Effect of Starvation,Refeeding and Hydrocortisone Administration on Turnover of Myofibrillar Proteins Estimated by Urinary Excretion of Nτ-Methylhistidine in the Rat

Quantitative changes in fractional catabolic and synthetic rates of the myosin-actin pool in rat muscle under starvation and refeeding, during growth or after treatment with hydrocortisone were studied by estimating urinary excretion of N^τ-methylhistidine (3-methyl- histidine; Me-His).

Following deprivation of food, urinary Me-His output increased from 0.35 mg/day to 0.45 mg/day during first 2 day in spite of decreasing body Me-His pool. This high rate of Me-His excretion was maintained for the following 4 days of starvation and then decreased. When rats were refed a 20% casein diet after 10 days of starvation, Me-His excretion continued to decrease even after 3 days of refeeding. On the fifth day of refeeding, it began to rise progressively. During starvation, fractional catabolic rate of myosin-actin was about 3.7 %/day in comparison with 2.6 %/day of fed rats. After refeeding, the fractional catabolic rate decreased rapidly to a minimum value of 1.7 %/day on the third day. After that, it reached to a value of 2.6 %/day of fed rats. On the other hand, fractional synthetic rate of myosin-actin dropped immediately after fasting and the low rate of about 0.4 %/day was maintained during starvation period. Fractional synthetic rate recovered quickly after refeeding.

Urinary output of nitrogen and creatinine rose quickly on the first day after administration of hydrocortisone and on the second day it fell to their normal value. While Me-His excretion increased after injection of hydrocortisone up to 0.52 mg/day on the second day and this high excretion rate remained until the following day. From these results, it was shown that administration of hydrocortisone to rats enhances catabolism and reduces synthesis of myosin-actin. The results also show that the effect of this hormone on myofibrillar protein catabolism appears to last longer than its effect on nitrogen metabolism in the whole body judged from urinary nitrogen output.

Fractional rates of catabolism and synthesis of rat myosin-actin were 3.3 %/day (half- life of 21 days) and 7.2%/day, respectively, at the growth stage of 129 g body weight. These rates were 2.3 %/day (half-life of 30 days) and 2.8 %/day, respectively, at the mature stage of 363 g body weight.

Under the dietary conditions in this experiment, fractional synthetic rate changed far more dramatically than catabolic rate. This suggests that mass of muscle protein is primarily regulated by the rate of synthesis, although the rate of catabolism should not be neglected.     

Studies on Continuous Enzyme Reactions

The kinetics of hydrolysis of acetyl-dl-methionine in DEAE-cellulose-aminoacylase (EC 3.5.1.14) column and DEAE-Sephadex-aminoacylase column was studied.

The rate of hydrolysis of substrate was shown to be dependent on the flow rate and independent to the dimension of the enzyme column. The rate of hydrolysis of the substrate was equal in cases of down-ward flow and of up-ward flow. The deteriorated aminoacylase columns by long period operation were reactivated by the recharge of aminoacylase to them. The continuous enzyme reaction using an aminoacylase column was superior to the batch enzyme reaction using native aminoacylase.

The enzymatic properties of the water-insoluble aminoacylase prepared by linking mold aminoacylase (EC 3.5.1.14) to DEAE-Sephadex were studied and compared with those of the native aminoacylase.

Optimum pH values for hydrolysis of several substrates by the DEAE-Sephadex-amino-acylase complex (DSA-complex) shifted about 0.5~1.5 pH units more to the acid side than those by the native enzyme. On the effects of metal ions and inhibitors, substrate specificity, optical specificity and kinetic constants, no marked difference was observed between the native enzyme and the DSA-complex. Heat stability, optimum temperature and resistance towards proteases were increased by conversion from the native form to the insoluble enzyme. It was also observed that the DSA-complex was activated by urea.     

Variations in the Lipid Compositions and in the Lipid Molecular Species of Lipomyces starkeyi Cultivated in the Glucose Sufficient and the Glucose Deficient Media

The lipid compositions, fatty acid compositions, positional distributions of fatty acids in glycerides, and molecular species of phospholipids of L. starkeyi, cultured in the glucose sufficient and the glucose deficient media were compared.

Under the glucose sufficient condition, the triglyceride content increased, accompanied by the remarkable increase of C_16:0–C_18:1–C_18:0. The phospholipid content also increased with the variations of the compositions of molecular species in phosphatidylethanolamine and phosphatidylcholine.

Under the glucose deficient condition, the triglyceride content remarkably decreased, especially in C_18:1–C_18:1–C_18:1. The compositions of phospholipid molecular species were considerably different from those of the glucose sufficient condition.     

Immobilized Aminoacylase on Porous Glass Beads

The preparation and properties of immobilized aminoacylase on porous glass by covalent binding [Porous glass-CVB-aminoacylase] and the continuous enzymatic reactions using such preparations are described.

Two types of porous glass-CVB-aminoacylase were prepared. One was aminoacylase covalently bound to alkylaminosilane derivative of porous glass with glutaraldehyde as a coupling agent [Alkylamino-porous glass-CVB-aminoacylase], and the other was aminoacylase covalently bound to arylaminosilane derivative of porous glass with nitrous acid as a coupling agent [Arylamino-porous glass-CVB-aminoacylase]. The enzyme activities of such immobilized aminoacylases were 3.2~13.0 units/ml glass for the former and 1.9~6.8 units/ml glass for the latter. Especially, alkylamino porous glass-CVB-aminoacylase showed excellent stability at pH 6~9 and temperature below 50°C, and was able to be stored for more than six months without appreciable loss of the activity.

The continuous enzyme reaction using the alkylamino porous glass-CVB-aminoacylase packed in a column was operated for 54 days at 37°C, and the half-life of the immobilized enzyme was calculated to be 78 days. From these results, it was recognized that such an immobilized aminoacylase on porous glass would be applicable in an industrial preparation of various l-amino acids from their dl-forms.     

Hyperoxia reverses glucotoxicity-induced inhibition of insulin secretion in rat INS-1 β cells

Chronic hyperglycemia has deleterious effects on pancreatic β-cell function, a process known as glucotoxicity. This study examined whether chronic high glucose (CHG) induces cellular hypoxia in rat INS-1 β cells, and whether hyperoxia (35% O₂) can reverse glucotoxicity-induced inhibition of insulin secretion. CHG (33.3?mm, 96?h) reduced insulin secretion, and down-regulated insulin and pancreatic duodenal homeobox factor 1 gene expression. CHG also increased intracellular pimonidazole-protein adducts, a marker for hypoxia. CHG also enhanced hypoxia-inducible factor 1α (HIF-1α) protein expression and its DNA-binding activity, which was accompanied by a decrease in mRNA expression of glucose transporter 2 (GLUT2), glucokinase and uncoupling protein-2 and an increase in mRNA expression of GLUT1 and pyruvate dehydrogenase kinase 1. Hyperoxia restored the decrease in insulin secretion and the gene expression except for GLUT2, and suppressed intracellular hypoxia and HIF-1α activation. These results suggest that glucotoxicity may cause β-cell hypoxia. Hyperoxia might prevent glucotoxicity-induced β-cell dysfunction and improve insulin secretion.     

Affinity-based screening of MDM2/MDMX–p53 interaction inhibitors by chemical array: Identification of novel peptidic inhibitors

MDM2 and MDMX are oncoproteins that negatively regulate the activity and stability of the tumor suppressor protein p53. The inhibitors of protein–protein interactions (PPIs) of MDM2–p53 and MDMX–p53 represent potential anticancer agents. In this study, a novel approach for identifying MDM2–p53 and MDMX–p53 PPI inhibitor candidates by affinity-based screening using a chemical array has been established. A number of compounds from an in-house compound library, which were immobilized onto a chemical array, were screened for interaction with fluorescence-labeled MDM2 and MDMX proteins. The subsequent fluorescent polarization assay identified several compounds that inhibited MDM2–p53 and MDMX–p53 interactions.     

Protease and Protease Inhibitor Activity in Rat Skeletal Muscle during Growth,Protein Deficiency and Fasting

Growing rats fed a protein deficient diet showed an increase in acid and alkaline protease activity and a decrease in protease inhibitor activity. Similar results were found after two and four day fasting periods. Animals on diets of 10% and 20% casein showed parallel protease and protease inhibitor activity when compared to each other at similar body weight periods.     

Lipid Molecular Species of Lipomyces starkeyi

The molecular species of glycerides and phospholipids of the yeast Lipomyces starkeyi IFO 0678 harvested at 60 hr, corresponding to the late exponential phase, were analyzed by gas chromatography-mass spectrometry. The major triglyceride was C_16:0–C_18:1–C_18:1. The major molecular species of phospholipid were 1–C_16:0–2–C_18:1 and 1–C_18:1–2–C_18:2. Although phosphatidylcholine and phosphatidylethanol amine were composed of several kinds of molecular species, 1–C_16:1–2–C_18:1, 1–C_18:1–2–C_18:2 and l-C_16:0–2–C_18:1, phosphatidylserine was composed of almost exclusively 1–C_16:0-2-C_18:1. The lipid and the fatty acid compositions of the yeast harvested at the different growth phases were also investigated.     

Use of Water-soluble Carbodiimide (EDC) for Immobilization of EDC-sensitive Dextranase

Water-soluble carbodiimide [EDC: (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide)] is a useful reagent for chemical modification of carboxyl group of various proteins. Model experiments to establish detailed conditions for the cross-linking reaction with EDC were conducted. Since the reactivity of hexamethylenediamine as a nucleophile was almost comparable to that of glycine ethyl ester, AH-Sephadex and the carboxyl group of aspartylphenylalanine methyl ester were coupled by EDC. From the hydrolyzate of the isolated gel, aspartic acid and phenylalanine methyl ester were identified. When bovine serum albumin (BSA) was incubated with AH-Sephadex and EDC, about 90 % of the BSA was coupled to the gel by 3 hr incubation. Moreover, BSA was effectively coupled with the carboxymethyl cellulose (CMC) after activation of the carboxyl groups of CMC with EDC followed by the removal of excess EDC. The latter case would be useful for cross-linking the enzyme molecules to the matrix because of the very mild reaction conditions. For example, endodextranase, which readily lost its activity upon being incubated with EDC (suggesting that a carboxyl group was essential for the enzyme activity), was effectively immobilized to CMC with EDC. This improved reaction step for the cross-linking seemed to be especially useful for the glycosylases, because in most of these enzymes carboxyl groups play a role in the catalytic residue.