Morphological changes in human gastric tumours after eradication therapy of Helicobacter pylori in a short-term follow-up.

BACKGROUND: It is controversial as to whether the development of gastric cancer is influenced by Helicobacter pylori eradication. If eradication itself influences the tumour morphology, this may affect the tumour discovery rate. AIM: To investigate the morphological changes in the gastric neoplasm after H. pylori eradication. METHODS: We studied 37 patients with eradication therapy. After a 1-month follow-up, endoscopic re-evaluation was performed and the appearance was compared with first image. All lesions were resected endoscopically, and were subjected to histological assessment and to immunohistochemistry. Serum gastrin levels were determined before and after eradication. RESULTS: Twenty-nine of 37 patients underwent successful eradication. The appearance of 11 lesions (33% of 33 lesions) became indistinct after successful eradication. All lesions were of the superficial-elevated type and the height of the lesions decreased. We detected normal columnar epithelium over the neoplasm in eight of the lesions. Higher expression of single-stranded deoxyribonucleic acid in the deep area was characteristic in tumours with an indistinct appearance. These changes did not correlate with the serum gastrin levels. CONCLUSIONS: The morphology of the gastric neoplasm change after eradication in the short-term. This may contribute to the decreased tumour discovery rate.

[Multiple myeloma with Sweet disease developing from monoclonal gammopathy of undetermined significance and Sjögren syndrome].

A 57-year-old woman was diagnosed as having monoclonal IgG kappa gammopathy of undetermined significance with Sjögren syndrome. Five years later, she was admitted with an increased level of serum IgG and diagnosed as having multiple myeloma. After admission, fever and painful erythema developed. Combined chemotherapy with adrenal cortical steroid diminished the skin lesions. Erythema recurred during treatment with granulocyte colony-stimulating factor for neutropenia due to chemotherapy. A biopsy specimen from the skin revealed dense neutrophilic infiltration in the dermis, and a diagnosis of Sweet disease was made.

[Burkitt's lymphoma originating in gluteal muscle tissue].

We report a case of Burkitt's lymphoma originating in gluteal muscle tissue. A 61-year-old Japanese man was admitted to our hospital due to painful swelling of the left femur and gluteal region in October 1996. A laboratory examination disclosed elevated levels of serum lactate dehydrogenase and soluble interleukin 2 receptor. Gallium scintigraphy demonstrated accumulations in the left femur and gluteal region. Magnetic resonance imaging yielded intense signals and disclosed swelling of muscle tissue in the same region. Pathological examination of biopsy specimens from the left femur revealed a starry-sky pattern, and a chromosomal analysis revealed t(2;8)(p11;q24). Heightened concentrations of antibody for Epstein-Barr virus were not detected. Non-African type Burkitt's lymphoma was diagnosed on the basis of these findings. CHOP therapy and irradiation of the affected region were initially effective, but the disease eventually became resistant to treatment. The patient died of cerebral hemorrhage. As far as we know, this is the first report in Japan of Burkitt's lymphoma originating in muscle tissue.

Endogenous protease-dependent replication of human influenza viruses in two MDCK cell lines.

Multi-cycle replication and plaque formation of influenza A and B viruses and cleavage activation of their hemagglutinin (HA) by an endogenous protease(s) were examined in two MDCK cell lines, MDCK(-) and MDCK(+). No exogenous trypsin was required for multi-cycle replication and plaque formation of all the influenza A viruses tested in the MDCK(+) cell, while those of the viruses in the MDCK(-) cell were completely trypsin-dependent. In both cell lines, on the other hand, influenza B viruses grew well in the absence of trypsin. The capability of multiple replication and plaque formation of the influenza viruses correlated with cleavage of the HA precursor (HA0) to HA1 and HA2, indicating that both cell lines express an HA activating endoprotease(s); that of the MDCK(+) cell activates the HA of influenza A and B viruses, and that of the MDCK(-) cell does only the HA of influenza B virus. Furthermore, the protease of the MDCK(+) cell was strongly suggested to be present on the cell surface and a serine protease. The MDCK(+) cell would be useful for isolation of influenza viruses from clinical specimens and for screening of protease inhibitors for anti-influenza virus drugs.

Preventative and therapeutic effects of AE0047 on renal injury in stroke-prone spontaneously hypertensive rats.

1. The present study was designed to investigate the preventative and therapeutic effects of AE0047 on renal injury compared with those of nitrendipine in stroke-prone spontaneously hypertensive rats (SHRSP). 2. In the preventative study, drug administration was started before the appearance of renal injury, such as proteinuria. Treatment for 6 weeks with AE0047 (1 and 3 mg/kg, p.o.) led to a dose-related reduction in systolic blood pressure (SBP). Nitrendipine, at doses of 10 and 30 mg/kg, also lowered SBP to a similar degree to that seen with AE0047 at 1 and 3 mg/kg, respectively. 3. In the vehicle-administered SHRSP group, urinary excretion of protein (Uprotein V) increased progressively from 14 weeks of age for another 6 weeks. AE0047 at both doses maintained Uprotein V within normal levels throughout the experimental period. However, the elevation of Uprotein V was only inhibited in the 30 mg/kg nitrendipine-treated group. Urinary N-acetyl-beta-D-glucosaminide (NAG) activity in the vehicle-treated SHRSP group was elevated. Urinary NAG activity remained at a low level only in AE0047-treated groups. 4. Histopathological examination revealed severe lesions (i.e. fibrinoid necrosis, proliferative vasculitis and glomerular lesions) of the kidney in SHRSP. AE0047 treatment at each dose attenuated the development of renal lesions in SHRSP. In contrast, nitrendipine, at 10 mg/kg, was ineffective against the development of renal lesions. Although nitrendipine at 30 mg/kg suppressed the development of renal lesions, this effect was still weaker than that seen with AE0047 at 1 mg/kg. 5. In the therapeutic study, drugs were administered to 17-week-old SHRSP with moderate renal damage for 10 days. Treatment with AE0047 (1 and 3 mg/kg) produced dose-dependent decreases in Uprotein V. In the nitrendipine-treated group, Uprotein V tended to decrease but the changes were not significant. 6. Histopathological studies revealed that 3 mg/kg AE0047 improved renal lesions, such as fibrinoid necrosis, proliferative vasculitis and glomerular lesions, whereas 30 mg/kg nitrendipine did not. 7. Taken together, the results indicate that AE0047 is capable of preventing proteinuria as well as renal lesions, in part via a mechanism independent of its depressor action on SBP. Furthermore, AE0047 improves proteinuria and renal lesions in proteinuria-established SHRSP. Thus, AE0047 may have therapeutic potential in suppressing either the development or the progression of renal disease in hypertensive patients.

Effects of the calcium antagonist AE0047 on the development of neurological deficit and infarction after middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats.

AE0047 [4-(4-benzhydrylpiperazino)phenethyl methyl 1,4-dihydro-2,6-dimethyl-4-(3-nitrophenyl)-3,5-pyridine dicarboxylate dihydrochloride] is a new dihydropyridine calcium antagonist with protective effects against cerebral ischaemia and the occurrence of stroke in several animal models. We investigated the effects of AE0047 on focal ischaemia induced by middle cerebral artery occlusion in stroke-prone spontaneously hypertensive rats. AE0047 at a dose causing 20 or 40% systemic hypotension (1 or 3 mg kg-1) was given orally twice, 15 min and 24 h after occlusion. The neurological status of animals was investigated 2, 24 and 48 h after occlusion. Infarct area of brain was measured 48 h after occlusion. Middle cerebral artery occlusion resulted in the progressive deterioration of neurological status and large infarction in middle cerebral artery territories with 40% mortality. AE0047 dose-dependently attenuated the deterioration of neurological status, and reduced mortality to 0 or 10%. AE0047 significantly reduced infarct size and left/right hemispheric area ratio, an index of brain swelling. These results suggest that AE0047 has the ability to ameliorate ischaemic cerebral stroke in hypertensive patients.

[Antihypertensive effects of a novel calcium antagonist, AE0047, in various hypertensive models].

The antihypertensive effects of oral or intravenous administration of AE0047, a novel 1,4-dihydropyridine-type calcium antagonist, were investigated in spontaneously hypertensive rats (SHR/crj), one kidney-one clip renal hypertensive rats (RHR), deoxycorticosterone acetate (DOCA)-salt hypertensive rats (DHR) and two kidney-one clip renal hypertensive dogs (RHD). AE0047 (1, 3, 10 mg/kg, p.o.) caused a dose-related reduction of systolic blood pressure (SBP) with low reflex tachycardia in SHR/crj and RHR. The effect reached its maximum at 2-4 hr after administration and was sustained for a long time. In DHR, AE0047 (0.3, 1, 3 mg/kg, p.o.) similarly showed the antihypertensive effects at 2-7 hr with no significant changes in heart rates (HR). The doses (ED30) of AE0047 required to decrease SBP by 30% were 2.6, 3.4 and 0.68 mg/kg in SHR/crj, RHR and DHR, respectively. In RHD, and AE0047 capsule (GJ-0956: 4, 8, 16, 32 mg/body, p.o.) produced dose-dependent and long lasting effects with a transient and slight increase in HR. Furthermore, the intravenous administration of AE0047 (10, 30, 100 micrograms/kg) produced the antihypertensive action slowly, reached a plateau 10 min later and then maintained for many hours. In contrast, nitrendipine (3-100 mg/kg, p.o., 3-30 micrograms/kg, i.v.) and nicardipine (1-30 mg/kg, p.o., 3-30 micrograms/kg, i.v.) exhibited a similar potency to AE0047, but these maximal effects were produced at 1-2 hr and 0.5-1 min in the case of oral and intravenous administration, respectively, with a rapid recovery in the above hypertensive rats. These results indicate that AE0047 exhibits an antihypertensive effect with a slow onset and long-lasting profile.

[Antihypertensive effects of long-term treatment with AE0047, a novel long-lasting calcium antagonist, in hypertensive models of rats and dogs].

The antihypertensive effects of AE0047, a novel 1,4-dihydropyridine-type calcium antagonist, were investigated in spontaneously hypertensive rats (SHR/crj) and two kidney-one clip renal hypertensive dogs (RHD). AE0047, which was orally administered at the dose of 0.3, 1 or 3 mg/kg once daily for 8 consecutive weeks to SHR/crj, exhibited a dose-related decrease in systolic blood pressure. The antihypertensive action was reinforced during the drug treatment at 0.3 and 1 mg/kg. At each dose, the trough-to-peak (T/P) ratio was above 0.50 two weeks later. Although the reflex tachycardia was observed at 1 or 3 mg/kg on the 1st day, it gradually weakened within 8 weeks. Long-term treatment with AE0047 led to the regression of left ventricular hypertrophy. Furthermore, AE0047 had no influences on lipid and glucose metabolism. In RHD, and AE0047 capsule (GJ-0956) containing 2 or 8 mg of the drug was administered for 2 weeks. GJ-0956 produced no reduction in blood pressure at 2 mg, but enhanced the antihypertensive effect starting at 8 mg. The T/P ratios were 0.52 and 0.67 for the systolic and diastolic pressure, respectively, on the 14th day. These results indicate that AE0047 may be expected to exhibit beneficial effects for the clinical treatment of hypertension.

Inhibitory effects of endothelin-3 on antidiuresis and norepinephrine overflow induced by stimulation of renal nerves in anesthetized dogs.

The effects of endothelin-3 (ET-3) on changes in renal hemodynamics, urine formation, and norepinephrine (NE) overflow induced by renal nerve stimulation (RNS) were examined in anesthetized dogs. RNS at a low frequency (0.5-2.0 Hz) produced significant decreases in urine flow (UF), urinary excretion of sodium (UNaV), and fractional excretion of sodium (FENa), and increased the NE secretion rate (NESR) without affecting systemic or renal hemodynamics. RNS at a high frequency (2.5-5.0 Hz), which diminishes renal hemodynamics by causing renal vasoconstriction, affected urine formation and NESR more potently than did low-frequency RNS. When ET-3 (2.0 ng/kg/min) was infused into the renal artery, there was a slight and transient increase in renal blood flow (RBF); this response was followed by a gradual reduction. ET-3 infusion tended to increase the basal levels of UF without affecting UNaV, indicating the excretion of hypotonic urine with administration of this peptide. During ET-3 infusion, low-frequency RNS-induced antidiuretic action was significantly attenuated. Simultaneously, increase in NESR elicited by low-frequency RNS was markedly suppressed. Qualitatively similar results were observed in the case of high-frequency RNS. In addition, high-frequency RNS-induced decreases in the glomerular filtration rate (GFR) and the filtration fraction (FF) were suppressed by ET-3 infusion. These findings suggest that ET-3 suppresses renal responses to stimulated renal noradrenergic neurotransmission by inhibiting the release of NE. These findings, together with our previous findings, suggest that ET-3 (and/or ET-1) functions as an inhibitory modulator of the renal noradrenergic nervous system through the prejunctional ETB-receptor mechanism.

Enhancement of norepinephrine and angiotensin II-induced renal effects by NG-nitro-L-arginine, a nitric oxide synthase inhibitor.

We investigated whether endogenous nitric oxide (NO) has a role as an inhibitory modulator of norepinephrine (NE)- and angiotensin II (Ang II)-induced renal effects in anesthetized dogs. Intrarenal arterial infusion of NE (100 ng/kg/min) or Ang II (10 ng/kg/min) decreased renal blood flow (RBF), glomerular filtration rate (GFR) and urine formation. The NE- or Ang II-induced renal effects were augmented by the intrarenal administration of a NO synthase inhibitor, NG-nitro-L-arginine (NOARG), at doses (10 and 40 micrograms/kg/min) which did not affect the mean arterial blood pressure and heart rate. The stimulating activity of NOARG on NE- or Ang II-induced renal effects were abolished by the simultaneous administration of L-arginine, a NO precursor. These findings suggest that endogenous NO, which is probably generated within the kidney, functions as an inhibitory modulator in NE- or Ang II-induced renal vasoconstriction and antidiuresis.

Interaction between nitric oxide and angiotensin II on antidiuresis and norepinephrine overflow induced by stimulation of renal nerves in anesthetized dogs.

We examined the effects of NG-nitro-L-arginine (NOARG) on antidiuresis and norepinephrine (NE) overflow in anesthetized dogs, induced by renal nerve stimulation (RNS), with or without blockade of an action of endogenous angiotensin II (AII) on the AT1 receptors by losartan. RNS (2.5-5.0 Hz) caused significant reductions in renal blood flow (RBF), glomerular filtration rate (GFR), filtration fraction (FF), urine flow (UF), and urinary excretion of sodium (UNaV) and increases in the differences in renal arteriovenous NE concentrations (NEC). Intrarenal arterial (i.r.a.) infusion of NOARG (40 micrograms/kg/min) significantly decreased RBF and UF, and increased FF, but did not alter GFR. When losartan 100 micrograms/kg/min was infused simultaneously, NOARG reduced RBF, UF, and GFR but had no effect on FF. With high-frequency RNS, NOARG enhanced the RNS-induced decreases in RBF, GFR, UF, and UNaV and the increases in NEC. During losartan infusion, NOARG-induced enhancements on renal actions in response to RNS were observed in a manner qualitatively similar to that without losartan. Most likely endogenous nitric oxide (NO) plays the role of inhibitory modulator of renal noradrenergic neurotransmission. Enhancement of renal noradrenergic neurotransmission induced by NO blockade is likely to be independent of an action of endogenous AII on the AT1 receptors.

The effects of NG-nitro-L-arginine, a nitric oxide synthase inhibitor, on norepinephrine overflow and antidiuresis induced by stimulation of renal nerves in anesthetized dogs.

We examined the involvement of endogenous nitric oxide (NO) in noradrenergic neurotransmission and renal function in anesthetized dogs, by using NG-nitro-L-arginine (NOARG), a NO synthase inhibitor. Renal nerve stimulation (RNS) produced the frequency-dependent increase in the rate of norepinephrine secretion. The low frequency RNS (0.5-2.0 Hz) decreased urine flow and urinary excretion of sodium, without affecting renal hemodynamics. High frequency RNS (2.5-5.0 Hz) caused a more potent antidiuresis and renal vasoconstriction that resulted in reductions in renal blood flow and glomerular filtration rate. Intrarenal arterial infusion of NOARG, at a dose (10 micrograms/kg/min) which had no effect on renal hemodynamics, significantly enhanced the RNS-induced reductions of urine formation and renal vasoconstriction and increments in norepinephrine secretion rate. Qualitatively similar results were observed with a higher dose of NOARG (40 micrograms/kg/min), although this dose did decrease basal levels of renal blood flow and urine flow. Enhancement of NOARG on RNS-induced actions was abolished by the simultaneous administration of L-arginine. Endogenous NO probably has a role as inhibitory modulator of renal noradrenergic neurotransmission.

Effects of Losartan, a nonpeptide angiotensin II receptor antagonist, on norepinephrine overflow and antidiuresis induced by stimulation of renal nerves in anesthetized dogs.

We examined the role of endogenous angiotensin II on renal noradrenergic neurotransmission in anesthetized dogs, using Losartan, a nonpeptide angiotensin II receptor (angiotensin subtype 1) antagonist. The renal nerve stimulation caused a frequency-dependent increase in renal norepinephrine secretion rate. The low frequency renal nerve stimulation (0.5-2.0 Hz) significantly decreased urine flow and urinary excretion of sodium, without affecting renal hemodynamics. The high frequency renal nerve stimulation (2.5-5.0 Hz) produced a more potent antidiuresis and a renal vasoconstriction that resulted in reductions of renal blood flow and glomerular filtration rate. Intrarenal arterial infusion of Losartan (10 and 100 micrograms/kg/min) did not affect the basal levels of norepinephrine secretion rate, although increased urine formation with some renal vasodilation were observed during infusion of the drug. The administration of Losartan had an inhibitory action on the decreased urine formation, renal vasoconstriction and enhanced norepinephrine secretion rate, in response to renal nerve stimulation. Based on these findings, we suggest that endogenous angiotensin II seems to regulate renal noradrenergic neurotransmission by facilitating norepinephrine release, through the prejunctional angiotensin subtype 1 receptor.

Identification, purification and reconstitution of thiamin metabolizing enzymes in human red blood cells.

Thiamin and its mono- (TMP), di- (TDP) and triphosphate (TTP) were assayed in adult human whole blood using high-performance liquid chromatography (HPLC). TDP and TTP were detected in red blood cells (RBC), but not in plasma. After incubation with 20 microM thiamin and 5 mM glucose for 2 h, the TDP and TTP contents of RBC increased from 111 to 222 and 0.6 to 2.2 nmol/l of packed RBC, respectively, suggesting enzymatic conversion of thiamin to TDP and then to TTP. Thiamin pyrophosphokinase (TPK, EC 2.7.6.2) had not been isolated before from human materials, nor had cytosolic adenylate kinase (AK1, EC 2.7.4.3) in human RBC been demonstrated to catalyze the phosphorylation of TDP to TTP, although AK1 from pig and chicken skeletal muscle possess TTP-synthesizing activity. TPK and AK1 in a human RBC lysate were therefore purified by a series of the conventional techniques. The specific activity of the purified TPK, which was obtained as a single protein, was 720 nmol TDP formed/mg protein per h at 37 degrees C. A partially purified AK1 preparation catalyzed the formation of TTP from TDP (specific activity, 170 nmol/mg protein per h at 37 degrees C) in addition to its proper reaction to form ATP from ADP. After incubation of the purified TPK and AK1 with 20 microM thiamin in the presence of ATP, ADP and Mg2+ at 37 degrees C for 48 h, the amounts of TDP and TTP synthesized were 465 and 54.0 pmol/250 microliters reaction mixture, respectively. Neither TDP nor TTP was formed when TPK was omitted from the reaction mixture and an omission of AK1 resulted in the formation of TDP alone. These results indicate that thiamin is converted to TDP by TPK and, subsequently, to TTP by AK1 in human RBC.

Properties of thiamin triphosphate-synthesizing activity of chicken cytosolic adenylate kinase and the effect of adenine nucleotides.

Cytosolic adenylate kinase synthesis thiamin triphosphate (TTP) from thiamin diphosphate (TDP) in vitro by a reversible reaction: TDP + ADP Mg2+ in equilibrium TTP + AMP. The backward (TTP----TDP) reaction rate was 3-times faster than the forward (TDP----TTP) reaction rate when all the substrate concentrations were 0.1 mM. This property of TTP-synthesizing activity of the enzyme did not explain the fact that the [TTP]/[TDP] molar ratio determined in chicken white skeletal muscle is 5.0 (Miyoshi, K., Egi, Y., Shioda, T. and Kawasaki, T. (1990) J. Biochem. 108, 267-270). To solve this problem, we have studied the properties of TTP-synthesizing activity of the purified recombinant chicken cytosolic adenylate kinase preparation and the effect of adenine nucleotides, especially of ATP. The backward reaction of the TTP synthesis did not proceed in the presence of 8.8 mM ATP, a physiological concentration in chicken white skeletal muscle, while the forward reaction proceeded at a reduced rate. The [TTP]/[TDP] ratio found after a long incubation period was 3.0 and 0.7, respectively, in the presence and absence of 8.8 mM ATP. These results indicate that the high [TTP]/[TDP] molar ratio found in chicken white muscle was demonstrated in vitro by the purified chicken cytosolic adenylate kinase and support in vivo TTP synthesis by this enzyme.

Synthesis and accumulation of thiamin triphosphate in Escherichia coli cells expressing chicken cytosolic adenylate kinase.

To examine whether cytosolic adenylate kinase (AK1, EC 2.7.4.3) catalyzes synthesis of thiamin triphosphate (TTP) in vivo, chicken AK1 was expressed in Escherichia coli, and cellular AK1 activity and TTP content were determined. E. coli harboring the vector plasmid was used as a control. Chicken AK1 was expressed in the producer strain at a high level (83 U/mg protein) even without inducers, and this expression was doubled (153 U/mg protein) by beta-D-isopropylthiogalactopyranoside (IPTG). TTP was accumulated in the producer cells at a high level (5.7 nmol/g dry weight) without IPTG and this was also doubled (10.2 nmol/g dry weight) by IPTG. TTP content in the control strain was very low (0.2-0.9 nmol/g dry weight) and was unaffected by IPTG. Neither bacterial growth curve nor cellular content of AMP, ADP, ATP, thiamin diphosphate or total thiamin (sum of thiamin and its phosphate esters) was different between the producer and the control strains. These results indicate that chicken AK1 expressed in E. coli catalyzed the synthesis and accumulation of TTP within the bacterial cells.

Evidence for in vivo synthesis of thiamin triphosphate by cytosolic adenylate kinase in chicken skeletal muscle.

We showed previously that cytosolic adenylate kinase (AK1) purified from pig skeletal muscle catalyzes in vitro formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP) and ADP in addition to ATP formation from ADP [Shikata, H. et al. (1989) Biochem. Int. 18, 933-942]. To obtain evidence for in vivo synthesis of TTP by AK1, changes in TTP content and AK1 activity were determined in chicken skeletal muscle during development after hatching. Thiamin phosphate metabolism in chicken skeletal muscle was also studied. i) An extremely high TTP content, 81% of total thiamin (thiamin plus thiamin phosphates), was detected in the white (fast-twitch) muscle of adult normal chicken (5th to 9th month) compared with a relatively high TTP content of 31% in the red (slow-tonic) muscle. Since approximately equivalent amounts of total thiamin were present in the two types of muscle, the ratio of TTP to TDP was high (5.0) in the white muscle and low (0.41) in the red muscle. ii) Rabbit anti-chicken AK1 antiserum against the purified chicken cytosolic AK1 preparation was obtained. Both AK1 activity and TTP-synthesizing activity in crude cytosol fraction of adult chicken white muscle were inhibited in parallel by the antiserum. iii) In the white muscle of normal chicken, the TTP content and AK1 activity responsible for forming either ATP or TTP were increased in a parallel manner up to day 16 after hatching, after which both remained constant. In the red muscle, on the other hand, both the TTP content and the AK1 activity were low in comparison with those in the white muscle, and were almost constant after hatching.(ABSTRACT TRUNCATED AT 250 WORDS)

Cytosolic adenylate kinase catalyzes the synthesis of thiamin triphosphate from thiamin diphosphate.

An attempt was made to purify a porcine skeletal muscle enzyme catalyzing the formation of thiamin triphosphate (TTP) from thiamin diphosphate (TDP), requiring ATP, Mg2+ and a cofactor (creatine). As the purification proceeded, the reaction requirements for ATP and creatine were lost and then a requirement for ADP was manifested. The activity responsible for TTP synthesis from TDP, ADP, and Mg2+ was found to be copurified with adenylate kinase [EC 2.7.4.3] activity, and was finally purified to a single band on SDS-PAGE. Antiserum obtained against the purified enzyme preparation inhibited both adenylate kinase activity and the TTP-synthesizing activity to exactly the same extent. These results indicate that adenylate kinase catalyzes TTP formation from TDP in vitro.

Properties of the thiamin triphosphate-synthesizing activity catalyzed by adenylate kinase (isoenzyme 1).

Adenylate kinase isozyme 1 (AK1) catalyzes thiamin triphosphate (TTP) formation from thiamin diphosphate (TDP) and ADP. The properties of the TTP-synthesizing activity of purified AK1 from porcine skeletal muscle were studied. The activity was found to require TDP, ADP, and Mg2+, and ATP was only 14.4% as active as ADP. Thiamin monophosphate (TMP) and thiamin were not utilized as substrates. ADP was specific as a phosphate donor; and CDP, UDP, and GDP supported TTP formation at rates less than 1% of that with ADP. Optimal pH and temperature for the TTP-synthesizing activity were 10.0 and 37 degrees C, respectively. The activity showed saturation kinetics for both substrates, and the Km values for TDP and ADP were calculated to be 0.83 mM and 43 microM, respectively. The enzyme catalyzed the reverse reaction (TTP + AMP----TDP + ADP) and stoichiometry between TTP and TDP was demonstrated in the forward and reverse reactions.

Identification of creatine as a cofactor of thiamin-diphosphate kinase.

Thiamin-diphosphate (TDP) kinase which catalyzes thiamin triphosphate formation from TDP requires a low-molecular-mass cofactor in addition to ATP and Mg2+. The cofactor was isolated in a crystalline form from pig skeletal muscle and identified as creatine by proton NMR, mass spectrometry, infrared spectrometry and elemental analysis. The isolated cofactor and authentic creatine supported the same activity of partially purified TDP kinase at identical molar concentrations. Neither creatine phosphate nor creatinine showed activity as a cofactor. This is the first report showing evidence of the existence of a creatine-dependent enzyme.