A membrane-bound metallo-endopeptidase from rat kidney hydrolyzing parathyroid hormone. Purification and characterization

A metallo-endopeptidase, which appears to be an integral membrane protein of rat kidney, was purified to homogeneity by a series of standard chromatographic procedures. This enzyme significantly hydrolyzed human parathyroid hormone [hPTH(1-84)] and a synthetic substrate Suc-Leu-Leu-Val-Tyr-Mec (Suc = succinyl, Mec = 4-methyl-coumarinyl-7-amide). The purified enzyme had apparent molecular masses of 250 kDa on gel filtration, and 88 kDa and 245 kDa on sodium dodecyl sulfate/polyacrylamide gel electrophoresis under reducing and non-reducing conditions, respectively. Its pH optimum for activity was 8.0-8.5 and its isoelectric point was pH 4.9. Its activity was inhibited by EDTA, EGTA and o-phenanthroline, but not by phosphoramidon. The metal-depleted enzyme was reactivated by the addition of metal ions. The enzyme was also inhibited by chymostatin and eglin C, and by thiol compounds. Of the synthetic substrates examined, the enzyme hydrolyzed only Suc-Leu-Leu-Val-Tyr-Mec, one of the synthetic substrates for alpha-chymotrypsin. It did not hydrolyze synthetic substrates with less than four amino acid residues with tyrosine in the P1 position. The enzyme hydrolyzed hPTH and reduced hen egg lysozyme but did not hydrolyze azocasein or [3H]methyl-casein. NH2-terminal amino acid sequence analyses of the degradation products of hPTH(1-84) and reduced hen egg lysozyme by the purified enzyme revealed that the enzyme preferentially cleaved these peptides at peptide bonds flanked by hydrophilic amino acid residues. Amino acid analyses showed that the main degradation products of PTH were hPTH(17-29), hPTH(30-38) and hPTH(74-84). The ability of the enzyme to hydrolyze peptide bonds flanked by hydrophilic amino acid residues and its inability to degrade azocasein distinguish it from several other kidney endopeptidases reported, such as endopeptidase 24.11 and meprin.     

Tryptase TL2 in the membrane of human T4+ lymphocytes is a novel binding protein of the V3 domain of HIV-1 envelope glycoprotein gp 120.

A novel membrane-bound serine esterase in cultured human T4+ lymphocytes, recently purified and named tryptase TL2, binds specifically to the external envelope protein gp 120 of HIV-1, interacting with its V3 domain. This binding was selectively blocked by inhibitors of tryptase TL2 with a GPCR sequence in their reactive site, synthetic peptides corresponding with the sequences of the V3 domains of various HIV-1 strains with the GPGR sequence, and antibody against tryptase TL2, or neutralizing antibody against the V3 domain of HTLV-IIIB. These findings suggest that tryptase TL2 is a binding protein of the V3 domain of HIV-1 envelope glycoprotein.     

Excretion of peroxidase from horseradish hairy root in combination with ion supplementation

Summary The effect of ion-supplemented medium on peroxidase excretion from horseradish (Armoracia rusticana) hairy roots was studied. Supplementation of mannitol instead of ions revealed that the excretion was stimulated not by osmotic pressure in the medium but by ionic properties. Extracellular peroxidase activity per dry cell was proportionally correlated with the ionic strength of the cations. CaCl₂ or MgCl₂ was found to be the most effective agent for excretion among other combinations. CaCl₂ supplementation at the beginning of the culture caused higher peroxidase production in the medium without a significant loss of final cell mass compared with CaCl₂ addition during the culture. Repeated batch culture with 50 mM CaCl₂ supplementation allowed a continuous retention of cell viability over 149 days and produced a great amount of extracellular peroxidase, 12-fold higher than that achieved in a 40-day-old batch culture with 50 mM CaCl₂ supplementation. Correspondence to: T. Kobayashi     

Growth of the hydrocarbon-rich microalga Botryococcus braunii in secondarily treated sewage

Summary A hydrocarbon-rich green microalga, Botryococcus braunii, was able to grow well in secondarily treated sewage (STS) from domestic waste-water in a batch system. The growth in STS from domestic waste-water was as good as in the common artificial medium of modified Chu 13 and its hydrocarbon contents were high enough at 53% and 40% compared with 58% in the case of the modified Chu 13 medium. B. braunii utilized nitrate from 7.67 or 4.48 mg/l to a level below detection of < 0.01 mg/l in STS. After this consumption of nitrate, nitrite was consumed, and ammonium was not. Phosphate, even at an extremely low concentration, was also consumed by B. braunii. These results show the possibility of using STS as a medium to grow B. braunii and for removal of nitrogen and phosphorus by algal consumption in STS.Correspondence to: S. Yokoyama     

Phosphoramidon inhibits the conversion of intracisternally administered big endothelin-1 to endothelin-1

It is suggested that endothelin-1 (ET-1), a potent vasoconstrictor peptide, is involved in the pathogenesis of cerebral vasospasm following subarachnoid hemorrhage (SAH). We examined the effects of intracisternal administration of big ET-1 on the cerebral arteries in the absence or presence of pretreatment with phosphoramidon, an inhibitor of ET converting enzyme, in anesthetized dogs. After intracisternal administration of big ET-1 (10 micrograms/dog), the caliber of the basilar artery on the angiogram was decreased to about 59% of the control. This was accompanied by a marked increase in immunoreactive ET in the cerebrospinal fluid. Systemic arterial pressure was markedly elevated following big ET-1 injection. All changes induced by big ET-1 were effectively prevented with phosphoramidon. These data suggest that intracisternally administered big ET-1 is converted to ET-1 and that the generated ET-1 produces cerebral vasospasm and hypertension. A phosphoramidon-sensitive metalloproteinase appears to contribute to this conversion.     

Growth of a human yolk sac tumor cell line with yolk sac-derived functions in selenium-supplemented chemically defined synthetic medium

Summary A human yolk sac tumor cell line, TG1, which was established from a testicular yolk sac tumor, was found to replicate continuously in a chemically defined medium supplemented with Na₂SeO₃ (ISRPMI). TG1 produced several plasma proteins and growth factors: albumin, alpha-fetoprotein (AFP), ferritin, carcinoembryonic antigen, beta-2-microglobulin, polyamine, neuron specific enolase, tissue polypeptide antigen, transferrin (Tf), epidermal growth factor, and platelet derived growth factor. By analysis of lectin (LcHA)-affinity electrophoresis, to examine the microheterogeneity of carbohydrate chains of synthetic glycoproteins, TG1 cells cultured with ISRPMI produced only LcHA reactive Tf and AFP based on core fucose attached to asparagine-linkedN-acetylglucosamine residues instead of LcHA-nonreactive Tf and AFP produced by TG1 cells cultured with fetal bovine serum (FBS)-containing medium.α1-6 Fucosyltransferase activity was significantly greater in the TG1 cells cultured with ISRPMI (39.9±1.5 pmol · h⁻¹ · mg⁻¹ protein) than cultured with FBS-containing media (18.2±1.2 pmol · h⁻¹ · mg⁻¹ protein). These results have indicated that the selective increase ofα1-6 fucosyltransferase occurred when the cells were cultured with the FBS-free synthetic media.     

Superoxide dismutases enhance the rate of autoxidation of 3-hydroxyanthranilic acid

The autoxidation of 3-hydroxyanthranilate to cinnabarinate at 37 degrees C and at pH 7.4 is hastened by superoxide dismutase (SOD). The Cu,Zn-containing enzyme from bovine erythrocytes and the Mn-containing enzyme from Escherichia coli were equally effective in this regard; whereas the H2O2-inactivated Cu,Zn enzyme was ineffective. Catalase appears to augment the effect of superoxide dismutase, because it prevents the bleaching of cinnabarinate by H2O2. It follows that O2-, which is a product of the autoxidation, slows the net autoxidation by engaging in back reactions and that SOD increases the rate of autoxidation by removal of O2- and thus by prevention of these back reactions.     

Inhibition of rabbit liver fructose 1,6-biphosphatase by AMP: effect of temperature and physiological concentrations of cations and anions

Turkey gizzard smooth muscle myofibrils, the actin of which is composed of 75% smooth muscle γ-isoactin and 25% nonmuscle β-isoactin, were separated into an actomyosin and a cytoskeletal fraction. Isoelectric focusing analysis of the actomyosin actin showed it was 80% γ-isoactin and 20% β-isoactin. It thus appears that the major actin in the tissue is also the major form involved in force generation. When the cytoskeletal material was extracted with low-ionic-strength solution for 18 h at 4 °C, the actin released was 95% γ and 5% β compared with the 75:25 ratio found in the original cytoskeletal material. The extracted material revealed the presence of F-actin filaments and high-molecular-weight aggregates. Little of the material was in a low-molecular-weight form. On the other hand, extraction of the cytoskeletal material with 0.6 m KI resulted in the two isoactins being extracted in the same proportions in which they were found in the original cytoskeletal material. However, when this KI-extracted material was subsequently chromatographed on Bio-Gel A-5m equilibrated with 0.6 m KCl, the γ-isoactin migrated predominantly as a very high molecular weight form while the β-isomer moved in the lower-molecular-weight range of the elution profile. This aggregation behavior displayed by the γ-isoactin was not observed with the γ-isoactin in the actomyosin fraction. These results show that the two gizzard isoactins in the cytoskeletal residue behave very differently in response to various extraction media, and are consistent with possible differential isoactin utilization in gizzard smooth muscle.     

Adductor muscles of the thigh of crab-eating monkeys (Macaca fascicularis)

On the basis of 44 hindlimbs of 14 male and 14 female crab-eating monkeys (Macaca fascicularis), the morphology of the adductor muscles of the thigh was described and some functional indices were calculated. The results obtained from this study agreed generally with those of otherMacaca species reported by various authors. For the classification and nomenclature of the adductors, the criteria proposed byUhlmann (1967, 1968) was well adapted to the crab-eating monkey. The adductors comprise the m. gracilis, m. pectineus, m. adductor longus, pars longa and pars brevis of m. adductor brevis, pars lata and pars minima of m. adductor magnus and m. obturatorius externus. In males, the adductors are generally inserted further down the femur, and the insertions of pars brevis of the m. adductor brevis and pars minima of the m. adductor magnus have longer attachments to the femur than in females. The arrangement of each adductor muscle and of each fasciculus of a thigh muscle may invoke a principle of organization.