Ingensin, a high-molecular-mass alkaline protease from rabbit reticulocyte

A high-molecular-mass protease, ingensin, was purified to homogeneity from rabbit reticulocytes by DEAE-cellulose, HPLC gel filtration, and hydroxyapatite chromatographies. By these procedures, ingensin activity was separated from the activities of two other unique aminopeptidases, one of which is activated by ATP. Ingensin had the following properties: the optimum activity was seen around pH 9.0 and at 50 degrees C; addition of 0.04% SDS and 1 mg/ml linoleic acid resulted in 8- and 4-fold increases in peptide-hydrolyzing activity, respectively. The molecular mass was found to be 700,000 +/- 100,000 daltons on gel filtration, but SDS electrophoresis revealed that the enzyme is composed of several subunits with molecular weights of less than 35,000. The N-terminal-blocked tyrosine- and arginine-MCA derivatives, but not Arg-MCA, were hydrolyzed rapidly by ingensin. The approximate Km values for the reaction of ingensin with Suc-Leu-Leu-Val-Tyr-MCA and Z-Ala-Arg-Arg-MCA were 0.32 and 0.12 mM, respectively. The degradation of several proteins in the reticulocyte extract was stimulated by the addition of SDS and linoleic acid. The activator concentrations necessary for stimulation of the protein hydrolysis are similar to those of the purified reticulocyte ingensin for synthetic substrates. Ingensin did not associate with either right-side-out or inside-out red cell membranes. These results suggest that ingensin is a cytosolic fatty acid-stimulated protease, which is involved in the protein turnover in reticulocyte extracts.     

Isolation and characterization of three alpha-glucosidases from the Japanese quail

We have defined one type of acid alpha-glucosidase and two types of neutral alpha-glucosidases from quail skeletal muscle on the basis of differences in the elution patterns on a DEAE-cellulose column. The appearance of the two neutral alpha-glucosidase isoenzymes was age-dependent. A decrease in acid alpha-glucosidase activity was demonstrated in Japanese quails with glycogenosis type II. The characteristics of these three alpha-glucosidase isoenzymes are described.     

Control of ventilation in extreme-altitude climbers

Ten climbers who participated in the Nepal-Japan Kangchenjunga Expedition (altitude, 8,478-8,586 m) in 1984 were examined for their hypercapnic and isocapnic hypoxic ventilatory responses (HCVR and HVR) at sea level before and after the expedition. Five climbers who reached an altitude higher than 8,000 m, [designated high-performance climbers (HPC)] exhibited significantly higher HVR than five climbers who did not [low-performance climbers (LPC)]. On the other hand, no significant difference in HCVR was seen between HPC and LPC. Our results were in agreement with the findings reported by Schoene et al. (J. Appl. Physiol. 56: 1478-1483, 1984) obtained in the American Medical Research Expedition to Everest in 1981. Significant depression in HVR in five climbers was found even 35-40 days after the expedition, which was accompanied by decreased arterial partial pressure of CO2 and increased pH at rest. Hence, the effect of altitude acclimatization in the climbers exposed to extreme altitude may have still persisted at the time of the postexpedition study. Our results confirmed that HRV evaluated at sea level may be used as an indicator of a climber's capability at great high altitude.     

Neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii

The neutral glycosphingolipids of ova of the fresh-water bivalve, Hyriopsis schlegelii were characterized. The most abundant glycolipid was ceramide monosaccharide, followed by ceramide trisaccharide, ceramide tetrasaccharide, and ceramide disaccharide. More complex neutral glycolipids accounted for almost one-third of the total. The total amount of these glycolipids was 0.59 mg/g of dry weight of the ova preparation, a yield which was one-seventh of that of spermatozoa neutral glycolipids. Structural analyses were performed by enzymatic hydrolysis of the glycolipids with exoglycosidases, permethylation experiments, and also immuno-chemical assays. The proposed structures are as follows: ceramide monosaccharides, Gal-Cer and Glc-Cer; ceramide disacharides, Gal(beta 1-4)Gal-Cer, Gal(beta 1-4)Glc-Cer, and Man(beta 1-4)Glc-Cer; ceramide trisaccharide, Man(alpha 1-3)Man(beta 1-4)Glc-Cer; ceramide tetrasaccharides, Man(alpha 1-3)[Xyl(beta 1-2)]Man(beta 1-4)Glc-Cer, GlcNAc(beta 1-2)Man(alpha 1-3)Man(beta 1-4)Glc-Cer, Man(alpha 1-3)[Gal(beta 1-2)]Man(beta 1-4)Glc-Cer, and Man(alpha 1-2?)Man(alpha 1-3)Man(beta 1-4)Glc-Cer. The latter two ceramide tetrasaccharides were new types of glycosphingolipids. The spectrum of ova glycolipids appeared to be more complicated than that of the spermatozoa glycolipids. The ova glycolipids characterized here, with the exception of ceramide tetrasaccharides, contained considerable amounts of 2-hydroxy fatty acids, which were not observed in the spermatozoa glycolipids. The major sphingosine base was C18-sphingenine in all the ova glycolipids as well as in the spermatozoa glycolipids. However, the content of anteiso type of sphingosine base was 2- to 3-fold higher in the ova than in the spermatozoa.     

Fluorescence polarization as a tool to study lectin-sugar interaction

The binding ofRicinus communis agglutinin andAbrus agglutinin to 4-methylumbelliferyl β-D-galactopyranoside was studied by equilibrium dialysis, fluo-rescence quenching and fluorescence polarization. The number of binding sites and the association constant value obtained by fluorescence polarization for bothRicinus communis agglutinin andAbrus agglutinin are in close agreement with those obtained by the other methods. This indicates the potential of ligand-fluorescence polarization measurements in the investigation of lectin-sugar interactions.     

Lectins from<Emphasis Type="Italic">Griffonia simplicifolia</Emphasis> seeds

The physical-chemical and carbohydrate binding specificity ofGriffonia simplicifolia I (GS I) isolectins, one of the 4 lectins isolated fromGriffonia simplicifolia seeds, are described.Association constants for the binding of methyl α- and β-D-galactopyranoside and methyl 2-acetamido-2-deoxy-α-D-galactopyranoside to the A₄, A₂ B₂ and B₄ isolectins are reported.Precipitation reactions of theGriffonia simplicifolia isolectins with guaran and type B blood group substance are described.The hypothesis that subunit B is a precursor of subunit A, a process involving proteolytic cleavage of the B subunit, was tested by conducting structural studies on the 2 subunits. The results indicated that the A and B subunits are probably products of 2 separate but closely related, possibly contiguous genes.     

Haemolysis of rabbit erythrocytes by a lectin from the seeds of<Emphasis Type="Italic">Croton tiglium</Emphasis>

The kinetics of haemolysis of rabbit erythrocytes byCroton tiglium lectin was studied as a function of concentration of the lectin and erythrocytes. The length of the prelytic period decreased with increasing lectin concentrations, indicating that the secondary events at the membrane which follow the binding of the lectin to cell surface carbohydrate receptors are accelerated at higher surface concentrations of the lectin. The rate or extent of haemolysis was not affected by the inclusion of ions like K⁺, Ca²⁺ and Mg²⁺ in the medium or by the substitution of ionic medium by a non-ionic medium. The inhibition of haemagglutination and haemolysis of rabbit red cells byCroton tiglium lectin by antilectin rabbit serum was observed. A possible mechanism of haemolysis by the lectin is discussed.     

Plant lectins specific for N-acetyl-β-D-galactosamine

N-Acetyl-D-galactosamine in β-linkage being ubiquitous in cell surface glycoproteins, their interaction with lectins specific for this sugar moiety may be a significant event in cell adhesion phenomena. This article discusses the common β-N-acetyl galactosamine-specific lectins, with particular stress on the lectin from winged beans (Psophocarpus tetragonolobus).     

Histochemical studies on the regeneration of aminergic nerves in rat cerebral artery after superior cervical ganglionectomy

The course of regeneration of aminergic nerves in rat cerebral arteries was studied by means of histochemical methods, after uni- or bilateral cervical sympathectomy. Degeneration of aminergic nerves started on day 1 and was complete between days 3 and 7 after surgery. Between weeks 4 and 6, regenerating nerves started to appear from the proximal internal carotid artery. Regenerated aminergic nerve fibres were generally unbeaded and intensity of fluorescence was weak. The circular nerves appeared earlier than the longitudinal ones. The number of regenerating nerves reached the maximum, between months 9 and 12, at about half the normal level. AChE activity of the cerebral arteries showed no significant changes at any stage.     

A putative gene of tobacco chloroplast coding for ribosomal protein similar to E. coli ribosomal protein S19.

A partial sequence of a cloned 3.2 Md BamHI fragment from tobacco chloroplast DNA revealed the occurrence of a putative gene for ribosomal protein. The putative gene is located on the left margin of the large single-copy region in the chloroplast DNA. The coding region contains 276 bp (92 codons). The amino acid sequence deduced from the DNA sequence shows 55% homology with that of E. coli S19 (91 amino acid residues).