Kohichi Kojima

Changes in Prolyl Endopeptidase During Maturation of Rat Brain and Hydrolysis of Substance P by the Purified Enzyme

: We determined changes in prolyl endopeptidase activity in developing rat brain. A new and highly sensitive fluorogenic substrate, 7‐(succinyl‐Gly‐Pro)‐4‐methylcoumarinamide, was used for determination of the enzyme activity. The enzyme activity per brain increased until 2 weeks of age, and then decreased during maturation. The enzyme was purified about 7800‐fold from the brain of the rat at 2 or 3 weeks of age. The enzyme has a pH optimum of 5.8 to 6.5, and an approximate molecular weight of 70,000. The enzyme activity was completely inhibited by low concentrations of diisopropylfluorophosphate and partially inhibited by high concentrations of phenylmethanesul‐phonylfluoride, which are potent serine protease inhibitors. Moreover, thiolblocking agents and some heavy metals also have a strong effect on the activity. Bacitracin was found to be a potent inhibitor, with an IC50 value of 2.5 × 10−6m at 0.5 mm of the substrate. The enzyme was proved to hydrolyze the NH2‐terminal tetrapeptide, Arg1‐Pro2‐Lys3‐Pro4, from substance P to produce the heptapeptide, Gln5‐Gln6‐Phe7‐Phe8‐Gly9‐Leu10‐Met11‐CONH2. The Km value of the hydrolysis of substance P was 1.0 mm. This enzyme may be related to the regulation of substance P in the brain, and to the development of neurones by forming the tetrapeptide because the tetrapeptide has almost the same effect as substance P on the neurite extension of neuroblastoma.

A new and highly sensitive fluorescence assay for collagenase-like peptidase activity.

Abstract A highly sensitive fluorescence assay for collagenase-like peptidase (CL-peptidase) has been developed using a newly synthesized substrate, (succinyl-Gly-Pro-Leu-Gly-Pro)-4-methylcoumaryl-7-amide (Suc-GPLGP-MCA). Suc-GPLGP-MCA was hydrolyzed at the Leu-Gly bond by CL-peptidase, (Gly-Pro)-4-methylcoumaryl-7-amide liberated by the enzyme was immediately hydrolyzed to Gly-Pro and 7-amino-4-methylcoumarin (AMC) by an excess of an auxiliary enzyme, X-prolyl dipeptidyl-aminopeptidase, and the fluorescence intensity of the AMC was measured at 460 nm with excitation at 380 nm. When assayed by this method, CL-peptidase partially purified from chick embryo showed a pH optimum at 8.0 and a K m value of 4.0 × 10 −4 m toward Suc-GPLGP-MCA. Under the optimum condition, the reaction proceeded linearly up to 4 h. The CL-peptidase activity was found in normal human sera by this method and the mean and standard deviation of the activity was 0.59 ± 0.10 nmol/min/ml of serum ( n = 10). This assay was also applicable for the CL-peptidase in human liver and kidney. The results suggest that the CL-peptidase assayed by this new substrate may be different from the “PZ-peptidase” which cleaves a synthetic substrate for collagenase-like peptidase, 4-phenylazobenzyloxycarbonyl (PZ)-Pro-Leu-Gly-Pro- d -Arg (PZ-peptide). The new peptide, Suc-GPLGP-MCA, was found not to be a substrate for specific collagenase from tadpole.

Simple purification of aromatic l-amino acid decarboxylase from human pheochromocytoma using high-performance liquid chromatography☆

We purified aromatic L-amino acid decarboxylase (AADC) homogeneously and rapidly from human pheochromocytoma using high-performance liquid chromatography. HPLC with gel permeation and hydrophobic columns was highly effective, and the entire purification could be finished within 3 days. Purified AADC showed a single band with an Mr of 50,000 on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and decarboxylated L-3,4-dihydroxyphenylalanine, L-5-hydroxytryptophan, and L-threo-3,4-dihydroxyphenylserine (a synthetic precursor of natural norepinephrine). Amino acid analysis of purified AADC was performed.

Rapid chromatogrpahic purification of dipeptidyl peptidase IV in human submaxillary gland

Pure dipeptidyl peptidase IV (X-prolyl dipeptidyl aminopeptidase), which did not contain aminopeptidase activity at all, was rapidly prepared from the human submaxillary gland by chromatography with concanavalin A-Sepharose and Gly-Pro-NH-(CH2)6-NH-Sepharose. The entire purification took only 3 days. Aminopeptidase, which was very difficult to separate from dipeptidyl peptidase IV by various chromatographic procedures, could be completely removed by chromatography with Gly-Pro-NH-(CH2)6-NH-Sepharose. On SDS gel electrophoresis the purified enzyme gave a single band with a molecular weight of 116,000. The apparent molecular weight of the enzyme was estimated to be 225,000 by gel filtration. Therefore, the enzyme consists of two identical subunits. It did not hydrolyze Ala p-nitroanilide at all, but the hydrolysis of the p-nitroanilides of Gyl-Pro, Lys-Pro and Arg-Pro at pH 8.0 was nearly specific.

Effects of repeated systemic administration of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) on striatal tyrosine hydroxylase activity in vitro and tyrosine hydroxylase content

We examined both in vitro tyrosine hydroxylase (TH) activity and TH content determined by a new enzyme immunoassay in the mouse striatum after repeated systemic injection of MPTP. Repeated systemic administration of MPTP to mice (30 mg/kg per day, subcutaneously for 8 days) caused an approximately 65% decrease of both TH activity and TH content in the striatum. The intensity of immunohistochemical staining of TH protein in the striatum was also reduced in MPTP-treated mice. These results indicate that the reduction of TH activity in vitro after the repeated administration of MPTP is due to reduction of TH protein as a result of nerve degeneration.

An integrated scheme for the simultaneous determination of biogenic amines, precursor amino acids, and related metabolites by liquid chromatography with electrochemical detection.

A new method using high-performance liquid chromatography with electrochemical detection (HPLC-ED) for the simultaneous determination of monoamines, their precursor amino acids, and related major metabolites in small samples of brain tissue weighing from 0.5 to 50 mg is described. The method is based on the preliminary isolation of monoamines (dopamine, norepinephrine, epinephrine, and serotonin), their precursor amino acids (tyrosine, 3,4-dihydroxyphenylalanine, tryptophan and 5-hydroxytryptophan), and their major metabolites (3-methoxytyramine, normetanephrine, 3,4-dihydroxyphenylacetic acid, homovanillic acid, vanillylmandelic acid, 3-methoxy-4-hydroxyphenylethyleneglycol, and 5-hydroxyindoleacetic acid) by chromatography on small columns of Amberlite CG-50 and Dowex 50W, and by ethyl acetate extraction. All the compounds in the four isolated fractions were measured by HPLC-ED on a reversed-phase column under four different conditions. The sensitivity was from 0.1 to 40 pmol, depending on the substances analysed. This newly established method was applied to the study of the effects of an aromatic L-amino acid decarboxylase inhibitor (NSD-1015) and a monoamine oxidase inhibitor (pargyline) on the levels of monoamines, their precursor amino acids and their major metabolites in brain regions of mice.

A sensitive and specific assay for dipeptidyl-aminopeptidase II in serum and tissues by liquid chromatography-fluorometry

A highly sensitive and specific method for the assay of dipeptidyl-aminopeptidase II (DAP II) in crude enzyme preparations such as serum and tissue homogenates has been established by using a newly synthesized fluorogenic substrate, 7-Lys-Ala-4-methylcoumarinamide. The enzymatically formed 7-amino-4-methylcoumarin was determined by high-performance liquid chromatography with fluorescence detection. The activities of other aminopeptidases in human serum and rat brain homogenates were completely inhibited by o-phenanthroline without any effect on DAP II activity to permit specific determination of DAP II. The limit of sensitivity for DAP II activity was about 300 fmol/30 min. DAP II activity was found to be increased in sera from cancer patients, in contrast to the decrease in serum DAP IV activity. DAP II activity was found to be unequally distributed in rat brain regions, and the highest activity was found in the hypothalamus.

Aromatic l-amino acid decarboxylase activities in human lung tissues: Comparison between normal lung and lung carcinomas

We measured the activity of aromatic L-amino acid decarboxylase with L-dihydroxyphenylalanine as a substrate (DOPA decarboxylase) in normal lung tissues and lung tumors obtained fresh at surgery. The activity in control human lung tissues was low and variable: 3.50 +/- 0.42 pmole/min/mg protein (n = 56, mean +/- SE, range 0.01-15), indicating the wide individual variations. Most of small cell carcinoma specimens showed very high activity, as compared with both control lung tissues and with other types of non-SCC lung cancers. Similar results were also obtained in the athymic mice heterotransplants of SCC. High activity was also observed using 5-L-hydroxytryptophan as a substrate (5-HTP decarboxylase) in nine SCC samples. Serotonin was not detected in any control lung tissues, but was detected in all the nine SCC samples, but dopamine was detected only in three out of nine SCC samples.

Effects of Systemic Administration of 1-Methyl-4-Phenyl-1,2,3,6-Tetrahydropyridine to Mice on Tyrosine Hydroxylase, l-3,4-Dihydroxyphenylalanine Decarboxylase, Dopamine β-Hydroxylase, and Monoamine Oxidase Activities in the Striatum and Hypothalamus

Abstract: The effects of 1‐methyl‐4‐phenyl‐l,2,3,6‐tetrahydropyridine (MPTP) (30 mg/kg subcutaneously per day for 8 days) to C57BL/6N mice were studied on tyrosine hydroxylase (TH), l‐3,4‐dihydroxyphenylalanine decarboxylase (DDC), and monoamine oxidase (MAO) activities in the striatum, and TH, DDC, dopamine‐β‐hydroxylase (DBH), and MAO activities in the hypothalamus. Treatment with MPTP led to a large decrease in TH activity and a parallel decrease in DDC activity in the striatum, as compared with the saline controls. In contrast, MPTP administration did not cause a decrease of the activities of TH, DDC, and DBH in the hypothalamus. There was also no reduction in MAO activities of striatum and hypothalamus. These data indicate that MPTP administration to mice results in specific degeneration of the dopaminergic nigrostriatal pathway and that DDC in the mouse striatum may mainly be localized in the dopaminergic neurons with TH.

β2-microglobulin decrease in cerebrospinal fluid from parkinsonian patients

A sandwich enzyme immunoassay (EIA) was established by using purified beta 2-microglobulin (beta 2-MG) as a standard protein and a polyclonal antibody raised against human beta 2-MG. The EIA was applied for the measurement of beta 2-MG levels in human cerebrospinal fluid (CSF) from parkinsonian patients and control patients devoid of neurological diseases. beta 2-MG contents in CSF of the control group and the parkinsonian group were 1.81 +/- 0.11 micrograms/ml CSF and 0.63 +/- 0.09 microgram/ml CSF, respectively. Thus, beta 2-MG content in CSF was reduced in parkinsonian patients to less than 35% of the control value (P less than 0.005). We had previously reported that the activity and content of dopamine beta-hydroxylase (DBH) were decreased in CSF from parkinsonian patients. A significant positive correlation (r = 0.87) was observed between the beta 2-MG content and DBH activity for CSF from 45 patients. These results suggest a probable link between an immunological change and the changes in catecholaminergic neurons in Parkinsons disease.