Hemisphaericin-D,a dialysable and polymerizable protease found in Bromelia hemisphaerica

Summary Proteolytic activity was detected outside dialysis bag filled with Bromelia hemisphaerica fruit juice. The dialysable protease was concentrated and purified from small molecular weight contaminants on Sephadex G-10 columns. Acrylamide gel electrophoresis of the dialysable protease, in the presence of SDS and 2-mercaptoethanol. demonstrated a single protein band of about 8000 daltons mol. wt. The same single band with identical mobility was shown with hemisphaericin, the enzyme retained inside the dialysis bag. The small protease, named Hemisphaericin-D was antigenic in rabbits and the antibodies cross-reacted fully with Hemisphaericin. Hemisphaericin-D appears not to be a degradation product of Hemisphaericin.This is contribution No. 22 from Departamento de Biología Experimental, Facultad de Medicina, UNAM, Mexico.Acknowledgments. We thank Irma Coria and Silvia Vizconde for skillful assistance. Interchange of information with Dr E. Toro-Goyco, University of Puerto Rico, School of Medicin, was most helpful.     

Presence of a protein disulfide isomerase (EC 5.3.4.1) in wheat germ]

The presence of a protein disulfide isomerase (rearrangease) in wheat embryo has been demonstrated by its ability in reactivating randomly cross-linked ribonuclease. This activity requires a dialysable cofactor; after dialysis, the activity is recovered by addition of reduced glutathione. The enzyme can be precipitated by 70% saturation ammonium sulfate.     

Peritoneal dialysis in small laboratory animals

Summary Healthy rats and guinea-pigs were treated with a simple method of continuous peritoneal dialysis for 12, 24 and 48 h. Increasing with time, both animal species developed severe hypoproteinemia and hemoconcentration due to protein loss into the dialyzate fluid. These changes were associated with a high mortality rate, when Sterofundin^® was used for dialysis. Therefore, protein loss should be substituted and the type of dialyzate must be considered in experimental long-term dialysis using these small laboratory animals.Supported by Deutsche Forschungsgemeinschaft, Bonn-Bad Godesberg, La 369/1.Acknowledgment. Our thanks are due to Jutta Otto and Ulrich Oberdieck for expert technical assistance.     

An improved method for flow dialysis studies with highly increased diffusion rates

Summary An improved flow dialysis procedure with highly increased diffusion rates has been developed allowing the study of small changes in the rate of diffusion. The application of the method described with only a few individual experiments, and with the use of small amounts of biological material, gives much information about binding systems.This work was supported by the Schweizerischer Nationalfonds zur Förderung der wissenschaftlichen Forschung (grant No. 3.1650.73).     

Naturally occurring pea diamine oxidase inhibitors obtained fromSorghum vulgare during germination

Summary During germination ofSorghum vulgare seeds, inhibitors of pea diamine oxidase appeared in the embryos. One was heat labile, dialysable and inhibited the enzyme in vitro, while another was heat stable and inhibited the enzyme synthesis when pea seeds were soaked and allowed to germinate in the extract containing the inhibitors.Acknowledgments. We thank Dr S.K. Srivastava for his helpful suggestions and access to his facilities.     

A dialysis rate method for the measurement of free iodothyronine and steroid hormones in blood

From the dialysis rate of a small amount of tracer hormone from a serum sample through a semipermeable membrane towards an identical serum sample, the free hormone fraction can be calculated. This method is free from a number of artefacts that may occur in equilibrium dialysis.     

Hachweis von individualspezifischen Gewebsantikörpern

Summary Injection of homologous tissue or tissue extracts and homologous transplantation of skin in rabbits provoke the production of antibodies. By means of a new nephelometric method, acquired antibodies against homologous tissue circulating in the serum were found. This tissue-antibody seems to be an individual-specific one, reacting with all organs of the donor. The antibody is thermolabile and not dialysable.

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Die Arbeit wurde mit Hilfe des Schweizerischen Nationalfonds zur Förderung der wissenschaftlichen Forschung durchgeführt.     

Spectral properties of methemoglobins prepared by the action of sodium nitrite and potassium ferricyanide

Summary Methemoglobin was prepared by the addition of sodium nitrite or potassium ferricyanide to oxy or deoxyhemoglobin. The spectral properties of these methemoglobins were studied before and after extensive dialysis. It is shown that the methemoglobin formed by sodium nitrite has substantial spectral differences in visible and Soret band compared to that formed by potassium ferricyanide. These differences are proportional to the excess of sodium nitrite only. This suggests that both methemoglobins are similar compounds.Supported by NIH grant No. 5R01AM 20181-03 and VA grant No. 5455-003.Acknowledgments. The technical assistance of Ms Carol A. Perry and the secretarial assistance of Ms Wanda Stewart are highly appreciated.     

尿毒症患者血清对内皮细胞小凹蛋白-1的影响及意义

目的体外观察尿毒症患者血液透析前后血清和健康人血清对人脐静脉内皮细胞(humanumbilicalveinendothelialcell, HUVECs)小凹蛋白 1(caveolin 1)表达的影响.方法取对数生长的 HUVECs,分为健康组(DMEM+健康人血清,n=8)、透前组(DMEM+尿毒症患者透前血清,n=18)、透后组(DMEM+尿毒症患者透后血清,n=18).采用 MTT法检测细胞活力,免疫组化SABC法和 Westernblot检测各组细胞内 caveolin 1蛋白的分布和含量.结果 MTT法筛选血清最佳干预时间和浓度分别为12小时、10%的血清干预浓度.与健康组比较,透前组细胞内 caveolin 1的蛋白表达水平明显下调(p<0.05),透后组改变不明显(p>0.05);而透后组细胞内 caveolin 1的蛋白表达水平较透前组细胞内 caveolin 1的蛋白表达水平上调(p<0.05).结论血液透析不能降低尿毒症患者动脉粥样硬化的形成的发生率,caveolin 1的变化可能是尿毒症血透患者动脉粥样硬化加速的原因之一     

Cytosine deaminase: structural modifications studies

Structural modification studies have been shown that a cysteine, a histidine and possibly an arginine residue are involved in the catalytic process. The enzyme gave a single band on polyacrylamide gel electrophoresis, and the amino acid analysis showed it to contain a high proportion of hydrophobic residues, which was in agreement with the chemical modification results.     

Cytosine deaminase: Structural modification studies

Summary Structural modification studies have shown that a cysteine, a histidine and possibly an arginine residue are involved in the catalytic process. The enzyme gave a single band on polyacrylamide gel electrophoresis, and the amino acid analysis showed it to contain a high proportion of hydrophobic residues, which was in agreement with the chemical modification results.Acknowledgment. We wish to thank Herts CC for a research assistantship (S.Y.) and SRC for a research fellowship (to M.J.G.)     

Myosin heavy chains in fast skeletal muscle of chick embryo

Summary The peptide map obtained by electrophoresis after digestion of purified myosin heavy chains from pectoralis muscle of embryonic chicken with theStaphylococcus aureus V8 protease, produces a peptide pattern very similar but not identical to that of adult fast myosin. In fact, some components that are present in a small amount in the map of slow adult myosin are visible in the embryonic pattern.Acknowledgments. I wish to acknowledge the enthusiastic encouragement of Dr Ugo Carraro and the help of Prof. Stefano Schiaffino's group and of Dr Marcello Cantini for embryonic tissue isolation. The skillful technical assistance of Mr Silvio Belluco is also gratefully acknowledged. This work was supported in part by a grant of Muscular Dystrophy Association of America to Prof. Massimiliano Aloisi.     

Specific cleavage of insulin-like growth factor-binding protein-1 by a novel protease activity

Insulin-like growth factor-binding protein-1 (IGFBP-1) is secreted in a highly phosphorylated form that binds IGF-I with high affinity and is resistant to proteolysis. We have purified IGFBP-1-specific protease activity from the urine of an individual with multiple myeloma. This protease efficiently cleaves both phosphorylated and non-phosphorylated IGFBP-1 at Ile130-Ser131, generating fragments that together have higher association and dissociation rates for IGFs compared with intact IGFBP-1. The proteolytic fraction contained azurocidin, a protease homologue hitherto considered inactive. After cleavage of IGFBP-1, there was a lower affinity, but higher capacity for IGF-I binding, suggesting both N- and C-terminal fragments may interact with ligand independently. There was decreased inhibition of IGF-II-stimulated cell growth and glucose uptake. Alone, proteolysed IGFBP-1 stimulated glucose uptake in muscle. We conclude that specific cleavage of IGFBP-1 at target tissues is important in cellular growth and metabolism and opens novel strategies for targeting IGFBP-1 in treatment of disease.     

Chymotrypsin-like and trypsin-like protease activities in the sea urchin (Hemicentrotus pulcherrimus) egg.

Proteolytic activities in extracts of sea urchin eggs were examined using SDS (sodium dodecyl sulphate)-polyacrylamide gels. In the unfertilized eggs, proteases were detected as bands corresponding to the molecular weights of 40 kD and 26 kD on the gelatin gel, and 35 kD and 30 kD on the casein gel. Using various protease inhibitors, it was found that 40 kD, 30 kD, and 26 kD are chymotrypsin-like proteases and that 35 kD is a trypsin-like protease. The activity of the 40 kD chymotrypsin-like protease was found to be almost completely lost after insemination.     

Chymotrypsin-like and trypsin-like protease activities in the sea urchin (Hemicentrotus pulcherrimus) egg

Proteolytic activities in extracts of sea urchin eggs were examined using SDS (sodium dodecyl sulphate)-polyacrylamide gels. In the unfertilized eggs, proteases were detected as bands corresponding to the molecular weights of 40 kD and 26 kD on the gelatin gel, and 35 kD and 30 kD on the casein gel. Using various protease inhibitors, it was found that 40 kD, 30 kD, and 26 kD are chymotrypsin-like proteases and that 35 kD is a trypsin-like protease. The activity of the 40 kD chymotrypsin-like protease was found to be almost completely lost after insemination.     

Widespread occurrence of inhibitors of melanoma tyrosinase in plant and mammalian tissues

Résumé Les inhibiteurs de tyrosinase ont été trouvés dans des extraits de foie, de rein, de rate et de cerveau de rats et de souris, ainsi que dans des tumeurs amélanotiques S91, des sérums humains, des extraits de foie, de rein, de rate, de sein, et de peau humaine. Il y a au moins deux inhibiteurs: le premier, stable à la chaleur et dialysable, tandis que le second est labile à la chaleur et non-dialysable.

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This investigation was supported by the Medical Research Council of Canada, and the Ontario Cancer Treatment and Research Foundation. One of us (H.F.H.) is a Fellow of the Ontario Cancer Treatment and Research Foundation. The authors thank Mrs.H. Li and MissE. Santos for their skilful technical assistance.     

Glucose does not influence the insulin-like growth factor (IGF) binding to carrier proteins (IGFBPs): Analysis of rat and human serum by western ligand blotting

The insulin-like growth factors (IGFs) circulate bound to specific proteins (termed IGFBP-1 through IGFBP-6) that modulate IGF bioactivity in tissues. The aim of this study was to analyse the effects of glucose on IGF binding to IGFBPs in rat and human serum by means of western ligand blotting. Serum samples were incubated with increasing concentrations of glucose (0 to 50 mmol/l), and EDTA (25 mmol/l) was added to inhibit protease activity. To analyse the effect of glucose on protection of IGFBPs from protease activity, serum from pregnant women (reported to be very rich in proteolytic activity against IGFBPs) was added to rat serum previously incubated with glucose. Glucose did not affect the¹²⁵I-IGF binding to rat and human serum IGFBPs. The intensity of IGFBP-3 bands decreased considerably during the incubation. This appeared to be due to endogenous protease activity, since the decrease was blocked by addition of EDTA. The incubationi of rat serum with pregnant human serum produced a marked attenuation of IGFBP-3 and disappearance of IGFBP-4 bands. In conclusion, our study shows that glucose does not influence the IGF binding to IGFBP-3 either in rat or in human serum, confirms the presence of endogenous proteolytic activity in normal non-pregnant serum, and demonstrates that glucose has no protective action against protease activity.     

Nachweis hoher Aktivitäten von Kreatin-Kinase bei dem ChaetognathenSagitta setosa

Summary Notably high activities of creatine kinase have been shown in tissues of Chaetognaths both by spectrophotometric assays and on agarose-gels, where the enzyme appears as a single band. Arginine kinase activity could not be detected. It is pointed out that these findings might be of significance in elucidating the systematic position of Chaetognaths.     

Regulated protein degradation in mitochondria

Various adenosine triphosphate (ATP)-dependent proteases were identified within mitochondria which mediate selective mitochondrial protein degradation and fulfill crucial functions in mitochondrial biogenesis. The matrix-localized PIM1 protease, a homologue of theEscherichia coli Lon protease, is required for respiration and maintenance of mitochondrial genome integrity. Degradation of non-native polypeptides by PIM1 protease depends on the chaperone activity of the mitochondrial Hsp70 system, posing intriguing questions about the relation between the proteolytic system and the folding machinery in mitochondria. The mitochondrial inner membrane harbors two ATP-dependent metallopeptidases, them- and thei-AAA protease, which expose their catalytic sites to opposite membrane surfaces and cooperate in the degradation of inner membrane proteins. In addition to its proteolytic activity, them-AAA protease has chaperone-like activity during the assembly of respiratory and ATP-synthase complexes. It constitutes a quality control system in the inner membrane for membrane-embedded protein complexes.