Calcium-activated neutral protease inhibitor from rabbit erythrocytes lacks the N-terminal region of the liver inhibitor but retains three inhibitory units

Endogenous inhibitors for calcium-activated neutral protease (CANP) were purified from rabbit erythrocytes and liver. The purified inhibitors showed single bands but with significantly different mobilities on sodium dodecylsulfate-polyacrylamide gel electrophoresis. Peptide mapping and sequencing analyses have revealed that the erythrocyte inhibitor (429 residues) retains the C-terminal three repetitive units of the liver inhibitor (639 residues), which contains four potential repetitive units for inhibition of CANP. The erythrocyte and liver inhibitors inhibited 3 and 4 moles of CANP on the basis of the molecular weights of 46,000 and 68,000, respectively.     

Analysis of water in Autonomous Biological Systems (ABS) samples.

Several soluble components, peptidase and amino acids, and carbon isotopic ratio in the water retrieved from flight experiments of Autonomous Biological Systems (ABS) as well as ground control samples are analyzed to interpret the condition, dynamics, material balance of the ABS ecosystems. Organic carbons in flight samples were found to be more abundant compared with the control ones, which suggested the uniform ecosystems in low gravity might easily dissolve more soluble components. The Mir-1997 flight sample showed higher C/N ratio probably because of the dissolution of carbon-rich plant materials.     

Aminoglutethimide effect on circadian rhythms in urinary variables in a patient with idiopathic hyperaldosteronism

Aminoglutethimide (AG: 750 mg/day) was administered to a patient with idiopathic hyperaldosteronism (IHA) and circadian rhythms in urinary excretion of sodium (UNaV), potassium (UKV), aldosterone (AER) and 17-OHCS were analyzed by the single cosinor method. Urine was collected every 4h for 24h on the day before and on the 1st, 3rd and 7th day of AG administration, and above variables in each sample were determined. Circadian rhythms of 14 patients with primary aldosteronism (PA) who served as controls were also analyzed. In the present case, circadian acrophases in UNaV and AER studied before AG administration occurred at 22(19) and 07(05), respectively. They were similar to those of preoperative PA-patients. Circadian acrophase in UNaV occurred earlier with AG administration and on the 7th day it was at 14(05), a value similar to that of postoperative PA-patients. Circadian mesor in AER decreased remarkably from 4.1 to 0.6 micrograms/4h with AG administration, as did circadian mesor in UKV, whereas circadian mesor and acrophase in 17-OHCS did not change. Thus, the circadian characteristics in urinary variables in the present IHA-case were pathophysiologically similar to those of PA.     

Identification of 4-Chloroindole-3-acetic Acid and Its Methyl Ester in Immature Seeds of Vicia amurensis (the Tribe Vicieae), and Their Absence from Three Species of Phaseoleae

The natural chlorinated auxins 4-chloroindole-3-acetic acid(4-Cl-IAA) and its methyl ester (4-Cl-IAA Me ester) were found,in addition to IAA and its Me ester, by gas chromatography-massspectrometry in immature seeds of Vicia amurensis, a Vicieaespecies. In contrast, only non-chlorinated, IAA and IAA Me esterwere present in immature seeds of three Phaseoleae species.These results are further evidence of the wide distributionof 4-Cl-IAA and its Me ester in various Vicieae. (Received October 3, 1986; Accepted December 22, 1986)     

Calcium-activated neutral protease and its endogenous inhibitor Activation at the cell membrane and biological function

The structures of calcium-activated neutral protease (CANP) and its endogenous inhibitor elucidated recently have revealed novel features with respect to their structure-function relationship and enzyme activity regulation. The protease is regarded as a proenzyme which can be activated at the cell membrane in the presence of Ca²⁺ and phospholipid, and presumably regulates the functions of proteins, especially membrane-associated proteins, by limited proteolysis. Protein kinase C is hydrolysed and activated by CANP at the cell membrane to a cofactor-independent form. These results are reviewed and the possible involvement of CANP in signal transduction is discussed.     

A monoclonal antibody raised to tumor-specific T cell-derived suppressor factors also recognizes T suppressor inducer factors of the 4-hydroxy-3-nitrophenyl acetyl hapten suppressor network

A monoclonal antibody (mAb), B16G, was raised from BALB/c mice immunized with affinity-purified T suppressor factors (TsF) specific for the murine mastocytoma P815. This mAb was found to bind to polyclonal TsF isolated from the spleens of tumor-bearing animals, and to the TsF released from a P815-specific T cell hybridoma. In this study, B16G was tested for its reactivity with TsF produced in the 4-hydroxy-3-nitrophenyl acetyl hapten system. The factors from three types of suppressor T cell hybridomas, each representing the immortalized analogues of the inducer T suppressor cell (Ts1), transducer suppressor cell (Ts2), and effector suppressor cell (Ts3) network populations, were tested. B16G was found to be reactive with two sources of TsF1 as assayed by enzyme-linked immunosorbent assay and delayed-type hypersensitivity bioassay. By contrast, TsF2 and TsF3 were nonreactive with B16G. These results indicate that B16G recognizes class-specific suppressor factor determinants, and that the transducer/effector factors of the network are apparently serologically distinct. Because the B16G mAb fails to recognize 4-hydroxy-3-nitro-phenyl acetyl-specific TsF3 that share idiotype-related determinants with TsF1 yet binds to TsF1 molecules that have interacted with antigen, the binding is apparently independent of the site of antigen recognition. Additionally, the results show that the tumor-specific TsF1 raised in one suppressor system share serologic determinants with anti-hapten TsF1 raised in another.     

Extensin Secreted into the Culture Medium by Tobacco Cells I. Purification and Some Properties

The medium of 12-day-old culturs of tobacco cells (Nicotianatabacum L., var Xanthi; line XD-6S) contain c.a. 160mg/literof protein, of which 14% of the constituent ami no acids werefound to be hydroxyproline. By sequential column chromatographiesand CsCl density-gradient centrifugation, a basic hydroxyproline-richglycoprotein was purified from the medium and found to havean amino acid composition typical of extensin; with a high levelof hydroxyproline (33mole%), tyrosine (13%), and lysine (14%).The glycoprotein contained 42% (w/w) of sugars, among whicharabinose was the major component (85%). The proportion of thisextensin in the proteins in the culture medium was estimatedto be much higher than that of arabino-galactan protein (about5 times higher) on a protein basis, with extensin comprisingbetween 25% and 41%, and probably about 37% of the proteinsin the medium. (Received September 19, 1988; Accepted December 26, 1988)     

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.     

Preparation and application of a fluorogenic substrate for endo-beta-xylosidase

The present paper describes a fluorometric assay for galactosaminoglycan-degrading endo-beta-xylosidase, utilizing glycosaminoglycan chains bearing a 4-methylumbelliferyl group at the reducing terminus as a substrate. This fluorogenic substrate is synthesized by human skin fibroblasts cultured in the presence of a fluorogenic xyloside, 4-methylumbelliferyl-beta-D-xyloside. The assay is based on measurement of the fluorescence of 4-methylumbelliferone, enzymatically liberated from the synthetic substrate by endo-beta-xylosidase. We examined the applicability of the assay for analysis of endo-beta-xylosidase activity.