Biomedical subjects

N Nakai

Publications and source records attributed to N Nakai.

At least 55 records · Page 3Linked to original sources

PubMed1997

Participation of leukotriene D4 and tumor necrosis factor on lipopolysaccharide-induced airway hyperresponsiveness in guinea pigs.

In guinea pigs, a marked increase in airway responsiveness to acetylcholine (Ach) was observed at 2 h after lipopolysaccharide (LPS) inhalation. To examine the mediators responsible for the airway hyperresponsiveness, the changes of peptide-leukotrienes (LTs), tumor necrosis factor (TNF), interleukin-1 (IL-1), histamine and 5-hydroxytryptamine (5-HT) levels in bronchoalveolar lavage fluid (BALF) were measured. Airway responsiveness to Ach reached a peak 2 h after LPS inhalation. The influx of neutrophil into BALF increased gradually and reached a peak 24 h after LPS inhalation. After the inhalation of LPS, LTD4 and TNF contents in BALF increased within the first 2 h after LPS inhalation. However, other mediators were not detected or increased 6 h after LPS inhalation. Aeroinhalation of LTD4 and murine recombinant TNF-alpha caused airway hyperresponsiveness in guinea pigs. In addition, a LTD4 antagonist, BAYx7195, and an inhibitor of TNF, pentoxifylline, inhibited the LPS-induced airway hyperresponsiveness. These results suggest that LTs and/or TNF play an important role in the onset of airway hyperresponsiveness in guinea pigs.

Acetylcholine↗

PubMed1996

Glycogen depletion of the intrafusal fibers in a mouse muscle spindle during prolonged swimming.

This study investigated the recruitment of different types of intrafusal fibers during prolonged swimming at 60-75% of VO2max. We used 56 male adult mice and examined depletion of glycogen in soleus (Sol) and extensor digitorum longus (EDL) muscle spindles by visual inspection and a newly developed optical scanning method. More than 80% of all spindles from six mice consisted of four fibers: one type I nuclear bag (bag1) fiber, one type II nuclear bag (bag2) fiber, and two nuclear chain fibers. Glycogen content was estimated in muscle fibers from groups of six mice that had rested or swum for either 0.5, 1, 2, 4, or 8 h. The optical scanning intensity of periodic acid Schiff (PAS)-stained sections was correlated with their biochemically determined glycogen content (r = 0.93). Both methods showed fundamentally the same result: each type of intrafusal fiber has its own typical recruitment pattern during exercise. In the initial phase (0-0.5 h), glycogen depletion was largest in nuclear bag1 fibers and insignificant in the bag2 and chain fibers. With the bag1 fibers having become fatigued, nuclear bag2 fibers mainly took over during the middle phase (2-4 h). During the last phase (4-8 h), only the glycogen content of chain fibers decreased significantly (4-8 h). There were significant correlations between the recruitment pattern of bag1 and extrafusal type I fibers in both Sol and EDL, between nuclear bag2 and type IIa fibers in Sol, and between nuclear chain and type IIb fibers in EDL. This suggests that, during moderately intense exercise, glycogen depletion occurs first in the slow, then the intermediate, and, finally, the fast intrafusal fibers.

Animals↗

PubMed1996

Exercise training prevents maturation-induced decrease in insulin sensitivity.

We examined the effects of exercise training initiated before maturation or after maturation on insulin sensitivity and glucose transporter GLUT-4 content in membrane fractions of skeletal muscle. Female Wistar rats (4 wk of age) were divided into sedentary and exercise-trained groups. At 12 wk of age, a subset of the trained animals (Tr) was killed along with a subset of sedentary controls (Sed). One-half of the remaining sedentary animals remained sedentary (Sed-Sed) while the other half began exercise training (Sed-Tr). The remaining rats in the original trained group continued to train (Tr-Tr). Euglycemic clamp (insulin infusion rate at 6 mU.kg body wt-1. min-1) was performed at 4, 12, and 27 wk. After euglycemic clamp in all animals except the 4-wk-old, hindlimb (gastrocnemius and part of quadriceps) muscles were removed for preparation of membrane fractions. In sedentary rats, glucose infusion rate (GIR) during euglycemic clamp was decreased from 15.9 mg.kg-1.min-1 at 4 wk of age to 9.8 mg.kg-1.min-1 at 12 wk of age and 9.1 mg.kg-1.min-1 at 27 wk of age. In exercise-trained rats, the GIR was not significantly decreased by maturation (at 12 wk) and further aging (at 27 wk). Initiation of exercise after maturation restored the GIR at 27 wk of age to the same levels as these for the corresponding exercise-trained rats. GLUT-4 content in plasma and intracellular membrane fractions of hindlimb muscle obtained just after euglycemic clamp showed the same trend as the results of GIR. These results suggest that exercise training prevented the maturation-induced decrease in insulin sensitivity. Improvement of insulin sensitivity caused by exercise training was attributed, at least in part, to the increase in insulin-sensitive GLUT-4 on the plasma membrane in skeletal muscle.

Age Factors↗

PubMed1995

Probing active galactic nuclei with H2O megamasers.

We describe the characteristics of the rapidly rotating molecular disk in the nucleus of the mildly active galaxy NGC4258. The morphology and kinematics of the disk are delineated by the point-like watervapor emission sources at 1.35-cm wavelength. High angular resolution [200 microas where as is arcsec, corresponding to 0.006 parsec (pc) at 6.4 million pc] and high spectral resolution (0.2 km.s-1 or nu/Deltanu = 1.4 x 10(6)) with the Very-Long-Baseline Array allow precise definition of the disk. The disk is very thin, but slightly warped, and is viewed nearly edge-on. The masers show that the disk is in nearly perfect Keplerian rotation within the observable range of radii of 0.13-0.26 pc. The approximately random deviations from the Keplerian rotation curve among the high-velocity masers are approximately 3.5 km.s-1 (rms). These deviations may be due to the masers lying off the midline by about +/-4 degrees or variations in the inclination of the disk by +/-4 degrees. Lack of systematic deviations indicates that the disk has a mass of <4 x 10(6) solar mass (M[symbol: see text]). The gravitational binding mass is 3.5 x 10(7) M[symbol: see text], which must lie within the inner radius of the disk and requires that the mass density be >4 x 10(9) M[symbol: see text].pc-3. If the central mass were in the form of a star cluster with a density distribution such as a Plummer model, then the central mass density would be 4 x 10(12) M[symbol: see text].pc-3. The lifetime of such a cluster would be short with respect to the age of the galaxy [Maoz, E. (1995) Astrophys. J. Lett. 447, L91-L94]. Therefore, the central mass may be a black hole. The disk as traced by the systemic velocity features is unresolved in the vertical direction, indicating that its scale height is <0.0003 pc (hence the ratio of thickness to radius, H/R, is <0.0025). For a disk in hydrostatic equilibrium the quadrature sum of the sound speed and Alfven velocity is <2.5 km.s-1, so that the temperature of the disk must be <1000 K and the toroidal magnetic field component must be <250 mG. If the molecular mass density in the disk is 10(10) cm-3, then the disk mass is approximately 10(4) M[symbol: see text], and the disk is marginally stable as defined by the Toomre stability parameter Q (Q = 6 at the inner edge and 1 at the outer edge). The inward drift velocity is predicted to be <0.007 km.s-1, for a viscosity parameter of 0.1, and the accretion rate is <7 x 10(-5) M[symbol: see text].yr-1. At this value the accretion would be sufficient to power the nuclear x-ray source of 4 x 10(40) ergs-1 (1 erg = 0.1 microJ). The volume of individual maser components may be as large as 10(46) cm3, based on the velocity gradients, which is sufficient to supply the observed luminosity. The pump power undoubtedly comes from the nucleus, perhaps in the form of x-rays. The warp may allow the pump radiation to penetrate the disk obliquely [Neufeld, D. A. & Maloney, P. R. (1995) Astrophys. J. Lett. 447, L17-L19]. A total of 15 H2O megamasers have been identified out of >250 galaxies searched. Galaxy NGC4258 may be the only case where conditions are optimal to reveal a well-defined nuclear disk. Future measurement of proper motions and accelerations for NGC4258 will yield an accurate distance and a more precise definition of the dynamics of the disk

Journal Article↗

PubMed1995

Matrix metalloproteinase 7 (matrilysin) from human rectal carcinoma cells. Activation of the precursor, interaction with other matrix metalloproteinases and enzymic properties.

Matrix metalloproteinase 7 (MMP-7) has been purified as an inactive zymogen of M(r) 28,000 (proMMP-7) from the culture medium of CaR-1 human rectal carcinoma cells. The NH2-terminal sequence of proMMP-7 is Lys-Pro-Lys-Pro-Gln-Glu, which is identical to that of matrilysin. The zymogen is activated by 4-aminophenylmercuric acetate (APMA), yielding an intermediate form of M(r) 21,000 and an active species of M(r) 19,000 which shows the new NH2-terminal sequence of Tyr78-Ser-Leu-Phe-Pro-Asn-Ser. Although trypsin fully activates the zymogen, the activation rate by plasmin or leukocyte elastase is confined to approximately 50%. ProMMP-7 can be activated by MMP-3 (stromelysin 1) to its full activity in a single-step mechanism and generates the same NH2 terminus obtained by APMA activation, whereas MMP-1 (tissue collagenase), MMP-2 (gelatinase A), and MMP-9 (gelatinase B) do not have such an effect. On the other hand, proMMP-1 is activated by MMP-7 to an activity similar to that obtained by APMA and the activation by MMP-7 is enhanced up to approximately 6.5 fold in the presence of APMA. This enhanced activity is donated by specific cleavage at the Gln80-Phe81 bond of proMMP-1. MMP-7 can also activate proMMP-9 up to approximately 50% of the full activity with a new NH2 terminus of Leu16-Arg-Thr-(Asn)-Leu. Incubation of proMMP-2 or proMMP-3 with MMP-7 results in no activation of these proMMPs. MMP-7 degrades type IV collagen, laminin-1, fibronectin, proteoglycan, type I gelatin, and insoluble elastin. These results suggest that in vivo MMP-7 may play a role in degradation of extracellular matrix macromolecules in concert with MMP-1, -3, and -9 under pathological conditions.

Collagenases↗

PubMed1995

Regulation by physical training of enzyme activity and gene expression of branched-chain 2-oxo acid dehydrogenase complex in rat skeletal muscle.

We examined the effects of short-term (5 weeks) and long-term (12 weeks) physical training on actual and total activities, protein content and mRNA abundance of branched-chain 2-oxo acid dehydrogenase complex in rat skeletal muscle. The actual and total activities were significantly increased approximately 60% and approximately 40%, respectively, by long-term training. No effects of short-term training on activities were observed. The increase in the total activity corresponded to increased protein content of the E1 alpha and E2 components of the complex. On the other hand, mRNA abundance for E1 alpha and E2 were not affected by the training, but that for E1 beta was slightly, but significantly increased by both short-term and long-term trainings. These divergent alterations of the message levels for the subunits of the complex suggest that posttranslational regulatory mechanisms determine the amount of the complex in skeletal muscle. Since the complex is located in the mitochondrial matrix space, mitochondrial biogenesis in response to the training was examined by determining the content of mitochondrial DNA in the muscle. The mitochondrial DNA was proportionally increased with the total activity as well as the protein content of the complex, suggesting that expression of branched-chain 2-oxo acid dehydrogenase complex in skeletal muscle in response to physical training is associated with mitochondrial biogenesis.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1995

Selective potentiation of IGE-dependent histamine release from rat peritoneal mast cells by stem cell factor.

Effect of stem cell factor on histamine release from rat peritoneal mast cells was studied. Although stem cell factor did not evoke histamine release by itself, it clearly potentiated histamine release from sensitized mast cells caused by antigen, anti-IgE and concanavalin A. However, stem cell factor did not affect histamine release caused by compound 48/80, calcium ionophore A23187 and substance P. Although maximum potentiation of antigen-induced histamine release by stem cell factor was accomplished after 1-10 minute-preincubation, potentiation was decline after a longer incubation period. Potentiation of histamine release by phosphatidylserine and non-mast cells in the rat peritoneal cavity was incubation time-dependent. Potentiation by stem cell factor was additive to that by phosphatidylserine or non-mast cells. These results indicate that stem cell factor selectively potentiates IgE-dependent histamine release from rat peritoneal mast cells, and suggest that the mechanism involved is distinct from that of phosphatidylserine or non-mast cells in the rat peritoneal cavity.

Animals↗

PubMed1995

Branched-chain alpha-keto acid dehydrogenase complex in rat skeletal muscle: regulation of the activity and gene expression by nutrition and physical exercise.

Branched-chain alpha-keto acid dehydrogenase complex is the rate-limiting enzyme in the catabolism of branched-chain amino acids in skeletal muscle. It is suggested that activation of this enzyme in the muscle during exercise plays an important role in the increased oxidation of branched-chain amino acids in the muscle. Evidence suggests that branched-chain alpha-keto acids, the substrates for the enzyme, regulate the activity state of the enzyme in the muscle during exercise through phosphorylation/dephosphorylation cycle of the enzyme protein. We propose a model for the mechanism of enzyme activation by exercise. In addition to this acute effect of exercise, we present evidence suggesting that exercise training modulates the enzyme activity and gene expression for the enzyme. Increases in the total activity as well as enzyme proteins by exercise training are suggested to be associated with mitochondrial biogenesis in the muscle.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1995

[Single dose toxicity studies of montirelin hydrate(NS-3) in mice, rats and dogs].

The single dose toxicity studies of montirelin hydrate (NS-3), a new drug for the treatment of disturbance of consciousness, were conducted in Slc:ddY mice, Slc:SD rats, and beagle dogs of both sexes. The drug was administered intravenously (i.v.) to mice, rats and dogs, and intramuscularly (i.m.) to mice and rats. The animals were observed for 14 days after administration. LD50 values were more than 500 mg/kg and 200 mg/kg in mice and rats, respectively, by the i.v. route, and more than 20 mg/kg in both animal species by the i.m. route. In dogs, the minimum lethal dose was more than 200 mg/kg by the i.v. route. Mice that received more than 125 mg/kg by the i.v. route showed tremor and a decrease in locomotor activity during administration and for 30 min thereafter. Mice that received more than 5 mg/kg by the i.m. route showed tremor 5 min after administration and for 2 hr thereafter. Rats that received more than 50 mg/kg by the i.v. route showed tremor, and those that received 200 mg/kg by the same route showed a decrease in locomotor activity and ataxic gait, during and immediately after administration. Rats that received more than 5 mg/kg by the i.m. route showed tremor, and those that received 20 mg/kg by the same route showed salivation 5 min after administration and for 30 min thereafter. Dogs that received more than 12.5 mg/kg by the i.v. route revealed excitement, biting, vocalization, mydriasis, salivation, urination, defecation, licking chops, vomiting, increase in heart rate, panting, hyperthermia, tremor and conjunctival injection during administration and for 6 hr thereafter. The body weight, food consumption and water consumption, and pathological findings showed no changes attributable to the dosing of montirelin hydrate in any animal.

Animals↗

PubMed1994

Purification and partial characterization of 3-hydroxyisobutyryl-coenzyme A hydrolase of rat liver.

An unusual feature of valine catabolism is a reaction in which an intermediate of its catabolic pathway, (S)-3-hydroxyisobutyryl-CoA, is hydrolyzed to give the free acid and CoA-SH. The enzyme responsible for this reaction, 3-hydroxyisobutyryl-CoA hydrolase (EC 3.1.2.4), was purified 7200-fold from rat liver in this study. The purified enzyme consists of a single polypeptide with an M(r) of 36,000 in the native and denatured forms. The hydrolase is highly specific for (S)-3-hydroxyisobutyryl-CoA and 3-hydroxypropionyl-CoA (Km, 6 and 25 microM, respectively) with optimal activity around pH 8. The turnover rate of the enzyme for (S)-3-hydroxyisobutyryl-CoA is 270 s-1, which is high relative to other enzymes of the valine pathway. Likewise, activity of the enzyme expressed on a wet weight basis is also very high in the major tissues of the rat. These findings suggest that rapid destruction of (S)-3-hydroxyisobutyryl-CoA produced during valine catabolism is physiologically important. We propose that the need for a mechanism to protect cells against the toxic effects of methacrylyl-CoA, which is maintained in equilibrium with (S)-3-hydroxyisobutyryl-CoA by crotonase, explains why valine catabolism involves this enzyme and why its tissue activity is so high.

Acyl Coenzyme A↗

PubMed1994

Enzymatic and genetic adaptation of soleus muscle mitochondria to physical training in rats.

To evaluate the effects of physical training on mitochondrial gene expression and mitochondrial biogenesis in slow-twitch muscle, adult female Sprague-Dawley rats were trained for 3, 6, and 12 wk by running on a motor-driven treadmill (speed of 25 m/min and duration of 90 min/day, 5 days/wk), and the activities of citrate synthase, ubiquinol-cytochrome-c oxidoreductase, cytochrome oxidase, mitochondrial cytochrome b mRNA (by Northern blot analysis), and mitochondrial DNA (by slot-blot and Southern blot analyses) were measured in rat soleus muscle. A DNA probe for detection of mitochondrial mRNA and DNA was prepared from a 1,500-bp fragment of human mitochondrial DNA that included the coding region of the cytochrome b gene. Training for 3, 6, and 12 wk significantly increased the activities of citrate synthase (31, 28, and 47%, respectively), ubiquinol-cytochrome-c oxidoreductase (61, 63, and 77%, respectively), and cytochrome oxidase (25, 26, and 32%, respectively) in muscle. The concentration of cytochrome b mRNA in the muscle was proportionally elevated with the enzyme activities. On the other hand, the mitochondrial DNA concentration in the muscle was not altered by training for 3 or 6 wk but increased significantly after training for 12 wk (35% in the slot-blot analysis and 31% in the Southern blot analysis). These results suggest that an increase in the oxidative capacity of slow-twitch muscle by the relatively short-term training is regulated at the pretranslational step in mitochondrial protein synthesis but that the increase by the long-term training involves mitochondrial replication.

Adaptation, Physiological↗

PubMed1992

[Age-related changes in hematological and serum biochemical values in cats].

Nineteen hematological and serum biochemical values were analyzed for 91 healthy cats of both sexes (aged 1 to 48 months) that were bred and reared in our laboratory. Age-related changes were found for many parameters. Red blood cell counts (RBC), hemoglobin (Hb), hematocrit (Ht), Mean corpuscular constants, GPT, total protein (TP) and albumin (ALB) initially were low but increased then stabilized. White blood cell counts (WBC), alkaline phosphatase (ALP), inorganic phosphorus (Pi), total bilirubin (TBil), total cholesterol (TC), glucose (GLU), and triglyceride (TG) initially were high, but decreased then stabilized. No age-related changes were found for GOT, blood urea nitrogen, or calcium. Of the parameters that changed with age, the mean corpuscular constants, GPT, GLU, and TG became stabilized during the first 3 to 4 months of life, but others (RBC, Hb, Ht, TP, ALB) became stabilized after 9 to 11 months, during which period body weight reached a plateau. Some parameters (WBC, ALP, TG, Pi) showed change up to 18 months of age. These results suggest that cats 9 to 11 months old can be regarded as adults; but for some parameters, cats aged 18 months, or older, are better regarded as adults. Sex-related differences in the values for mean corpuscular volume, mean corpuscular hemoglobin, and WBC that were found after 11 months of age were higher in females. ALB was higher in males.

Age Factors↗

PubMed1991

Phylogenetic affiliation of ancient and contemporary humans inferred from mitochondrial DNA.

Nucleotide sequence analysis of the major non-coding region of human mitochondrial DNA (mtDNA) from three major races was extended with data from 27 contemporary Mongoloids (20 from southeast Asia, seven from America) and 11 Ancient Japanese bones (five from Jomon Age; 3000-6000 years BP, six from the early modern Ainu; 200-300 years BP). In both cases, the sequence was determined directly from the polymerase chain reaction products. Based on a comparison of the 482 base pair sequences from a total of 128 contemporary humans, the nucleotide diversity is estimated to be 1.46%, which is three times higher than the corresponding value estimated from restriction-enzyme analysis of the whole mtDNA genome. The phylogenetic tree revealed that all lineages are classified into at least five clusters designated as C1-C5. C1 consists exclusively of Africans, and most Asians and Europeans formed C2, C3, C5 and C4, respectively. Phylogenetic analysis also indicated that part of the Asians, including the Japanese, subsequently diverged from the majority of Africans, and that Asians can therefore be separated into two distinct groups. Native Americans, however, appeared only in C3 and C5, suggesting that the size of the founder population was not so large during the peopling of American. Nucleotide sequences derived from ancient bones in a highly polymorphic region were also compared with those of contemporary humans. The nucleotide diversity among the 139 sequences in the region was estimated to be 2.26%. A group of ancient Japanese, including both Jomon peoples and the Ainu, showed a close phylogenetic affiliation with one group of contemporary Japanese and southeast Asians.(ABSTRACT TRUNCATED AT 250 WORDS)

Asian People↗

PubMed1988

[Hepato-portal-splenic dynamics after hepatectomies].

The present study was performed to clarify the regeneration and splenic changes following hepatectomy. The processes were compared between the three classes (L greater than 50%, 30 less than M less than 50%, S less than 30%) categorized by the resection rate (%) calculated by CT scan in the 26 cirrhotics (LC) and 22 non-cirrhotics (N). Hepato-splenic volumes were serially measured by CT scan. The regenerative speed (cm3/day) of the remaining liver were significantly higher in the N group than LC and in the larger resection class. Liver functions tended to return to the initial levels behind the time of restoration of the liver volumes. The delay was partially caused by the posttransfusion hepatitis (PTH), which developed more frequently with an increase of fresh frozen plasma transfusion given. The changing pattern of splenic size was strongly regulated by the massiveness of hepatectomy. Newly developed posthepatectomy esophageal variceal ruptures were endoscopically determined in the 10% of the 129 late deaths after hepatectomies for hepatomas (1973-1987) with or without recurrences and its occurrence was found to be enhanced in the massive resection in the LC group ended with the poor regeneration of the liver and persistent postoperative splenic enlargement.

Esophageal and Gastric Varices↗

PubMed1986

Mechanism of enzymic isomerization and epimerization of D-erythrose 4-phosphate.

The mechanism of the enzymic isomerization and epimerization of D-erythrose 4-phosphate (Ery4P) by an enzyme preparation from bovine liver was investigated with the use of 2H2O. The incorporation of 2H was quantitatively determined by a procedure using gas chromatography-mass spectrometry. About one atom of 2H was incorporated per molecule of the enzymic epimerization reaction product of Ery4P (D-threose 4-phosphate) or that of D-ribulose 5-phosphate. Computer simulation of the Ery4P isomerization reaction indicated that the 2H of 2H2O was not directly incorporated into the enzymic reaction product (D-erythrulose 4-phosphate). Instead, intramolecular transfer of hydrogen atoms had occurred.

Carbohydrate Epimerases↗

PubMed1985

Aldose reductase inhibitors: flavonoids, alkaloids, acetophenones, benzophenones, and spirohydantoins of chroman.

The inhibitory activity of various compounds, including 12 flavonoids, 10 alkaloids, 15 benzophenones, 5 acetophenones, and 7 spirohydantoins of chroman, was tested on rabbit lens aldose reductase, an enzyme involved in complications of diabetes. Almost all compounds tested were found to inhibit the enzyme at low concentrations (10(-5) M). The most potent inhibitor was 2R,4S-6-chloro-2-methylspiro(chroman-4,4'-imidazo-lidine+ ++)-2',5'-dione with an I50 value of 4.7 x 10(-8) M; other spirohydantoins showed similar potency. Polyhydroxybenzophenones were also potent inhibitors with an I50 value of about 10(-7) M. The possible structure-inhibitory activity relationships of the compounds tested are discussed.

Acetophenones↗

PubMed1984

Change in kinetic property of M1-type pyruvate kinase from hyperbolic type to sigmoidal type by limited proteolysis with an acid fraction from rabbit liver.

An acid extract of rabbit liver contained M1-type pyruvate kinase inactivating activity, and was separated to three fractions. The optimal inactivation of the enzyme with Fraction II (Mr 42,000) was observed at pH 5.5, and this inactivation was completely prevented by leupeptin and antipain, but not by pepstatin. With Fraction III (Mr 22,000), on the other hand, optimal inactivation of the enzyme was observed at pH 8-9, and was not prevented by these inhibitors. The kinetic properties, with phosphoenolpyruvate, of the enzyme were changed from hyperbolic type to sigmoidal type by the limited proteolysis with Fractions II and III. The subunit molecular weight of the enzyme (57,300) was decreased to 55,800 via 56,400 in the former case and to 56,400 in the latter case.

Animals↗

PubMed1984

Photo-oxidation of histidine residues in rat M1- and L-type pyruvate kinases.

Rat M1- and L-type pyruvate kinases were inactivated by photo-oxidation mediated by methylene blue at pH 8.0 and O degrees C according to first-order kinetics. The pH profiles of the inactivation rates of these isozymes showed that amino acid residues having a pK value of 7.0-7.5 were involved in the inactivation. Three histidine residues per subunit of M1- or L-type enzyme were destroyed by the photo-oxidation with complete loss of the enzyme activities. However, two of the three photo-oxidized histidine residues in the L-type enzyme were more important in the inactivation reaction. The kinetics of the partially inactivated L-type enzyme suggests that complete inactivation is achieved via an intermediate form having low affinity for phosphoenolpyruvate (PEP). These observations revealed the involvement of essential histidine residues of two different kinds in the catalytic mechanism of the L-type enzyme. In the photo-oxidation of M1-type enzyme, no intermediate form was observed. Addition of PEP or pyruvate to the reaction mixture markedly prevented the photo-oxidative inactivation of only the M1-type enzyme in the presence of K+ and Mg2+; the addition of ADP or ATP was ineffective even in the presence of both metal ions. This protective effect of PEP was counteracted by further addition of ATP but not by ADP. However, photo-oxidative inactivation of the L-type enzyme was not prevented even by the addition of PEP in the presence of both metal ions, owing to the low affinity for PEP at 0 degrees C, in spite of the presence of fructose 1,6-bisphosphate (Fru-1,6-P2).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗