Hemoglobin Hijiyama: a new fast-moving hemoglobin in a Japanese family.

A variant of hemoglobin A, named Hb Hijiyama, found in two generations of a Japanese family living in Hiroshima, Japan, has a higher anodal electrophoretic mobility than hemoglobin A; a gain of two negative charges per molecule is indicated. Fingerprinting and amino acid analysis showed the biochemical anomaly to be in the beta chain at residue 120, where lysine is replaced by glutamic acid. In the heterozygote carriers of the abnormal hemoglobin there is no apparent association with clinical or hematologic abnormalities.

Hemoglobin Hiroshima (beta-143 histidine--aspartic acid): a newly identified fast moving beta chain variant associated with increased oxygen affinity and compensatory erythremia.

During a survey for hemoglobinopathies in over 9000 residents of Hiroshima Prefecture, Japan, a fast moving hemoglobin was identified in eight members of three generations in a Japanese family. The abnormal hemoglobin, named Hb Hiroshima, constitutes about 50% of the total hemoglobin in hemolysates from the carriers who have a mild erythremia but are otherwise apparently clinically unaffected. All preparations of Hb Hiroshima have increased affinity for oxygen, by either tonometric or oxygen electrode determinations. At pH 7.0, the oxygen pressure, P(50) required to half saturate an unfractionated hemolysate from a carrier was one-half that of Hb A, and the P(50) of a purified sample containing no Hb A was one-fourth that of Hb A. The pH dependence of the oxygen equilibrium (Bohr effect) is below normal, as shown by the absolute value of the Bohr effect factor which is about half that of Hb A, in the pH range between 7.0 and 7.4. The Hill constant, n, for Hb Hiroshima between pH 7.0 and 7.4 is 2-2.4, compared to 2.8-3 for Hb A under the same conditions, indicating reduction of, but not complete abolition of heme-heme interaction. Urea dissociation and canine hybridization tests located the biochemical lesion in the beta chain. Fingerprints (Ingram), carboxypeptidase digestion, and amino acid analysis demonstrated that the substitution was at residue 143 in the beta chain, where histidine was replaced by aspartic acid.In contrast to other recently described high oxygen affinity mutants that show intact Bohr effects, all three of the major characteristics of the reversible combination of hemoglobin with oxygen (oxygen equilibrium, heme-heme interaction, and pH dependence) are affected in Hb Hiroshima. A tentative interpretation of these effects, relating structure to function, is offered in terms of recently developed models of normal hemoglobin.

HCE, a constituent of the hatching enzymes of Oryzias latipes embryos, releases unique proline-rich polypeptides from its natural substrate, the hardened chorion.

HCE, a constituent protease of the hatching enzymes of Oryzias latipes embryos [1,2], releases unique proline-rich polypeptides from its natural substrate, the hardened chorion. The polypeptides consist of repeats of Pro-X-Y, mainly Pro-Glx-X. In addition, the polypeptides contain abundant gamma-glutamyl epsilon-lysine isopeptides which are regarded to be responsible for chorion hardening. These findings suggest that HCE recognizes specific site(s) of the chorion, releases the proline-rich polypeptides from it, and makes the substrate accessible to LCE, another protease of the hatching enzymes.

Presence of Active Hatching Enzyme in the Secretory Granule of Prehatching Medaka Embryos: (hatching enzyme/secretory granule/hatching gland/medaka/fish).

Secretory granules of hatching gland were isolated from a 0.3 M sucrose homogenate of whole medaka embryos at prehatching stage by differential centrifugation, followed by a Percoll density gradient centrifugation. The obtained preparation was almost free of melanosomes and composed exclusively of the secretory granules of hatching gland (hatching enzyme granules), as judged by morphological as well as enzymological criteria. The aqueous extracts of the purified secretory granules showed a specific choriolytic activity as high as about 40 times that of a partially purified secretory granule preparation, P1,000 , and represented a single protein band with molecular weight of about 21,000 on SDS-polyacrylamide gel electrophoresis. It was also revealed that a major component of the hatching enzyme preparation (P II-0.3 enzyme, 13) purified from the hatching liquid was identical with the 21,000 molecular weight band. These results suggest that the hatching enzyme is present in the secretory granules of prehatching embryos in an active molecular form.

Purification and partial characterization of 76 kDa transglutaminase in the egg envelope (chorion) of rainbow trout, Oncorhynchus mykiss.

Transglutaminase (TGase), responsible for crosslinking between proteins, is known to be localized exclusively in the egg envelope (chorion) of rainbow trout, Oncorhynchus mykiss, and probably participates in the post-fertilization chorion hardening. We purified the TGase from unfertilized egg chorions by sequential chromatography using SP-Sepharose, Q-Sepharose, and TSK-gel G3000SWXL columns. The purified enzyme was a monomeric protein having the molecular mass of 76 kDa. It promoted incorporation of monodansyl-cadaverine into chorion protein and catalyzed the polymerization of chorion subunit proteins. The effect of various reagents suggested that the chorion TGase is a Ca2+-dependent SH-enzyme similar to the well-characterized TGases of various animals. The highest activity was observed at pH 6.0. The amines examined in the present study inhibited the TGase activity of the purified enzyme. However, they did not necessarily cause effective inhibition of its activity. These properties of the chorion TGase were essentially consistent with our previous observations on polymerization of chorion proteins, resulting in chorion hardening. We compared the amino acid composition of the purified TGase with those of the previously characterized TGases of fishes, such as chum salmon and red sea bream. The results suggest that the chorion 76 kDa TGase is not homologous with those liver TGases in terms of amino acid composition.

Enzyme responsible for egg envelope (chorion) hardening in fish: purification and partial characterization of two transglutaminases associated with their substrate, unfertilized egg chorion, of the rainbow trout, Oncorhynchus mykiss.

From our previous studies, we have suggested that "egg envelope (chorion) hardening-enzyme" (first proposed by Zotin [J. Embriol. Exp. Morph. 6, 546-568 (1958)]) in the rainbow trout, Oncorhynchus mykiss, is transglutaminase (TGase), and that it coexists with its substrate, the unfertilized egg chorion, and forms epsilon-(gamma-glutamyl)lysine cross-links between the chorion proteins. In the present study, we extracted the TGase activity from the isolated chorions by homogenization with isotonic saline (143 mM NaCl-10 mM Tris.HCl, pH 7.2) and fractionated the extract by Toyopearl HW55S gel filtration with the isotonic saline containing 5 mM CaCl2 and 5 mM 2-mercaptoethanol (2-ME). One peak of TGase activity (P2) was obtained. When the eluates were dialyzed against 5 mM CaCl2/5 mM 2-ME/10 mM Tris.HCl (pH 7.2), another peak of the activity (P1) appeared. P1 TGase activity, which becomes apparent in a medium of low ionic strength, is involved in acceleration of the chorion hardening after egg activation in fresh water, so-called water activation. We purified the two TGases, P1 and P2, by SP-Sepharose, Q-Sepharose, and TSK-gel column chromatography. The molecular mass of the native form of P1 TGase was estimated as 103 kDa by Toyopearl HW55S gel filtration and as 100 kDa by the TSK-gel filtration. SDS-PAGE analysis showed that it consisted of heterogeneous 86- and 76-kDa proteins. However, these proteins closely resembled each other in amino acid composition, which was characterized by high content of Thr, Gly, and Pro residues as compared with P2 TGase. In contrast, the P2 TGase was isolated as a homogeneous 76-kDa protein and characterized by high content of Glx (Glu/Gln) and His residues. Neither of the chorion TGases of rainbow trout, P1 and P2, was similar to the liver-type TGase of red sea bream or the tissue-type TGase of chum salmon in amino acid composition. Examination of susceptibility to various inhibitors, reactivation by CaCl2, pH dependency, and activity of the polymerization of chorion proteins suggested that the P1 and P2 TGases were essentially similar to each other in enzymatic properties.

Purification and partial characterization of high choriolytic enzyme (HCE), a component of the hatching enzyme of the teleost, Oryzias latipes.

The hatching enzyme is an embryo-secreted enzyme(s) which digests the egg envelope, allowing the embryo to emerge at the time of hatching. The hatching enzyme of the fish, Oryzias latipes, has recently been found to consist of two kinds of proteases which may digest the inner layer of chorion (egg envelope) cooperatively [Yasumasu, S. et al. (1988) Zool. Sci. 5, 191-195]. In the present study, one of them, high choriolytic (egg envelope digesting) enzyme (HCE) was purified and some biochemical and enzymological properties were examined. The enzyme was a basic protein with a molecular weight of about 24 kDa, and exhibited choriolytic activity as well as proteolytic (caseinolytic) activity. The results of inhibitor studies and metal analyses strongly suggested that it was a zinc-protease. The purified HCE consisted of two probable isomers, HCE-1 and HCE-2. Both of them were markedly similar in amino acid composition, specific activities of choriolysis and proteolysis, and substrate specificity as determined using MCA-peptides. Moreover, they were not separable on SDS-PAGE, electrofocusing PAGE, or ultracentrifugal analysis, but were discriminated only on HPLC with a CM-300 column. Thus, the mixture of HCE-1 and HCE-2 could be regarded as almost a single enzyme, HCE. When it acted on an intact chorion, the purified HCE caused a remarkable swelling of its inner layer with concomitant release of peptides from it. Once the inner layer of chorion was swollen, the enzyme hardly digested it.

Isolation and some properties of low choriolytic enzyme (LCE), a component of the hatching enzyme of the teleost, Oryzias latipes.

One of the two component proteases of the hatching enzyme of the fish, Oryzias latipes, low choriolytic enzyme (LCE), was isolated from the hatching liquid and partly characterized. The enzyme was a basic protein with molecular weight of about 25.5 kDa. Like high choriolytic enzyme (HCE), the other component of the O. latipes hatching enzyme [Yasumasu, S. et al. (1989) J. Biochem. 105, 204-211], LCE was considered to be a zinc-protease from the results of inhibitor studies and metal analyses. However, LCE was found to be distinct from HCE not only in some biochemical characteristics such as molecular weight, amino acid composition, and isoelectric point, but also in some enzymological properties such as substrate specificity, heat stability, and mode of action toward their natural substrate, chorion (egg envelope). Although LCE was almost incapable of digesting the inner layer of intact chorion, it very efficiently digested the inner layer of chorion that had been swollen previously by the action of HCE. Taking account of the fact that HCE swells the inner layer of intact chorion by partial proteolysis but does not efficiently digest the swollen chorion any more [Yasumasu, S. et al. (1989) J. Biochem. 105, 204-211], the present results demonstrated an essential role of LCE in choriolysis, in cooperation with HCE.

Formation of mature egg envelope subunit proteins from their precursors (choriogenins) in the fish, Oryzias latipes: loss of partial C-terminal sequences of the choriogenins.

The inner layer of egg envelope of the medaka, Oryzias latipes, comprises two major groups of glycoprotein subunits, ZI-1,2 and ZI-3. Their precursor proteins, choriogenin H (Chg H) and choriogenin L (Chg L), respectively, are synthesized in spawning female liver. In the present study, the primary structures of the precursors and the corresponding mature subunits were compared by peptide mapping and amino acid sequencing to find what difference in their molecular structures is relevant to the assembly of the soluble precursors into the insoluble inner layer. The primary structures of the solubilized subunits were mostly identical to those of the respective precursors, but they lacked C-terminal partial sequences that their precursors possessed, namely, ZI-1,2 subunit was shorter than Chg H by 34 amino acid residues and ZI-3 was shorter than Chg L by 27 residues. In addition, a consensus amino acid sequence, Arg-Lys-X-Arg, was found at the putative cleavage sites in the C-terminal region of the precursors. It is conjectured that the truncation of the precursor proteins is prerequisite for formation of mature chorion subunit proteins and their assembly into chorion.

Diffuse palmoplantar keratoderma with deafness.

Two brothers with diffuse palmoplantar keratoderma (Thost-Unna type) also were deaf. Of the 38 members of the patients' family, five had a similar disorder and ten had only hearing loss. The mode of inheritance of the dermatosis is regarded as autosomal dominant and is diagnostically distinguished from the other dermatoses associated with the disturbance of keratinization and deafness. To our knowledge, this is the second report of diffuse palmoplantar keratoderma (Thost-Unna type) with deafness, which is considered to be a new variant of the keratodermatoses.

Nonsense mutation in exon 2 of the butyrylcholinesterase gene: a case of familial cholinesterasemia.

A point mutation that causes a silent phenotype for human serum butyrylcholinesterase (BChE) was proved by DNA analyses of a 64-year-old Japanese female who visited the hospital because of a common cold. The propositus and her two siblings showed extremely low BChE activity, but other family members (six individuals) manifested from intermediate to normal values of BChE activity. An immunological method revealed that the propositus and her two siblings showed absence of the BChE protein in serum. DNA sequence analysis of the propositus identified a point mutation at codon 400 (TGC-->TGA), resulting in the production of a stop codon. This alteration exists upstream of the Cys571 of the subunit, which forms a disulfide bridge with the Cys571 of another partner subunit.

Identification of a point mutation associated with a silent phenotype of human serum butyrylcholinesterase--a case of familial cholinesterasemia.

A point mutation which caused a silent phenotype of human serum butyrylcholinesterase (BChE) was identified in the DNA of a 47-year-old Japanese woman who visited our hospital complaining of hypertension. The propositus exhibited an unusually low level of BChE activity, whereas her younger sister and her daughter had intermediate levels of BChE activity and her elder sister a normal level. Immunologically, the amount of BChE protein in the serum of the propositus was normal. DNA sequence analysis of the propositus identified a point mutation at codon 199 (GCA --> GTA), resulting in a Ala --> Val substitution. This alteration is one downstream codon from the catalytic active site (Ser, 198). A family study showed her younger sister and her daughter to have the same mutation.

Gene analysis of genomic DNA from stored serum by polymerase chain reaction: identification of three missense mutations in patients with cholinesterasemia and ABO genotyping.

We established a method to determine the butyrylcholinesterase genotype associated with a BCHE deficiency directly using multiple PCR from stored serum, which was stored at -70 degrees C for more than 30 years. PCR products from sera of six propositi were used for DNA sequence analysis. All of these BChE variants were characterized by a single nucleotide substitution. Four of them were homozygotes and demonstrated a C-->T single nucleotide point mutation at codon 100 from CCA (Pro) to TCA (Ser). The fifth case was a heterozygote of this mutation. The remaining one was a compound heterozygote showing a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and a G-->C transversion mutation at codon 365 from GGA (Gly) to CGA (Arg). Furthermore we developed a method to determine the ABO genotype from the same serum. These results indicated that serum is useful as a starting material for amplification of genomic DNA when fresh blood samples are not available.

Evaluation of solubilizing methods of the egg envelope of the fish, Oryzias latipes, and partial determination of amino acid sequence of its subunit protein, ZI-3.

The inner layer of most teleostean egg envelopes, especially those after hardening, is almost insoluble in ordinary solvent, and therefore the inner layer of only the unhardened egg envelope has been subjected to solubilization with some potent solvents. We comparatively evaluated the methods of solubilization of the inner layer of egg envelope of medaka, Oryzias latipes, with SDS, urea and guanidium chloride (GuHCI). Analysis of the solubilized samples by SDS-polyacrylamide gel electrophoresis, comparison of their amino acid compositions or peptide maps using high-performance liquid chromatography and partial determination of their amino acid sequences showed that SDS and GuHCI were appropriate for solubilization and characterization of the envelope. Urea solubilization resulted in some artificial modifications of lysine and/or cysteine residues of envelope proteins. Partial determination of amino acid sequence of a subunit, ZI-3, isolated from the SDS-or GuHCI-solubilized envelope strongly suggested the identity of the envelope subunit, ZI-3, and its precursor, L-SF.

Identification of missense mutation (G365R) of the butyrylcholinesterase (BCHE) gene in a Japanese patient with familial cholinesterasemia.

A point mutation which caused a silent phenotype of human serum butyrylcholinesterase (BChE) was identified in the genomic DNA of a 57-year-old Japanese woman who visited our hospital because of pneumonia. The propositus exhibited an unusually low level of BChE activity, whereas her son and daughter had an intermediate level. Immunologically, there was an absence of BChE protein in the propositus's serum. DNA sequence analysis of the propositus demonstrated a point mutation at codon 365 (GGA-CGA), resulting in a Gly-Arg substitution. A family study showed her son and daughter to have the same mutation.

[Determination of gene mutation of silent serum cholinesterase and its epidemiologic characters in the Japanese].

We detected 121 individuals with silent type of serum cholinesterase from 36 families in Japan. DNA analysis totaling 37 members of eleven blood unrelated families were carried out by four useful methods, namely, 1) PCR-SSCP analysis, 2) dot blot hybridization (DBH) with the use of synthetic oligonucleotide probe, 3) restriction endonuclease analysis (REA) and 4) direct sequencing analysis. Their mutations were classified into four groups, namely, 1) a G-->C transversion at codon 365, 2) a frameshift mutation with insertion of an extra A at codon 315, 3) an A-->G transition at codon 128 and 4) a C-->A transition at codon 400. The three procedures including (PCR-SSCP, DBH, REA) without the use of radio labeled materials (non-RI) are recommendable for the analyses. However, the direct sequencing analysis of bases with RI might be, at present, necessary for the final identification.

[Identification of two different genetic mutation associated with silent phenotypes for human serum cholinesterase in Japanese].

Two different gene mutations associated with the silent phenotype for human serum cholinesterase were demonstrated. DNA from five individuals with silent gene phenotype of three unrelated Japanese families was amplified by the polymerase chain reaction (PCR) and analyzed by direct sequencing. The first instance demonstrated a G----C transversion at codon 365 from GGA (Gly) to CGA (Arg), which was seen in three individuals of the two families. This mutation was resulted to create a new Taq 1 restriction site (TCGA). The second mutation was shown by a double heterozygous condition with two different silent gene mutations in two members of remaining one family. These mutations were as follows: 1) one type was a frameshift mutation, in which an extra A was inserted in codon 315 (ACC----AACC) to create a new stop codon at position 322 and 2) the other was the same point mutation at codon 365 as seen in the first instance. These results indicated that many silent variants can be distinguished by direct sequence analyses of genomic DNA.

[Missense mutations of the butyrylcholinesterase gene in six Japanese patients with low cholinesterasemia: genetic analysis using sera stored in a freezer].

Six serum samples with no detectable butyrylcholinesterase (BCHE) activity had been stored at -70 degrees C for more than 10 years. These sera were used for amplification of BCHE gene using polymerase chain reaction (PCR) and for nucleotide sequence analysis. Five of them demonstrated a C-->T transition at codon 100 (CCA-->TCA), resulting in a Pro-->Ser substitution. The other one was a compound heterozygote as revealed a T-->C transition mutation at codon 203 from TCA (Ser) to CCA (Pro) and G-->C transversion at codon 365 from GGA (Gly) to CGA (Arg). These results showed sera stored in a freezer could be used as a starting material for amplification of genomic DNA when it is not possible to obtain fresh blood samples.