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Biomedical subjects

H Kihara

Publications and source records attributed to H Kihara.

At least 181 records · Page 10Linked to original sources
PubMed1980

Purification and properties of phospholipase A from venom of Trimeresurus flavoviridis (Habu snake).

Phospholipase A was purified from venom of Trimeresurus flavoviridis (Habu snake) via three steps consisting of Sephadez G-100, CM-cellulose, and DEAE-cellulose column chromatographies. The apparent molecular weights determined by gel filtration on Sephadex G-75 and by sodium dodecyl sulfate-polyacrylamide gel electrophoresis were 28,000 and 14,000, respectively, suggesting that the enzyme is composed of two identical subunits. The isoelectric point was 7.9. The enzyme is characterized by high contents of aspartic acid, glycine, tyrosine, and lysine and contains one residue each of histidine and methionine, three or four tryptophans, and eight disulfide bonds per subunit. The enzyme was inactivated by reaction with p-bromophenacyl bromide following pseudo first order kinetics. The loss of activity was accompanied by loss of histidine, indicating that a single histidine residue is essential for activity. Oxidation with N-bromosuccinimide decreased the enzymic activity. Tryptophan residues appear to play some role in catalysis.

Amino Acids↗
PubMed1980

Proton nuclear-magnetic-resonance and resonance Raman studies of thermophilic cytochrome c-552 from Thermus thermophilus HB8.

The pH and temperature dependences of the 270-MHz proton nuclear magnetic resonance and resonance Raman spectra of Thermus thermophilus cytochrome c-552 were studied. Observation of the NMR methyl signal of the iron-bound methionine indicates that a methionine residue is the sixth ligand of heme iron in both ferric and ferrous states, although the environment of this methionine is not similar to that in mitochondrial cytochrome c. The NMR methyl signal of the coordinated methionine in the ferrous state was observed even at 87 degrees C, indicating the retention of the methionine ligand at the sixth coordination position. None of resonance Raman lines in ferrous cytochrome c-552 at higher temperatures showed a prominant temperature-dependent frequency shift, which implies that the heme iron was still bound with strong ligands and retained the low-spin state. In either redox state overall thermal denaturation did not occur even at 87 degrees C, although the ferric form existed in thermal spin mixture of the low-spin and high-spin species at higher temperatures. The hyperfine-shifted NMR resonances of the ferric form indicated rapid exchange of the sixth ligand at alkaline pH in the process of a single-step alkaline isomerization.

Cytochrome c Group↗
PubMed1980

Prenatal diagnosis of metachromatic leukodystrophy in a family with pseudo arylsulfatase A deficiency by the cerebroside sulfate loading test.

Prenatal diagnosis was requested by a family at risk for metachromatic leukodystrophy (MLD). An examination of the family leukocyte arylsulfatase A profile revealed that the mother had pseudo arylsulfatase A deficiency. Cultured amniotic fluid cells were deficient in arylsulfatase A, so two possibilities were indicated: the fetus was affected with MLD or had the pseudodeficiency phenotype. The only known biochemical test to differentiate the two enzyme deficient phenotypes is cerebroside sulfate loading of growing fibroblasts. The pseudodeficient cells hydrolyze the incorporated sulfatide as efficiently as control cells, whereas MLD cells show no hydrolysis. Application of this test to the at risk cultured amniotic fluid cells resulted in appreciable uptake of the sulfolipid, but no hydrolysis. Control amniotic fluid cell cultures hydrolyzed 82 to 95% of the incorporated sulfatide. Therefore, an affected fetus was indicated. Fibroblasts derived from the aborted fetus showed a deficiency of arylsulfatase A and a similar inability to hydrolyze cerebroside sulfate in the loading test. The loading technique allowed the prenatal diagnosis of MLD when the arylsulfatase A analysis was equivocal.

Amniotic Fluid↗
PubMed1979

The activity of arylsulfatase A and B on tyrosine O-sulfates.

L-Tyrosine O-sulfate was hydrolyzed by pure human arylsulfatase A (arylsufate sulfohydrolase, EC 3.1.6.1). The rate of hydrolysis was 1/20 of the rate with nitrocatechol sulfate, but was comparable to the rate with cerebroside sulfate. The reaction was optimal at pH 5.3--5.5 and displayed zero order kinetics with time and enzyme concentration. The Km was about 35 mM. The enzyme showed no stereospecificity and hydrolyzed D-tyrosine O-sulfate with Km and V similar to those for the L-isomer. Arylsulfatase B was less than 5% as effective as arylsulfatase A in catalyzing the hydrolysis of the tyrosine sulfates. The daily urinary excretion of tyrosine sulfate by a patient with metachromatic leukodystrophy (arylsulfatase A deficiency) was comparable to the excretion by control subjects. The biological relevance of the tyrosine sulfatase activity of arylsulfatase A remains uncertain.

Cerebroside-Sulfatase↗
PubMed1979

Metachromatic leukodystrophy without arylsulfatase A deficiency.

Two siblings of consanguinous parents were noted to have a neurologic syndrome marked by developmental delay, regression of psychomotor performance, marked spasticity and progressive central nervous system degeneration. Markedly delayed nerve conduction times and a sural nerve biopsy which demonstrated changes typical of metachromatic leukodystrophy (MLD) were evident. Impairment of sulfated glycolipid metabolism was documented by analysis of glycospingolipid in urinary sediment. In spite of these findings, activities of arylsulfatase A and cerebroside sulfatidase in white blood cells and cultured skin fibroblasts were near normal. However, when intact growing fibroblasts were loaded with 35SO4-sulfatide a clear defect in sulfatide cleavage, comparable to that seen in MLD patients, was observed. Thus, these patients represent a new form of sulfatide storage disease -- MLD characterized by intact enzyme activity in cell homogenates but defective sulfolipid metabolism in vivo and in intact fibroblasts.

Adult↗
PubMed1979

Arysulfatase A modulation with pH in multiple sulfatase deficiency disorder fibroblasts.

It has been observed that multiple sulfatase deficiency disorder (MSDD) fibroblasts contained from profoundly deficient to near normal amounts of arylsulfatase (ARS) A depending on the medium in which they were cultured. Our present findings show that the major factor determining the enzyme level is the pH of the medium during growth. In media which became acidic or was maintained at low pH (less than 7), the cells expressed the enzymopathy, while in high pH media (7.4), the cells produced enzyme. The high and low enzyme states were reversible. The ARS A deficiency in MSDD must, therefore, be a secondary manifestation of a mutation in another system.

Cerebroside-Sulfatase↗
PubMed1978

Alkaline isomerization of thermoresistant cytochrome c-552 and horse heart cytochrome c studied by absorption and resonance Raman spectroscopy.

The structure of the thermoresistant cytochrome c (552, Thermus thermophilus) has been investigated at neutral and alkaline pH by absorption and resonance Raman spectroscopy and compared with that of horse heart cytochrome c. The ligands of the ferricytochrome c-552 at neutral pH are considered to be histidine and methionine, whereas the ligands of ferrocytochrome c-552 are histidine and another nitrogen base, histidine or lysine. Ferric cytochrome c-552 undergoes an alkaline isomerization with a pK of 12.3 (25 degrees C), accompanied by a ligand exchange. Horse heart cytochrome c has at least three isomerization states at alkaline pH (pK 9.3, 12.9 and greater than 13.5 at 25 degrees C). The replacement of the sixth ligand may not be involved in the second isomerization. The thermodynamic parameters for the isomerization were also estimated. The entropy change upon isomerization of cytochrome c-552 is negative, whereas for that of horse heart cytochrome c the entropy change is positive.

Animals↗
PubMed1978

Kinetic studies on redox reactions of hemoproteins. II. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ascorbic acid.

The reductions of thermoresistant cytochrome c-552 and horse heart cytochrome c by ascorbic acid were studied by the stopped-flow method between pH 4 and 10. The results were as follows (1) The reduction of horse heart cytochrome c showed two relaxation decays above pH 8.5, one of which was pseudo-first order, as was the case below pH 8, while the other was nearly concentration-independent. These results were consistent with those reported by Greenwood and Palmer (J. Biol. Chem. (1965) 240, 3660-3663). (2) For the reduction of cytochrome c-552, only a single relaxational decay that obeyed pseudo-first order kinetics was observed. (3) It seems most reasonable to assume that the concentration-independent relaxation process can be attributed to the isomerization reaction accompanying ligand exchange, since it is known that only horse heart cytochrome c exhibits ligand exchange, involving a residue with pK 9.3.

Animals↗
PubMed1978

Iduronate sulfatase analysis of hair roots for identification of Hunter syndrome heterozygotes.

Iduronate sulfatase, the enzyme deficient in Hunter syndrome, can be readily measured in individual hair roots. Samples from Hunter syndrome hemizygotes had activities at or near the limits of detection. Samples from two mothers of Hunter syndrome patients, one an obligate heterozygote, had lower average iduronate sulfatase activity than the normal mean, and a significant number of hair roots had activity in the pathognomic range. A third mother showed a normal distribution of enzyme activity, and no hair roots were in the range of those from an affected individual. These results are similar to studies on the distribution of other X-linked enzymes in individual hair root samples from heterozygotes. This suggests that hair root iduronate sulfatase assessment is useful in the detection of Hunter syndrome carrier status, but further refinement of the test system is necessary.

Female↗
PubMed1978

Presence of arylsulfatase A (ARS A) in multiple sulfatase deficiency disorder fibroblasts.

Multiple deficiency disorder fibroblasts cultured in MEM-CO2 showed deficiencies of arylsulfatase A(ARS A) comparable to the deficiency in metachromatic leukodystrophy fibroblasts. However, the MSDD fibroblasts cultured in MEM-HEPES contained near normal levels of ARS A. Moreover, the enzyme from the latter fibroblasts was indistinguishable from ARS A of control fibroblasts on DEAE-cellulose chromatography, ratio of activity with several substrates, thermal inactivation, sensitivity to inhibitors, and precipitation by antiserum to human ARS A. These data support the conclusion that the ARS A genome is intact in MSDD fibroblasts and, by extension, in MSDD patients. Other sulfatases were present at levels ranging from mildly deficient to near normal but never as low as seen in the corresponding specific sulfatase deficient disorders.

Cell Line↗
PubMed1977

Kinetic studies on redox reactions of hemoproteins. I. Reduction of thermoresistant cytochrome c-552 and horse heart cytochrome c by ferrocyanide.

The oxidation-reduction reaction of horse heart cytochrome c and cytochrome c (552, Thermus thermophilus), which is highly thermoresistant, was studied by temperature-jump method. Ferrohexacyanide was used as reductant. (Formula: see text.) Thermodynamic and activation parameters of the reaction obtained for both cytochromes were compared with each other. The results of this showed that (1) the redox potential of cytochrome c-552, + 0.19 V, is markedly less than that of horse heart cytochrome c. (2) deltaHox of cytochrome c-552 is considerably lower than that of horse heart cytochrome c. (3) deltaSox and deltaSred of cytochrome c-552 are more negative than those of horse heart cytochrome c. (4) kred of cytochrome c-552 is much lower than that of horse heart cytochrome c at room temperature.

Animals↗