Biomedical subjects

D Mayer

Publications and source records attributed to D Mayer.

At least 91 records · Page 5Linked to original sources

PubMed1996

Dehydroepiandrosterone-induced lipid peroxidation in rat liver mitochondria.

Administration of dehydroepiandrosterone (DHEA), a steroid hormone of the adrenal cortex which acts as a peroxisome proliferator and hepatocarcinogen in the rat, caused an increase in NADPH-dependent lipid peroxidation in mitochondria isolated from the liver, kidney and heart, but not from the brain. The effect of DHEA on rat liver mitochondrial lipid peroxidation became discernible after feeding steroid-containing diet (0.6% w/w) for 3 days, and reached maximal levels between 1 and 2 weeks. DHEA in the concentration range 0.001-0.02% did not significantly increase lipid peroxidation compared to the control. Lipid peroxidation was significantly enhanced in animals given a diet containing > or = 0.05% DHEA. The addition of DHEA in the concentration range 0.1-100 microM to mitochondria isolated from control rats had no effect on lipid peroxidation. It seems, therefore, that the steroid effect is mediated by an intracellular process. Our data indicate that induction of mitochondrial membrane lipid peroxidation is an early effect of DHEA administration at pharmacological doses.

Animals↗

PubMed1996

Isolation of Mycoplasma conjunctivae from conjunctival swabs of Alpine ibex (Capra ibex ibex) affected with infectious keratoconjunctivitis.

Mycoplasma conjunctivae was isolated four times from the eyes of nine Alpine ibex (Capra ibex ibex) suffering from keratoconjunctivitis. The animals examined were affected in two different outbreaks in the Swiss Alps. Parasitological and bacteriological studies, including investigations for chlamydia and mycoplasmas, were performed. The results indicate that M. conjunctivae is the primary pathogenic agent causing infectious keratoconjunctivitis in this species.

Animals↗

PubMed1996

Eosinophil VLA-4 binding to fibronectin augments bronchial narrowing through 5-lipoxygenase activation.

We examined the effect of ligation of human eosinophils activated by platelet-activating factor (PAF) to soluble human fibronectin (FN) on the augmented contractile response of human bronchial explants. Styrene microplate wells were FN-coated and eosinophils were allowed to adhere in the presence of 1) buffer control, 2) 20 micrograms/ml monoclonal antibody (HP2/1) to the alpha 4 beta 1 ligand (VLA-4) on the eosinophils, 3) 20 micrograms/ml anti-CD18 R15.7, 4) 20 micrograms/ml anti-CD16 3G8, or 5) 10(-6) M A63162, a 5-lipoxygenase inhibitor. Sixty minutes later, treated cells were activated with either buffer or 10(-6) M PAF. Airway luminal diameter was assessed by computerized videomicrometry as a function of pixel number, and activation of eosinophils was confirmed by measurement of leukotriene C4 (LTC4) secretion. Ligation with FN caused an increase in PAF-stimulated LTC4 secretion from 276 +/- 75.6 pg/10(6) cell at baseline to 606 +/- 90.2 pg/10(6) cell (P < 0.01). This corresponded to augmented luminal narrowing of human bronchial explants from 25.3 +/- 9.39% (PAF activation alone) to 42.9 +/- 8.0% (PAF-activated eosinophils + FN) (P < 0.01). Both augmented airway luminal narrowing and increased LTC4 secretion caused by PAF-activated cells after FN ligation were blocked completely by anti-VLA-4 MAb (P < 0.05 vs. control). Pretreatment with 10(-6) MA63162 inhibited completely the PAF-stimulated LTC4 secretion to baseline level ( P < 0.001). Inhibition of 5-lipoxygenase similarly blocked luminal narrowing caused by eosinophils stimulated by PAF by > 95% (P < 0.001). We demonstrate that the binding of human eosinophils to the matrix protein FN causes augmented secretion of LTC4 which, in turn, causes augmented luminal narrowing of explanted human bronchi in vitro. We also demonstrate that the augmented activity is blocked selectively by pretreatment with specific monoclonal antibody against VLA-4 and blockade of eosinophil 5-lipoxygenase inhibits both LTC4 secretion and airway narrowing after PAF-stimulation.

Acetamides↗

PubMed1996

[Helicobacter pylori eradication therapy with bismuth citrate/amoxycillin combination therapy].

The efficacy of a new combination preparation containing bismuth citrate and amoxicillin in one tablet was compared with the efficacy of bismuth citrate monotherapy in a randomised double-blind study on the eradication of Helicobacter pylori. The study involved 70 H. pylori positive (antrum biopsies showing a positive urease test) patients with non-ulcer dyspepsia and chronic gastritis. The treatment period was 14 days; 35 patients in group 1 received 2 tablets tid containing the bismuth citrate amoxicillin combination (BIAM tablet; 250 mg amoxicillin base and 120 mg bismuth); 35 patients in group 2 were treated with 2 tablets tid containing bismuth citrate (BI tablet; 120 mg bismuth). Total daily dose was therefore 1500 mg amoxicillin + 720 mg bismuth in group 1 patients resp. 720 mg bismuth in group 2 patients. 4 weeks after therapy H. pylori could not be histologically detected in the antrum of 22 patients (63%) in group 1 and 8 patients (24%) in group 2. Thus in group 1 (BIAM) a significantly higher eradication rate (p < 0.001) was shown than in group 2 (BI). Inflammation characterized by the infiltration of polymorphonuclear cells was significantly (p < 0.01) less pronounced in group 1 (BIAM) than in group 2 (BI) 4 weeks after the end of treatment. Gastrointestinal distress was quantified by evaluation of 13 different symptoms using a fourpoints scale at the beginning of the study and after 2 and 6 weeks. The sum of scores decreased by 81% in group 1 (BIAM) and 71% in group 2 (BI) after 6 weeks.

Adult↗

PubMed1996

Biotransformation of the aerosol propellant 1,1,1,2,3,3,3-heptafluoropropane (HFA-227): lack of protein binding of the metabolite hexafluoroacetone.

The biotransformation of the aerosol propellant 1,1,1,2,3,3,3-heptafluoropropane (HFA-227) was investigated in rats in vivo and in rat and human liver microsomes. In the urine of rats exposed to 5000 ppm HFA-227 for 6 hr, very small amounts of hexafluoroacetone trihydrate were identified as an HFA-227 metabolite by 19F-NMR. Fluoride concentrations in the urine samples (0-48 hr after the end of the exposure) from exposed animals were not significantly different from those found in samples from nonexposed rats. In rat and human liver microsomes, fluoride and hexafluoroacetone trihydrate formation from HFA-227 was detected in very low levels only in liver microsomes from pyridine-treated rats and in two of eight human liver microsome samples, which exhibited the highest cytochrome P4502E1 activities. Because some aldehydes may covalently bind to proteins and the formation of fluorinated protein adducts has been implicated in immune-mediated hepatitis induced by halothane, the binding of hexafluoroacetone trihydrate to proteins was also investigated. Hexafluoroacetone trihydrate also gave only a very small resonance in fluorine NMR experiments when binding to human serum albumin was studied in comparison with the acylating agent S-ethyltrifluoroacetate. Moreover, no fluorine-containing products were formed by the reaction of hexafluoroacetone trihydrate with N alpha-acetyl-L-lysine, and hexafluoroacetone trihydrate was not metabolized to fluorine-containing metabolites or inorganic fluoride in rats. Comparative studies in human liver microsomes demonstrated that a halothane metabolite may covalently bind to proteins; in contrast, metabolism and covalent binding of HFA-227 could not be demonstrated. In summary, these data indicate that HFA-227 is biotransformed at very low rates to hexafluoroacetone trihydrate but irreversible binding of hexafluoroacetone trihydrate cannot be demonstrated, even with the application of very sensitive methods, and is considered unlikely, based on the combination of the results obtained.

Acetone↗

PubMed1996

[Risk of hyperthyroidism in examinations with contrast media].

Investigations with contrast media inevitably lead to the patient being exposed to large amounts of iodine. Under certain preconditions this gentails danger for the patient by causing either iodine-induced hyperthyroidism, which is difficult to treat, or even a thyrotoxic crisis. Patients with normal thyroid function and size have only minute changes of thyroid hormones and TSH within the normal range and are not at risk. Patients with unknown hyperthyroidism--independent of the etiological form--and patients with functional autonomy are at risk of exacerbation of pre-existing hyperthyroidism or development of iodine-induced hyperthyroidism. This development depends on two factors: a) the volume of autonomous tissue and b) the quantity of iodine exposure. Besides contrast media, other sources of iodine excess are possible, such as iodine-containing disinfectants, secretolytic agents, antiarrhythmics like amiodarone, eye drops and ointments, geriatrics, skin ointments, toothpaste etc. The development of hyperthyroidism can be prevented by combined treatment with antithyroid drugs and perchlorate in the case of preexisting hyperthyroidism or the urgent clinical suspicion of thyrotoxicosis or with perchlorate alone, when the patients is euthyroid and does not have a large nodular goitre.

Contrast Media↗

PubMed1995

The immunomodulatory effect of leflunomide in rat cardiac allotransplantation.

Leflunomide, a novel immunosuppressant, has been the subject of recent preclinical studies using solid organ allo- and xenotransplantation models. The objectives of this study were to evaluate the efficacy and toxicity of leflunomide using a rat cardiac allotransplant model in two different strain combinations (DA x PVG and DA x Lew). Leflunomide, at doses ranging between 5 and 30 mg/kg, prolonged graft survival in both strain combinations as effectively as CsA and FK506 1 mg/kg (P < 0.05). A dose-dependent effect was seen only after a longer treatment course. When ongoing rejection was intercepted early (postoperative day 2), 5 mg/kg was as effective as 1 mg/kg FK506 (P > 0.05) but was inferior to CsA in the DA x PVG combination (P < 0.05). However, in the DA x Lew combination, leflunomide was equally as efficacious as 15 mg/kg CsA and 1 mg/kg FK506 (P > 0.05). If ongoing rejection was treated at postoperative day 4, 10 mg/kg leflunomide was not only as effective as 15 mg/kg CsA and 1 mg/kg FK506, but demonstrated a dose-dependent increase in graft survival in both strain combinations. The toxicity of leflunomide at doses of especially 5-20 mg/kg was minimal in comparison to therapeutic doses of CsA and FK506 using body weight and biochemical parameters of renal and liver function. These in vivo observations convincingly show leflunomide to be equally as potent an immunosuppressant as CsA and FK506 in transplant rejection. It is also well tolerated on long-term administration and, by virtue of this fact, is a potentially suitable candidate for clinical transplantation.

Animals↗

PubMed1995

FK 506 versus cyclosporin in the prevention of renal allograft rejection--European pilot study: six-week results.

FK 506 was compared with cyclosporin in a randomised trial in good-risk cadaveric renal transplant recipients. The objective was to evaluate whether oral FK 506 dosing was viable and whether blood concentrations in the range 10-20 ng/ml would prove to be practical. Thirty-one adult patients were randomised to FK 506 and 16 to cyclosporin. Both groups received an identical regimen of azathioprine and corticosteroids. Serum creatinine concentrations decreased rapidly in both groups with mean values below 200 mumol/l within 2 weeks. One graft in the cyclosporin group was lost due to renal vein thrombosis. During the 6-week study period, 19.4% of patients on FK 506 and 31.3% on cyclosporin experienced acute rejection. One patient in each group experienced corticosteroid-resistant rejection that responded to anti-lymphocyte therapy. Infections were reported in 51.6% of the FK 506 group compared with 37.5% of the cyclosporin group. The spectrum of adverse events was similar in both groups. However, minor neurological disorders were more common in the FK 506 group (54.8% versus 6.3%) whereas hypertension was less common (48.8% versus 75.0%). The results indicate that oral FK 506 rapidly achieves therapeutic blood concentrations and is an effective immunosuppressant for the initial treatment of renal allograft recipients.

Adolescent↗

PubMed1995

Biliary complications after paediatric liver transplantation: Birmingham's experience.

Between 1983 and 1992, 112 children underwent liver transplantation. Of 138 grafts, 60 (43.4%) were whole livers, 77 (55.6%) were reduced livers, and 1 (0.7%) was a split liver. Biliary complications (BC) were defined as any abnormality, even minor, related to the biliary tract. Results were analysed with a minimum follow-up of 9 months. Some 36 grafts (26.1%) in 34 patients (30.4%) presented with BC: bile leaks (17 grafts), biliary obstructions or dilatations (16 grafts), and other complications (3 grafts). Management was mainly surgical with biliary reconstruction via a Roux-en-Y loop. Interventional radiology had an increasing role in recent years. BC were associated with a mortality of 1.8% (2/112), a graft loss rate of 4.3% (6/138), and significant morbidity. Among the various factors whose association with BC was studied, the date of transplantation, the use of reduced grafts and the use of gallbladder conduits appeared to be the main determining factors for BC. From multivariate analysis the use of reduced grafts emerged as the most important factor in reducing BC. We therefore conclude that BC are associated with significant morbidity, but general improvements in both surgical and medical management seem to account for better results in recent years.

Adolescent↗

PubMed1995

Rat heart-aorta cluster transplantation: a novel model to study transplant rejection.

The purpose of this study was to develop a microsurgical cluster model of heart plus entire thoracic aorta transplantation and to compare it to the isolated model of heart transplantation as a tool to study transplant rejection. Thirty-six syngeneic (DA x DA and Lew x Lew) and allogeneic (DA x PVG and DA x Lew) cluster heart-aorta transplants were compared to 43 syngeneic and allogeneic isolated heart grafts. Graft survival, recipient survival and histological data on myocardial and aortic tissues were assessed. There was no statistically significant difference in graft survival between the two models studied (P > 0.05). In the cluster transplants, the aortic component was spared the severity of acute rejection noted for the myocardial counterpart. In conclusion, the results demonstrated that the cluster model was technically feasible and highly reproducible. Additionally, it was possible to apply this model to the study of experimental allograft rejection using novel immunosuppressants. The success of the cluster model in strongly mismatched transplant strain combinations underscores its potential for application in slower rejection combinations, making it particularly suited for chronic rejection studies. The inherent capacity for sampling a broader range of vessel sizes in one animal makes the cluster model more suitable than the isolated models of aorta or heart for application to experimental protocols.

Animals↗

PubMed1995

Central processes in gustatory adaptation.

In two experiments the adapting stimuli at the periphery of the taste system were of a constant objective intensity but, using mixture suppression and spatial summation, their perceived intensity was varied. The results have shown that, in spite of the constancy in stimulus concentration, the adaptation degree changed with the perceived intensity of the stimulus. The adaptation to the compounds when in binary mixtures proved to be significantly less effective than the adaptation to the same equimolar compounds when unmixed. Similarly, the adaptation effects of solutions of a constant concentration, when applied to a small tongue area, were significantly smaller than when applied to a larger area. This adaptation dependence upon the taste sensation intensity suggests that in the gustatory adaptation, peripheral as well as central processes take part.

Attention↗

PubMed1995

Transport of dehydroepiandrosterone and dehydroepiandrosterone sulphate into rat hepatocytes.

The purpose of the present study was to characterize the transport of dehydroepiandrosterone (DHEA) and dehydroepiandrosterone sulphate (DHEAS) into hepatocytes at physiological and pharmacological concentrations. Hepatocytes were isolated from female Sprague-Dawley rats by collagenase perfusion. Uptake of [3H]DHEA and [3H]DHEAS at increasing concentrations (3.5 nM-100 microM) was measured by the rapid filtration technique at 30 s intervals up to 120 s. The uptake of DHEAS by hepatocytes was saturable (Km = 17.0 microM; Vmax = 3.7 nmol/min/mg cell protein). In contrast, a specific saturable transport system for DHEA could not be detected in rat hepatocytes. It is suggested that DHEA enters the cell by diffusion. The uptake of DHEAS could be inhibited by antimycin A, carbonylcyanide-m-chlorophenylhydrazone, and dinitrophenol (inhibitors of the mitochondrial respiratory chain), by dinitrofluorobenzene and p-hydroxymercuribenzoate (NH2- and SH-blockers, respectively), and by monensin (Na(+)-specific ionophore). No inhibition was seen in the presence of ouabain (inhibitor of Na(+)-K(+)-ATPase) and phalloidin (inhibitor of cholate transport and actin-blocker). Interestingly, DHEAS uptake was inhibited by bile acids (cholate, taurocholate and glycocholate). Conversely, [3H]cholate uptake was strongly inhibited by DHEAS, which indicates a competition for the same carrier. Replacement of sodium ion with choline markedly decreased uptake velocity at pharmacological DHEAS concentrations. The results suggest that DHEAS uptake is a saturable, energy-dependent, carrier-mediated, partially Na(+)-dependent process, and that DHEAS may be taken up via the multispecific bile acid transport system.

Animals↗

PubMed1995

Identification of the transcriptional activator controlling the butanediol fermentation pathway in Klebsiella terrigena.

The gene budR, whose product is responsible for induction of the butanediol formation pathway under fermentative growth conditions in Klebsiella terrigena, has been cloned and sequenced. This gene is separated from the budABC operon by a nontranslated region of 106 bp and transcribed in the opposite direction. budR codes for a protein of molecular weight 32,124, the sequence of which exhibits characteristics of regulators belonging to the LysR family. When transferred into the heterologous host Escherichia coli, budR activates expression of budA'-lacZ transcriptional and translational fusions with a regulatory pattern identical to that in K. terrigena, namely, induction by acetate, low pH, and anaerobiosis. Induction by acetate was specific, indicating that it is the physiological inducer. Primer extension analysis located the start site of transcription to two positions, 23 and 24 bp upstream of the budR initiation codon, and also showed that BudR strongly autoregulates its own expression. The products of fhlA, arcA, hip, ntrA, and katF did not influence expression of the bud operon. A mutation in fnr, however, led to a threefold increase in expression, indicating that Fnr acts as a repressor. The results support the notion that BudR coordinates the activity of the energy-conserving, nonreductive, but acidifying acetate formation pathway with the expression of the non-energy-conserving, reductive, but nonacidifying butanediol pathway.

Amino Acid Sequence↗

PubMed1995

Treatment of pain in chronic pancreatitis by inhibition of pancreatic secretion with octreotide.

It has been suggested that pancreatic ductal hypertension, secondary to pancreatic outflow obstruction, is a cause of pain in chronic pancreatitis. This study investigated the effect of inhibiting pancreatic secretion with octreotide in chronic pancreatitis pain. Ten patients with chronic alcoholic pancreatitis and severe daily pain were included in an intraindividual double blind crossover study. All patients received octreotide (3 x 100 micrograms/day subcutaneously) and placebo (3 x 0.9% saline solution subcutaneously) for three days at random. Between both treatment phases a two day washout period was interposed. Intensity of pain (visual analogue scale) and analgesic consumption were carefully registered. Pancreatic secretion was monitored daily by measuring faecal chymotrypsin concentration. It was found that during the administration of octreotide, pancreatic secretion was strongly inhibited (faecal chymotrypsin mean (SD) 1.7 (0.6) U/g) with respect to placebo (9.6 (4.2) U/g) and washout (7.6 (3.1) U/g) periods (p < 0.001). Pain score (29.6 (4.5) v 28.7 (5.8)) and consumption of analgesics were no different during the octreotide and placebo periods. It is concluded that short term inhibition of pancreatic secretion does not result in pain relief in patients with chronic pancreatitis. This finding is in contrast with the hypothesis that outflow obstruction of pancreatic secretion with consequent ductal hypertension is an important cause of severe persistent pain in chronic pancreatitis.

Adult↗

PubMed1995

Assessment of agonist- and cell-mediated responses in airway microsections by computerized videomicrometry.

The objective of this investigation was to develop a method for real-time measurement of changes in luminal area in microexplants of airways during pharmacological and physiological interventions. After guinea pigs were killed, tracheal rings (1- to 2-mm thick) were excised and placed in 300-microliters chambers. The area of the airway lumen was calculated as pixel number with the use of computerized videomicrometry. In 29 epithelium-intact airways, 10(-3) M acetylcholine (ACh) caused decrease in luminal area of 38.1 +/- 2.80% (P < 0.001 vs. 10(-9) M). Spontaneous tone also was demonstrated in 34 preparations from 4 guinea pigs; decrease in area of 17.0 +/- 1.45% after 60-min incubation in buffer alone was blocked completely by 10(-5) M indomethacin (P = 0.01). Luminal narrowing caused by < or = 10(-6) M ACh was reversed completely by 10(-6) M albuterol (P = 0.002). Addition of 100,000 activated human eosinophils caused 24.7 +/- 4.41% decrease in luminal area vs. 7.24 +/- 5.51% for nonactivated cells (P = 0.048). We demonstrate a real-time method for the assessment of auxotonic changes in airway caliber that utilizes microsections of explanted airways and permits the use of extremely small numbers of isolated cells to achieve physiological activation. Concentration-response characteristics and spontaneous tone are similar to those of large chamber preparations, and narrowing is reversed by beta 2-adrenoceptor activation.

Acetylcholine↗

PubMed1995

Paradoxical blockade of beta adrenergically mediated inhibition of stimulated eosinophil secretion by salmeterol.

Salmeterol (SALM) is a long-acting beta 2 adrenoceptor agonist that causes prolonged relaxation of airway smooth muscle. To determine whether this agent also causes prolonged inhibition of stimulated eosinophil secretion, we studied interactions between SALM and albuterol (ALB) in inhibiting eosinophil peroxidase (EPO) secretion in human eosinophils in vitro. Peripheral blood eosinophils were isolated from 18 human volunteers by negative immunoselection, and secretion of EPO was elicited with 10(-6) M formyl-met-leu-phe (fMLP) + 5 micrograms/ml cytochalasin B (CytB) in aliquots of 10(5) cells. Eosinophils were pretreated with either 10(-8) M ALB, 10(-8) M SALM or SALM + ALB for 5 min to 18 hr at 37 degrees C. Pretreatment with ALB for 5 min caused inhibition of stimulated secretion of EPO to 783 +/- 210 ng/10(6) cells vs. 1475 +/- 286 ng/10(6) cells for eosinophils not treated with ALB (P < .05; n = 5). Inhibition of EPO secretion caused by ALB was sustained for 30 min (924 +/- 160 ng/10(6) cells; P < .05 vs. fMLP + CytB; n = 5). By contrast, SALM had no inhibitory effect on fMLP-induced secretion after incubation for 5 min to 18 hr. In cells obtained from four separate isolations, pretreatment with 10(-8)M SALM before addition of ALB blocked the inhibition of EPO release caused by 10(-8)M ALB alone (486 +/- 28 ng/10(6) cells for ALB alone vs. 902 +/- 32 ng/10(6) cells for SALM + ALB; P < .01; n = 4).(ABSTRACT TRUNCATED AT 250 WORDS)

Adrenergic beta-Agonists↗

PubMed1994

Differences in expression and intracellular distribution of hexokinase isoenzymes in rat liver cells of different transformation stages.

The activity, intracellular distribution and mRNA expression of hexokinase isoenzymes were studied in normal rat liver, and in epithelial liver cells at different stages of neoplastic transformation, including non-tumorigenic and tumorigenic cell lines. In contrast to liver, all transformed cells exhibited only hexokinase I and II, which both showed significantly increased activity, hexokinase II being the more abundant form. In parallel, the mRNA expression of the two isoenzymes was elevated, indicating transcriptional control of gene expression. Hexokinase I and II were found in the cytosol and bound to mitochondrial membranes; the percentage of membrane-bound enzyme activity increased with the grade of transformation from 32% of total activity in normal liver up to 69% in dedifferentiated tumor cells. The ratio of hexokinase I/II was higher in the membrane fraction than in the cytosol. In all tissues studied hexokinase II could be resolved in two subtypes IIa and IIb by hydrophobic interaction chromatography. The relative proportion of cytosolic IIa and IIb varied significantly between normal liver (1:1) and transformed cells, and among cells of different transformation stages (4:1 to 1:10). IIa demonstrated the main activity in the more differentiated, IIb in the less differentiated cell lines. IIa-activity showed a good correlation with the intracellular glucose 6-phosphate concentration of the cells. The data indicate that neoplastic cell transformation is accompanied by progressive alterations in the proportion and subcellular distribution of hexokinase isoenzymes I and II.

Animals↗

PubMed1994

Microheterogeneity of cytosolic and membrane-bound hexokinase II in Morris hepatoma 3924A.

Phosphorylation of glucose by hexokinase is the key step in glucose and energy metabolism of the cell. In the Morris hepatoma 3924A, hexokinase II is the predominant hexokinase isoenzyme and occurs in the cytosol as well as bound to membranes. Hexokinase II was isolated by DEAE-cellulose chromatography from both the cytosolic and the mitochondria-enriched fractions and further resolved by hydrophobic-interaction chromatography on phenyl-Sepharose into two components designated hexokinase IIa and IIb. In both the soluble and the mitochondria-enriched fractions, type IIb was the predominant form, but the IIb/IIa ratio was higher in the particulate (6-8) as compared with the cytosolic fraction (1.5-2.0). Binding of the isolated forms of the enzyme to rat liver mitochondria resulted in a 2-10-fold activation of both subtypes. Biochemical characterization showed that both subtypes are closely related to the isoenzyme commonly referred to as hexokinase II, and that the microheterogeneity was not a consequence of contamination with hexokinase I or III. Both subtypes had a molecular mass of 110 kDa, they were inhibited by Pi at concentrations higher than 5 mM, and activated by the detergent CHAPS. The two subtypes differed in electrophoretic mobility (IIa > IIb), in Km values for glucose (IIa, 0.109 mM; IIb, 0.216 mM), in Ki values for glucose 6-phosphate (IIa, 25 microM; IIb, 0.106 mM), and in Ki values for glucose 1,6-biphosphate (IIa, 12.2 microM; IIb, 5.5 microM). An artificial proteolytic cleavage as cause of the hexokinase II microheterogeneity can be excluded, since both subtypes show the same molecular mass and the ability to bind to mitochondria and phenyl-Sepharose. In addition, the relative proportions of the two subtypes did not vary markedly between several enzyme preparations. Northern-blot analysis with a hexokinase II-specific cDNA probe revealed two distinct mRNA transcripts of 5.2 and 6.3 kb in length, which offers the possibility that hexokinase II microheterogeneity is due to differential RNA transcription and/or processing.

Animals↗