Purification and immobilization of human carbonic anhydrase B by using polyacrylamide gel

Summary Human erythrocyte carbonic anhydrase B was purified and immobilized in polyacrylamide gel. As compared to the soluble enzyme, the immobilized enzyme was considerably more resistant to heat and sulphanilamide action.Acknowledgments. I thank Prof. R. L. Brahmachary for providing me all the laboratory facilities to carry out the investigation.     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K+ or NH+4, increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K+, respectively.     

Manipulating the activity of immobilized enzymes with different organo-smectite complexes

Summary Enzymes may be immobilized on hydrophobic surfaces of organo-smectite complexes. The immobilized enzyme may be active, partially active, or inactive depending on the nature of the organic surface. These materials may be useful as models for enzymes in natural systems, and in medicine and industry.This work was partially supported by the National Science Foundation under Grant No. CHE-8306583. Journal Article No. 11603 of the Michigan Agricultural Experiment Station.     

Immobilization of chicken liver fructose 1,6-bisphosphatase on CNBr-activated Sepharose

Summary Chicken liver fructose 1,6-bisphosphatase is readily immobilized on CNBr-activated Sepharose. The immobilization alters some enzymatic properties. They include broader pH activity curve, loss of activation by K⁺ or NH ₄ ⁺ , increased resistance to inactivation by trypsin, decreased sensitivity to AMP inhibition, and loss of cooperative interaction among AMP-binding sites. The immobilized enzyme retains about 38% or 19% of the specific activity of the native enzyme when the activity is measured in the absence or presence of K⁺, resepctively.This work was supported by grant RR-8006 from the General Research Branch, Division of Research Resources, NIH (USA).     

Deoxyribonuclease immobilized on the erythrocytes

Summary Bovine pancreatic deoxyribonuclease (DNase I) was immobilized on human erythrocytes with several procedures. DNase immobilized on the erythrocytes by chromic chloride showed DNase activity in vitro. Other binding procedures inhibit the immobilized DNase activity.     

Evidence for dissociation of ferrihemoglobin by poly-L-lysine.

Circular dichroism and absorption spectra of ferrihemoglobin were shown to be altered upon binding with poly-L-lysine at alkaline pH. When ferrihemoglobin immobilized to Sepharose gel was treated with poly-L-lysine, hemoglobin subunits were released from the gel. These results suggest that ferrihemoglobin was dissociated into subunits by poly-L-lysine.     

Coupling of fructose-1,6-P2 to aminated agarose by Schiff base reduction. Affinity chromatography of yeast aldolase.

Fructose-1,6-P2 was immobilized by sodium borohydride reduction of the Schiff base formed with aminated agarose (AH-Sepharose 4B). The coupling occurs with high yield (25 mumoles immobilized fructose-1,6-P2 per ml packed gel) at neutral pH and room temperature. Schiff base reduction thus provides a convenient and mild coupling prodecure for sugar phosphates preserving their labile phospho ester bonds. As exemplified by a new isolation procedure for fructose-1,6-P2 aldolase from yeast, sugar phosphates insolubilized in this manner may be used for affinity chromatography of the corresponding enzymes, provided that contaminating unspecific phosphatases are removed in a preceding fractionation step.     

New lectins and other putative adhesins in Aeromonas hydrophila

The ability of strains of Aeromonas hydrophila to bind 125I-labelled collagen types I and IV, fibronectin, laminin, lactoferrin, and immobilized mucins and orosomucoid on latex beads was found to be a property common to all the isolated strains. The binding was specific, was inhibited by homologous unlabelled glycoproteins, and was protease sensitive. The nature of the binding is discussed.     

A bioreactor for continuous treatment of waste waters with immobilized cells of photosynthetic bacteria

Summary A bioreactor for assaying immobilized cells of photosynthetic bacteria for hydrogen production under optimal operating conditions was made. High and lasting H₂-photoevolutions were obtained using waste waters as substrates.     

Evidence for dissociation of ferrihemoglobin by poly-L-lysine

Summary Circular dichroism and absorption spectra of ferrihemoglobin were shown to be altered upon binding with poly-L-lysine at alkaline pH. When ferrihemoglobin immobilized to Sepharose gel was treated with poly-L-lysine, hemoglobin subunits were released from the gel. These results suggest that ferrihemoglobin was dissociated into subunits by poly-L-lysine.We thank Dr A. Ichihara and K. Aki for their valuable discussions and encouragement during the course of these studies.     

The morphology of the Schwann cells and the unmyelinated fibers of a nerve supplying an immobilized muscle

Summary Hind limbs of cats were immobilized in the resting position for varying periods and the nerve supplying the medial head of the gastrocnemius muscle was studied while it was undergoing immobilization atrophy. Degenerative changes in the unmyelinated fibers and the Schwann, cells, followed by an abundant increase in collagen, were noticed after prolonged immobilization. Electron microscopic evidence that Schwann cells produce the collagen is discussed.     

Intracellular behaviour of Leishmania enriettii within murine macrophages

Both promastigotes and amastigotes of Leishmania enriettii were readily ingested by mouse peritoneal macrophages (MPM). Promastigotes after their entry within MPM were rapidly immobilized and their multiplication was never observed. Microscopic examination revealed that ingested promastigotes were degraded with MPM. Nonmotile amastigotes of L. enriettii taken up by MPM, on the other hand, multiplied intracellularly and eventually destroyed the infected cells.     

Harmaline-induced tremor and cerebral activities: labeling with 14C-2-deoxyglucose in the rat]

The brain structures specifically involved in harmaline-induced tremor have been identified in the Rat by using the 14C-2-deoxyglucose marking Method. The results obtained in an animal treated with tremogenic doses of harmaline, but immobilized with Faxedil, have been compared with those of another animal, not treated, but submitted to the same experimental procedure. The most specifically marked structure was the inferior olive. Secondarily marked structures were the posterior part of the lateral reticular formation, the caudato-putamen area and the prefrontal and frontal cortex.     

Chemische Kopplung von Hexokinase aus Hefe an Sephadex

Summary Yeast hexokinase was coupled to Sephadex by means of cyanogen bromide. The activity of the fixed enzyme was 2% of that before coupling. The activity of the noncoupled part of the enzyme was also decreased to the same degree. This decrease cannot be caused by the coupling reaction itself. The Michaelis-constants and the pH-activity curve of the coupled enzyme do not differ significantly from that of the free enzyme.     

Characterization of a soluble form of dipeptidyl peptidase IV from pig liver

Soluble dipeptidyl peptidase IV (EC 3.4.14.5) was purified from the 100,000 X g supernatant fraction of pig liver homogenate. The purified enzyme had the same properties as, and immunological identity with, the membrane-bound enzyme which was described previously. However, the purified enzyme had a pattern of molecular heterogeneity different from the membrane-bound enzyme; this was shown by isoelectric focusing. Carbohydrate analysis revealed that the soluble enzyme contained glucose, which is not found in the membrane-bound one, and less fucose, mannose, and sialic acid than the latter. From these results, we conclude that the soluble form of dipeptidyl peptidase IV in pig liver is closely related to the membrane-bound enzyme, but is not simply a proteolytically solubilized product of it.     

Intracellular behaviour ofLeishmania enriettii within murine macrophages

Summary Both promastigotes and amastigotes ofLeishmania enriettii were readily ingested by mouse peritoneal macrophages (MPM). Promastigotes after their entry within MPM were rapidly immobilized and their multiplication was never observed. Microscopic examination revealed that ingested promastigotes were degraded within MPM. Nonmotile amastigotes ofL. enriettii taken up by MPM, on the other hand, multiplied intracellularly and eventually destroyed the infected cells.     

The change of vagal activity evoked by spinal cord thermal stimulation in anesthetized rabbits

Summary Vagal activity decreased significantly during spinal cord warming and increased significantly during spinal cord cooling in anesthetized, immobilized rabbits. The result provide the first direct proof of changes in parasympathetic activity during spinal thermal stimulation.     

Characterization of a soluble form of dipeptidyl peptidase IV from pig liver

Summary Soluble dipeptidyl peptidase IV (EC 3.4.14.5) was purified from the 100,000×g supernatant fraction of pig liver homogenate. The purified enzyme had the same properties as, and immunological identity with, the membrane-bound enzyme which was described previously. However, the purified enzyme had a pattern of molecular heterogeneity different from the membrane-bound enzyme; this was shown by isoelectric focusing. Carbohydrate analysis revealed that the soluble enzyme contained glucose, which is not found in the membrane-bound one, and less fucose, mannose, and sialic acid than the latter. From these results, we conclude that the soluble form of dipeptidyl peptidase IV in pig liver is closley related to the membrane-bound enzyme, but is not simply a proteolytically solubilized product of it.We would like to thank Miss S. Fukushima for technical assistance.     

Coupling of fructose-1,6-P2 to aminated agarose by Schiff base reduction. Affinity chromatography of yeast aldolase

Summary Fructose-1,6-P₂ was immobilized by sodium borohydride reduction of the Schiff base formed with aminated agarose (AH-Sepharose 4B^®). The coupling occurs with high yield (25 moles immobilized fructose-1,6-P₂ per ml packed gel) at neutral pH and room temperature. Schiff base reduction thus provides a convenient and mild coupling procedure for sugar phosphates preserving their labile phospho ester bonds. As exemplified by a new isolation procedure for fructose-1,6-P₂ aldolase from yeast, sugar phosphates insolubilized in this manner may be used for affinity chromatography of the corresponding enzymes, provided that contaminating unspecific phosphatases are removed in a preceding fractionation step.This work was supported by the Swiss National Science Foundation, grant No. 3.620-0.75. A preliminary account has been presented at the 10th Int. Congress of Biochemistry, Hamburg, 1976, Abstracts. p. 194.Acknowledgment. The excellent technical assistance of Alice Gasser is gratefully acknowledged     

Enzyme immunometric assay for the determination of pregnancy associated plasma protein A (PAPP-A) with the antigen as solid phase (conjoint IEMA)

Purified pregnancy associated plasma protein A (PAPP-A) can be effectively bound to polystyrene microtitre plates. This immobilized antigen competes with the added serum PAPP-A of unknown concentration for the limited amount of peroxidase-labeled monospecific anti-PAPP-A antibody incubated simultaneously. The sensitivity is 0.1 WHO unit/ml and non-specific binding is 1.0%.