Jacob A. Verpoorte

Purification and characterization of glycoprotein prom human erythrocyte membranes

Abstract 1. 1. Glycoprotein has been purified from human erythrocyte membranes and its homogeneity is established. 2. 2. Gelelectrophoresis and ultracentrifugation studies indicate association. Dodecylsulfate reverses association and gives a single component with molecular weight 23,500. 3. 3. Matrix rank analysis and curve fitting of circular dichroism data indicate 32 % α-helix and 23 % β-structure. Similar studies on neuraminidase treated glycoprotein show that sialic acid residues, which make up 23 % of the dry weight, do not significantly interfere.

Structural Stability of Glycophorin - Effects of Heat and Guanidine.Hcl

The effects of guanidine hydrochloride and high temperature on human glycophorin and sialic acid-free glycophorin were monitored by circular dichroism, viscosity, and fluorescence of 1-anilino-8-naphthalane sulfonate (ANS). The following observations were made: 1. Glycophorin and its sialic acid-free counterpart are unusually stable to both guanidine . HCl and heat. 2. CD and viscosity measurements indicate that guanidine . HCl neither causes a cooperative unfolding nor generates a random coil. 3. The ANS binding site is much more sensitive to guanidine . HCl than the ellipticity at 220 nm (theta 220). 4. The effect of temperature on CD is reversible whereas the effect of guanidine . HCl is not. 5. The carbohydrate moiety influences the viscosity, and also contributes to the changes in theta 220 when solutions of glycophorin are heated. These unusual properties indicate a complex mechanism of unfolding for this structurally stable macromolecule.

Comparative studies on the structures of the heat labile β-N-acetylhexosaminidases from human plasma and placenta

Abstract 1. 1. Human plasma and placenta β - N -acetylhexosaminidase A have been purified by a combination of salt precipitation, DEAE-cellulose, Concanavalin-Sepharose and Sephadex ehromatography. 2. 2. Comparative studies using rabbit antisera against plasma enzyme indicate similarity but not identity of the enzymes. Amino acid analyses before and after tyrpsin treatment of the enzymes confirm the immunological findings. 3. 3. Heat denaturation and circular dichroism studies also suggest structural similarity of the enzymes. 4. 4. Matrix analysis of circular dichroism data indicate that combinations of basis spectra, for α-helix, β-structure and random chain cannot fit the observed spectra of the enzymes.

A comparative study of phospholipids bound to erythrocyte membrane proteins

Abstract 1. 1. The phosphoprotein and total phosphoinositide contents of solubilized erythrocyte membrane proteins were similar in human, dog, rabbit and sheep blood. Diphosphoinositide, a prominent component of human preparations, was found in trace quantities in the other species. The converse was true for phosphatidylinositol. 2. 2. During storage of human blood the triphosphoinositide was partially converted to diphosphoinositide. In stored dog blood the triphosphoinositide was degraded to phosphatidylinositol while little change was detected in rabbit blood. 3. 3. Solubilized membrane protein preparations from all species exhibited the same amino acid composition but there were differences in carbohydrate content. 4. 4. The polyphosphoinositides and phosphoproteins were metabolically active in all species.

Comparative studies on salivary kallikrein from cystic fibrosis patients and controls

ABSTRACT: Kallikrein (EC 3.4.21.8) has been purified from the saliva of cystic fibrosis (CF) patients and from healthy individuals. The yields of enzyme are the same for both kinds of saliva. The CF and normal kallikrein have similar physical-chemical properties, such as amino acid composition, electrophoretic mobilities in polyacrylamide gels when different conditions are used, intrinsic fluorescence, and circular dichroism. The enzymes have no ahelix structure but large amounts of pleated sheet structure. CF and normal salivary kallikrein have also similar enzymatic properties, for example both enzymes show maximum activity at pH 8.2. An identical value of Km = 0.4 mM has been found for both enzymes with N-benzoyl arginine ethyl ester as substrate, despite the fact that the kallikreins are inhibited by high substrate concentrations. The results of this investigation show that the salivary kallikrein in CF is normal and that it has normal activity. This leads us to suggest that the salivary kallikrein is not the cause of the observed abnormalities in CF saliva.

The purification and partial characterization of human salivary kallikrein

The major arginine esterase activity in human saliva has been purified. This enzyme lowers the blood pressure of a rabbit and produces kinins in acid treated dog plasma. It is therefore a kallikrein. The kallikrein has an unusual amino acid composition: aspartic acid and glutamic acid comprise 40% of the residues; the total number of basic residues is less than 5%; glycine and proline together make up more than 40% of the residues. The enzyme has a pI of 4.0 and an Mr of 27 000 as determined by dodecyl sulfate gel electrophoresis. On the other hand, sedimentation equilibrium data and the amino acid composition give an Mr value of only 9600. The enzyme could be a rather asymmetric molecule. The circular dichroism spectrum shows a minimum at 200 nm with [theta] = - 28 000 deg X cm2 X dmol-1. The spectrum suggests that the enzyme structure contains polyproline form II helix together with beta-turns. This structure is stable in the presence of dodecyl sulfate.

The purification of kallikrein from human whole saliva

A quick and simple procedure is described for purifying kallikrein from human whole saliva. The enzyme has been purified about 2700-fold with a yield of approx. 30%. The procedure is based on the immediate fractionation of saliva by ion exchange chromatography. This is followed by a combination of affinity and high performance liquid chromatography. The results indicate that another protein component binds to the enzyme at pH 8.0. The homogeneity of the enzyme has been demonstrated by gel electrophoresis in the absence as well as in the presence of sodium dodecylsulfate. A mol. wt of 40,100 +/- 1800 has been calculated from gel electrophoresis experiments. Sedimentation equilibrium in an analytical ultracentrifuge gave a mol. wt of 39,700. The amino acid composition has been determined and it confirms that the enzyme has a low isoelectric point. The presence of tryptophan has been demonstrated by absorption and fluorescence spectroscopy.

The precipitation of human IgG and its subunits with heparin

Abstract 1. 1. About 40% of IgG is precipitated when heparin is added to human plasma at pH 5.4, while only a little IgM and no IgA are precipitated. 2. 2. The precipitation of purified IgG by heparin is pH-dependent and follows a sigmoid curve between pH 7.0 and 5.0. 3. 3. The precipitate has a constant molar ratio heparin: IgG, independent of pH or the amount of heparin that is added. 4. 4. The precipitate does not redissolve at high heparin concentrations. 5. 5. The heavy chains of IgG precipitate also at pH 5.4, but this precipitate redissolves in excess heparin. 6. 6. Light chains do not precipitate and the F ab and F c fragments are only partly precipitated.

Phospholipid-induced changes in the circular dichroism of glycophorin and its association with 8-anilino-1-naphthalene sulfonate.

Previous studies have shown that polyphosphoinositides represent most of the metabolically active phosphorus that is bound to erythrocyte membrane protein in a proteolipid complex [1,2]. Glycophorin is the principal intrinsic sialoglycoprotein of human erythrocyte membranes [3]. Acidic phospholipids, particularly phosphatidylserine [4] and diphosphoinositide [5] remain bound to glycophorin during isolation by the diiodosalicylate method [6]. Therefore, certain acidic phospholipids may be specifically associated with glycophorin in the membrane, although non-specific binding can not be excluded. In this communication we report the interaction of glycophorin with various phospholipids as monitored by circular dichroism (CD) and the fluorescence of 8-anilino-l-naphthalene sulfonate (ANS). Diand triphosphoinositides or lysophospholipids increased the ellipticity (0220) of both native and sialic acid-free glycophorin. The polyphosphoinositides also decreased the fluorescence of ANSglycophorin complexes. Other diacyl phospolipids had little or no effect.

Physicochemical and immunological homogeneity of spinin, the subunit-protein of bacterial spinae

Bacterial spinae from marine bacterium D71 are multi-subunit structures of a single protein. This protein, called spinin, is homogeneous by immunodiffusion and immunoelectrophoresis, amino acid composition, polyacrylamide gel electrophoresis with a number of buffer systems, sedimentation velocity and diffusion boundary analysis. Sedimentation equilibrium gives Mr = 19,000, while phosphate polyacryl-amide gel electrophoresis in presence of dodecyl sulfate gives Mr = 32,000. The lower Mr estimate for spinin is supported by sedimentation equilibrium in 6 M guanidine . HCl, and covalent cross-linking with dimethyl suberimidate or glutaraldehyde. The higher Mr value probably arises from an anomalous spinin-dodecyl sulfate interaction. Isoelectric focusing in polyacrylamide gel gives pI = 3.45; however, the focusing pattern also contains three distinct bands that may arise from hydrolysis of the spinin protomer during anodic migration. This study presents the first extensive physicochemical characterization of spinin and provides the basis for investigating the subunit assembly of spinae.