Effects of amino acid replacements around the reactive site of chicken ovomucoid domain 3 on the inhibitory activity toward chymotrypsin and trypsin.

We have previously shown that replacing the P1-site residue (Ala) of chicken ovomucoid domain 3 (OMCHI3) with a Met or Lys results in the acquisition of inhibitory activity toward chymotrypsin or trypsin, respectively. However, the inhibitory activities thus induced are not strong. In the present study, we introduced additional amino acid replacements around the reactive site to try to make the P1-site mutants more effective inhibitors of chymotrypsin or trypsin. The amino acid replacement Asp-->Tyr at the P2' site of OMCHI3(P1Met) resulted in conversion to a 35000-fold more effective inhibitor of chymotrypsin with an inhibitor constant (K(i)) of 1. 17x10(-11) M. The K(i) value of OMCHI3(P1Met, P2'Ala) indicated that the effect on the interaction with chymotrypsin of removing a negative charge from the P2' site was greater than that of introducing an aromatic ring. Similarly, enhanced inhibition of trypsin was observed when the Asp-->Tyr replacement was introduced into the P2' site of OMCHI3(P1Lys). Two additional replacements, Asp-->Ala at the P4 site and Arg-->Ala at the P3' site, made the mutant a more effective inhibitor of trypsin with a K(i) value of 1. 44x10(-9) M. By contrast, Arg-->Ala replacement at the P3' site of OMCHI3(P1Met, P2'Tyr) resulted in a greatly reduced inhibition of chymotrypsin, and Asp-->Ala replacement at the P4 site produced only a small change when compared with a natural variant of OMCHI3. These results clearly indicate that not only the P1-site residue but also the characteristics, particularly the electrostatic properties, of the amino acid residues around the reactive site of the protease inhibitor determine the strength of its interactions with proteases. Furthermore, amino acids with different characteristics are required around the reactive site for strong inhibition of chymotrypsin and trypsin.     

One-dimensional food chain equations and their generalization

Two equations describing one-dimensional food chains are known to possess soliton solutions. It is demonstrated that both equations are embraced within another equation, which arises in the theory of chains of enzymic reactions. We find an elliptic function solution to this equation. We obtain a one-soliton solution from it and re-derive the elliptic function solutions of the two ecological equations.     

Effect of the sugar moiety on complex formation of cycloamyloses with p-nitrophenyl glycosides

Circular diochroism (CD) spectra of four p-nitrophenyl glycosides and their cycloamylose complexes were investigated at various concentrations of cycloamylose and at temperatures ranging from 20 to 60°C. The CD spectra of p-nitrophenyl glycosides changed remarkably in the presence of cycloamyloses, and significant differences in spectral shape and intensity were observed between the cyclohexaamylose complex and the cycloheptaamylose complex. The difference CD spectra between the free guest and its complex indicates that the nitrophenyl group is included in the cycloamylose cavity but its disposition is different between the complexes with cyclohexaamylose and cycloheptaamylose. Values of enthalpy and entropy of the cyclohexaamylose complex are considerably larger than those of the corresponding cycloheptaamylose complex, although the free energy differs only slightly. It is suggested that the nitrophenyl group is more loosely bound to the cycloheptaamylose cavity than to the cyclohexaamylose cavity, and has much more flexibility in its disposition.     

A case of insulin autoimmune syndrome associated with small insulinomas and rheumatoid arthritis

Twenty five cases of insulin autoimmune syndrome including this case has been reported so far without having the pathogenesis clarified. This paper describes a case which suggests one aspect of pathogenesis. The patient, a housewife concurrently had insulinoma and severe rheumatoid arthritis, complaining of hypoglycemic syncope attacks. During the attacks her blood sugar levels ranged from 19 to 22 mg%. Her serum extractable immunoreactive insulin (IRI) and insulin binding antibody levels were 557 microunits/ml and 0.390 mU/ml, respectively. gamma-Globulin-bound insulin was also measured electrophoretically. Bio-Gel P 10 column chromatography eluted almost all IRI at the void volume at pH 7.4 and a smaller but significant IRI peak also at pH 3.0. Selective angiography revealed a tumor-like staining in the pancreas body. Pancreatectomy relieved her of hypoglycemic attacks. Histology disclosed two small insulinomas. Insulinoma, rheumatoid arthritis and insulin autoimmune syndrome coexisted in this case, suggesting some causal relationship among them.     

Two opposite types of sister chromatid differential staining in BUdR-substituted chromosomes using tetrasodium salt of EDTA

The direct staining of BUdR-substituted Chinese hamster chromosomes in a 4Na-EDTA-Giemsa solution resulted in a B-dark type of sister chromatid differential staining (SCD) in which bifilarly substituted chromatids stained dark. On the other hand, when BUdR-substituted chromosomes were pretreated with a 4Na-EDTA solution and then stained with Giemsa, a B-light type SCD was obtained in which bifilarly substituted chromatids stained light.     

Chemical characterization of a new Japanese variant of carbonic anhydrase I,CA INagasaki 1 (76 Arg→Gln)

A new inherited variant of carbonic anhydrase I (CA I), designated CA I_{Nagasaki 1} (CA I_{NGS 1}), was discovered during a survey of hemolysates from 5852 individuals from the cities of Hiroshima and Nagasaki in Japan. Analysis of the amino acid composition of a tryptic peptide from the CA I_{NGS 1} variant indicated that a glutaminyl residue was substituted for an arginyl residue at position 76. Heat degradation studies showed that the CA I_{NGS 1} variant was less stable than normal CA I. The CO₂ hydrase and esterase activities of the normal and variant carbonic anhydrases I, as well as the relative amounts of the two enzymes in heterozygotes, were similar.This work was supported in part by Contract E(11-1)-1552 with the Energy Research and Development Administration, Washington, D.C. (to J. V. Neel), and by U.S. Public Health Service Grant GM-24681.     

Studies of oligosaccharide linkages by simultaneous determination of component aldehydes in dialdehyde fragments as (2,4-dinitrophenyl)hydrazones

The component aldehydes in dialdehyde fragments formed by periodate oxidation of oligosaccharides were converted quantitatively into the corresponding (2,4-dinitrophenyl)hydrazones by the simple procedure of treatment with excess (2,4-dinitrophenyl)hydrazine hydrochloride in 1,2-dimethoxyethane. Then, by chromatographic separation of the hydrazones on a small column of silica gel and subsequent spectrophotometric analysis, it was possible to determine the position of glycosidic substitution in μmolar amounts of various types of glucobioses, oligosaccharides of senega, and some synthetic (1→6)-β-D-gluco-oligosaccharides.     

Endocrine function in a case of beta-adrenergic hyperdynamic circulatory state.

Endocrine functions were investigated in a case of "beta-adrenergic hyperdynamic circulatory state". This state was diagnosed by (1) typical symptoms of cardiac awareness, (2) physical findings (increments of pulse rate and blood pressure by changing positions or walking), (3) increase in cardiac output (5.25 l/min leads to 14.03 l/min) and decrease in circulatory time (10.8 sec leads to 5.5 sec) by isoproterenol infusion (0.02 mug/min/kg body weight), (4) rapid loss of symptoms and above findings by propranolol treatment (30 mg per os daily) and reappearance by discontinuing medication. The mechanism of insulin response to glucose has been a controversy as to whether the secretion is transmitted by beta-receptor or independent glucose receptor. And in this physiologic beta-adrenergic state, it was found that insulin responses in IVGTT and OGTT were within normal limit. When beta-adrenergic condition was corrected by propranolol treatment, insulin responses were shown lowered, though in the normal range. This could be reproduced by discontinuing medication. Insulin, glucagon and growth hormone secretions caused by arginine were also found normal, but during the period the patient was on propranolol therapy, all responses were decreased, within the normal range. These results do not positively support the idea that glucose receptor is linked to beta-receptor. They do not either agree with the contention that secretions of insulin, glucagon and growth hormone induced by arginine are mediated through beta-receptors.     

SGIP1alpha is an endocytic protein that directly interacts with phospholipids and Eps15

SGIP1 has been shown to be an endophilin-interacting protein that regulates energy balance, but its function is not fully understood. Here, we identified its splicing variant of SGIP1 and named it SGIP1alpha. SGIP1alpha bound to phosphatidylserine and phosphoinositides and deformed the plasma membrane and liposomes into narrow tubules, suggesting the involvement in vesicle formation during endocytosis. SGIP1alpha furthermore bound to Eps15, an important adaptor protein of clathrin-mediated endocytic machinery. SGIP1alpha was colocalized with Eps15 and the AP-2 complex. Upon epidermal growth factor (EGF) stimulation, SGIP1alpha was colocalized with EGF at the plasma membrane, indicating the localization of SGIP1alpha at clathrin-coated pits/vesicles. SGIP1alpha overexpression reduced transferrin and EGF endocytosis. SGIP1alpha knockdown reduced transferrin endocytosis but not EGF endocytosis; this difference may be due to the presence of redundant pathways in EGF endocytosis. These results suggest that SGIP1alpha plays an essential role in clathrin-mediated endocytosis by interacting with phospholipids and Eps15.