Machiko Nagai

Aminopeptidase activities on the surface of mammalian cells.

Activities of hydrolytic enzymes on the surface of monkey kidney, canine kidney, L. FM3A and various tumor cells were determined and compared with those in the cell homogenate. Although aminopeptidase (EC 3.4.11.-) activities were always detected on the surface membrane in mammalian cells, trypsin, chymotrypsin and elastase activities were not detected while slight glycosidase activity was detected in a suspension of cultured cells. The activities of alanine-, leucine-, methionine- and phenylalanine-aminopeptidases were rather high but aminopeptidase A, proline-, valine-, glycyl propline dipeptidyl-and glycyl propyl leucine-tripeptidyl-aminopeptidases showed relatively low activities. Aminopeptidase activity was also demonstrated in the isolated membrane fractions. The specific activities of enzymes in these membrane fractions were not significantly greater than in cell homogenate so it was concluded that these enzyme activities were rather loosely bound to the cell membrane. Further evidence for the localization of the aminopeptidase activities on the cell surface was obtained by using glass-bead-bound substrate and detecting the release of the terminal residues. When bestatin, a specific inhibitor against aminopeptidase B and leucine aminopeptidase, was included in the assay system for the enzyme activities on the cell surface, the enzymes were commonly inhibited in all types of cells.

Release of a plasma membrane-bound triaminopeptidase activity from mammalian cells by thermolysin.

Abstract Aminopeptidase and other enzyme activities on cellular surface were determined in the presence and absence of endopeptidases. Gly-Pro-Leu-AP activity was specifically released into the medium by thermolysin treatment, while the other activities retained on the cellular surface were markedly decreased. A similar phenomenon was also observed in rat liver membrane, mouse FM3A, spleen lymphocyte and other cells. Structural rearrangement of some protein components in the cell plasma membrane was suggested.

Two different modes of enzymatic changes in serum with progression of Duchenne muscular dystrophy

Nineteen serum enzymes from patients with Duchenne muscular dystrophy and asthma, and normal subjects were studied. These enzymes include aminopeptidases, cathepsin C, angiotensin-converting enzyme, serine proteinase, sulphatase, phosphatase, esterases and ribonuclease. The enzymatic changes in dystrophic patients were related to two parameters: severity of the disease as judged from symptomatology, and duration of the disease. Most of the enzyme levels tested were increased in milder cases, but they tended to decrease with severity of the disease. On the other hand, there was a group of enzymes showing just opposite tendencies: serine proteinase, cathepsin C and ribonuclease. Even when viewed from the relationship to duration of the disease, the above mentioned grouping of enzymes was generally valid. Most of the enzyme levels, including those routinely applied as clinical parameters, tended to decrease, logarithmically, with an increase in duration of the disease. On the contrary, some others, including serine proteinase, cathepsin C and ribonuclease, tended to increase toward their control levels. Such tendencies were not found in the patients with asthma. The discrepancy between the above two groups of enzymes may have some implications for the process of protein degradation in dystrophic patients.

Role of intramuscular enzymatic changes in the development of muscular weakness in rats with experimental allergic neuritis

We investigated the role of intramuscular enzymatic changes in the development of muscular weakness in rats suffering from experimental allergic neuritis. At an initial stage without apparent clinical symptoms, enzymatic changes of similar types occurred in the muscles of the forelimbs and hind limbs. At a later stage when the weakness appeared in the hind limb but not in the forelimb, dissociation of the pattern of the enzymatic changes occurred between the two limbs. Comparison of the intramuscular enzymatic changes between the two stages and between the two limbs suggested that the increased activities of aminopeptidases and endopeptidases play some important roles in the development of muscular weakness in this experimental model. Low molecular weight protease inhibitors may thus be worthy of a trial in this disease condition.

Relative deficiency of serine proteinase activities in spleen of aged mice

We examined the relation of hydrolytic enzymes in spleen to the aging process in mice over a period of 30 months. When the enzymatic activities were expressed as activities per milligrams protein, those of serine proteinases and dipeptidyl peptidase IV (Gly-Pro-AP) significantly decreased with age, whereas that of L-leucine aminopeptidase (Leu-AP) increased significantly. However, when expressed as total activities, the enzymatic activities in spleen generally tended to increase with age, except in the case of serine proteinases, because of the age-related increase in spleen weight. The results were taken to indicate that the activities of serine proteinases become relatively more deficient in the spleen as age increases. The results of a multivariate study maintained this peculiarity of serine proteinases in comparison with other enzymes. The relative deficiency of serine proteinases in spleen may be somehow related to immunodeficiency in aged animals, as judged from similar findings in animal models of systemic erythematodes.