acetylglucosamine or mannose trasfer reaction. On the basis of this, and other data, it is concluded that the three sugar transfer reactions utilize separate enzymes.

Postnatal developmental changes in hapatic microsomal UDP-glucuronyltransferase were studied in the rat. The previously reported postnatal decline in the capacity of microsomal fractions to glucuronidate p-nitrophenol was found to be observable in unperturbed preparations only at non-saturating concentrations of the substrate UDP-glucuronic acid. At saturating concentrations of UDP-glucuronic acid, activity is identical in newborns and adults. Kinetic analysis revealed that the enzyme from liver of newborns has a much higher affinity for UDP-glucuronic acid than does the enzyme in adults, but the same activity at Vmax. On the other hand, the enzyme from adult liver microsomal fractions can be activated by the physiological allosteric effector UDP-N-acetylglucosamine, whereas the enzyme from newborns is largely unaffected by it. Thus it appears that the number of enzyme active sites is not changing; rather, the enzyme is maturing to a more highly regulable form. There were also differences between the enzymes in newborns and adults in their response to perturbation of the membrane-lipid environment by detergent and phospholipase A. Possible interpretations of these differences are discussed.