Robert W. Bernlohr

Enzyme therapy II purified human α-galactosidase A: Stabilization to heat and protease degradation by complexing with antibody and by chemical modification

Abstract Methods were investigated for the stabilization of human splenic α-galactosidase A (α- d -galactoside galactohydrolase, EC 3.2.1.22). Anti-α-galactosidase A antiserum was produced in a goat by repeated immunization with highly purified α-galactosidase A. When this antiserum was incubated at 37°C with α-galactosidase A in varying enzyme: antiserum ratios, a significant increase in the thermal (50 °C) stability and in the resistance to protease digestion of these mixtures was observed compared to appropriate controls. The enzyme was also treated with the bifunctional cross-linking reagent, hexamethylene diisocyanate, and the thermal stability and protease resistance of the cross-linked derivative were increased compared to the native enzyme treated with the monofunctional reagent, butyl isocyanate.

Threonine dehydratase of Bacillus licheniformis: I. Purification and properties

Abstract Threonine dehydratase (threonine hydro-lyase (deaminating), EC 4.2.1.16) has been purified about 50-fold from extracts of Bacillus licheniformis by heat treatments, ammonium sulfate fractionation, and gel filtration. The purified enzyme exhibits properties similar to the enzyme in crude extracts. The enzyme is stabilized in i M potassium phosphate at pH 8.0 by pyridoxal phosphate and l -isoleucine against denaturation at 0° and against heat inactivation. l -Threonine protects the enzyme against inactivation by dilution. l -Threonine and l -serine can serve as substrates, but threonine is deaminated 5–8 times more rapidly than the other substrate. The pH optimum of the reaction is between 9.0 and 9.5. The rate of reaction at increasing substrate concentration shows a slightly sigmoidal curve with an apparent Km of 3·10−3 M. l -Isoleucine inhibits the enzyme to about 50% at a threonine to isoleucine ratio of 1000:1. This inhibition is pH dependent and is overcome in part by l -α-aminobutyrate, although by itself l -α-aminobutyrate does not show any activating effect. The treatment of the enzyme with HgCl2 results in partial inactivation, but the remaining enzyme activity is completely insensitive to the inhibition by l -isoleucine. The extent of inactivation of the enzyme by urea is dependent on the urea concentration and the temperature.

Control of aspartokinase during development of Bacillus licheniformis

Extracts prepared from late exponential phase Bacillus licheniformis cells contain an aspartokinase (ATP:l-aspartate 4-phosphotransferase, EC 2.7.2.4) activity that is 4-fold greater than that from early exponential phase cells. This increase occurs in cells grown on several different media. After growth has ceased, the specific activity decreases at a rate much greater than would be expected by turnover. n nThe enzyme activity is subject to feedback inhibition by l-lysine and l-aspartic-β-semialdehyde. l-Threonine has no effect on the activity when added alone but produces a concerted inhibition with l-lysine. The asparticβ-semialdehyde effect may be an example of sequetial control.

Postlogarithmic phase metabolism of sporulating microorganisms: III. Breakdown of arginine to glutamic acid

Abstract During growth of Bacillus licheniformis , the biosynthesis of arginine proceeds from glutamic acid via ornithine and other intermediates typical of prokaryotic systems. In contrast, when the growth of this microorganism ceases and cells initiate sporulation, arginine is no longer synthesized from glutamic acid, but is degraded. An arginase is induced, converting arginine to ornithine. Part of the ornithine is then used in the biosynthesis of the peptide antibiotic, bactracin. The remainder is oxidized to glutamic acid, part of which is subsequently converted to CO 2 . The activities of the two enzymes responsible for the conversion of ornithine to glutamic acid, ornithine Δ-transaminase and Δ 1 pyrroline-5-carboxylate dehydrogenase, are increased at least 10-fold in extracts of sporulating cells when compared with extracts from vegetative cells.

Purification and characterization of phosphofructokinase of Bacillus licheniformis

Abstract The phosphofructokinase (PFK) of Bacillus licheniformis was purified about 50–65-fold and examined for a number of enzymatic and physical characteristics. The enzyme is quite unstable under normal assay conditions, but Mg 2+ , K + , adenosine-5′-diphosphate, phosphoenolpyruvate (PEP), and fructose-6-phosphate (fru-6-P) are fairly effective stabilizing agents. Saturation functions for ATP and fru-6-P were hyperbolic. Several attempts to induce positive cooperative binding of fru-6-P were unsuccessful. However, “sigmoidal” saturation kinetics for fru-6-P could be observed under assay conditions that permitted an irreversible inactivation of the PFK during assay. Several divalent cations could support the catalysis of B. licheniformis PFK and the enzyme was activated by both NH 4 + and K + ions. B. licheniformis PFK is inhibited by citrate, ATP, PEP, Ca 2+ , and several other metabolic intermediates, but the inhibition caused by citrate and ATP at high fru-6-P concentration and by calcium can be relieved by Mg 2+ addition while PEP inhibition is specifically relieved by fru-6-P. There are at least three binding sites for PEP on the PFK molecule. The active form of this PFK has a molecular weight of about 134,000 daltons. In the presence of Mg 2+ , adenosine-5′-triphosphate (ATP), and PEP, at 0 °C, the PFK molecule is rapidly dissociated to an inactive form with a molecular weight of about 68,000 daltons. Association of these subunits to yield the active form of PFK occurs spontaneously, and rapidly, when the temperature is raised to 30 °C. Ninety percent of the original activity is recovered after activation. Growth of B. licheniformis on several different substrates resulted in minor variations of PFK activity. In a parallel fashion, sporulation involved no irreversible inactivation of PFK and the level of the activity was about the same throughout the life cycle. Control of this enzyme during sporulation could be affected by any or all of the cell constituents found to regulate PFK activity in vitro , but it is considered likely that the most significant in vivo negative effector is PEP, with this inhibition being reversed by fru-6-P.

The regulation of aspartokinase inBacillus licheniformis

Abstract The specific activity of aspartokinase (ATP: l -aspartate 4-phosphotransferase, EC 2.7.2.4) decreases rapidly to a 10 to 20-fold lower level at the end of the growth phase in Bacillus licheniformis grown on a minimal glucose-salts medium. Concurrent with this decrease in specific activity is a loss of sensitivity to feedback inhibition by l -lysine and a slower decrease in the concerted inhibition caused by l -lysine and l -threonine. Growth of the cells in the minimal medium plus l -lysine and l -threonine caused the production of aspartokinase with characteristics similar to those of enzyme from cells harvested 4 h after the end of growth. Growth in the presence of either l -lysine or l -threonine did not affect the specific activity of the enzyme but did alter its inhibition sensitivity. The addition of l -lysine to the medium during growth on the minimal medium caused a rapid loss of sensitivity to lysine inhibition, 50% desensitization occurring in 15 min. These latter conditions did not produce an alteration in the specific activity of the enzyme or in the concerted inhibition by lysine and threonine. Despite the marked alteration in feedback-inhibition properties, changes in physical or kinetic properties of purified aspartokinase were not observed. Throughout the purification steps there was no evidence that more than one aspartokinase was present in B. licheniformis cells. It is concluded that this enzyme is unusually plastic and assumes alternate forms depending on the physiology of the cell.

Ornithine Transcarbamylase Enzymes: Occurrence in Bacillus licheniformis

Two separate ornithine transcarbamylase enzymes have been found in extracts of late exponentialphase cells of Bacillus licheniformis grown on glucose and L-arginine. One enzyme presumably has a biosynthetic function and is repressed by arginine. The other is induced by arginine, is relatively heat-stable, and can be separated from the first by diethylaminoethyl chromatography.

Enzyme Transplantation in Fabry's Disease

Fabrys disease,1 heretofore a rare and esoteric diagnostic rubric, has become an important focus for research by biochemists, enzymologists, geneticists, clinical investigators and transplant surgeons.2 As noted by Clarke and his colleagues in this issue of the Journal, several centers have transplanted normal kidneys into patients with renal failure secondary to Fabrys disease. Of the nine patients throughout the world who have received transplants, four have had normal renal function for periods of six months to over three years.3 , 4 These successful transplants have had documented clinical improvement, with subjective relief of their frequent excruciating pain, return of sweating and .xa0.xa0.

The regulation and kinetics of the two ornithine transcarbamylase enzymes of bacillus licheniformis

1. n1. The regulation of the two ornithine transcarbamylase enzymes (carbamol-phosphate; l-ornithine carbamoyltransferase, EC 2.1.3.3) of Bacillus licheniformis was studied. Ornithine transcarbamylase I is repressed by l-arginine, l-citrulline and l-ornithine during growth on glucose. Ornithine transcarbamylase II is induced by l-arginine (but not by l-citrulline or l-ornithine) in post-logarithmic phase cells or in cells growing on glutamate. Glucose addition represses this induction in the glutamate medium. n n2. n2. The ornithine transcarbamylase I was purified about 10 fold and ornithine transcarbamylase II about 8 fold. n n3. n3. The two ornithine transcarbamylases have the same pH optimum (8–9) and no significant differences could be detected in Michaelis-Menten constants for either substrate. n n4. n4. The back reaction (citrulline → ornithine) was not catalyzed by either ornithine transcarbamylase. n n5. n5. Arginine deiminase was not detected in extracts of these cells. n n6. n6. The physiological role of the inducible ornithine transcarbamylase (ornithine transcarbamylase II) is not known.

FABRY'S DISEASE

Publisher Summary This chapter focuses on Fabrys disease. Fabrys disease is an X-linked inborn error of glycosphingolipid catabolism. Transplantation of enzymatically active renal tissue into patients with Fabrys disease has resulted in decrease to normal of the accumulated glycosphingolipid substrate in plasma. The diagnosis of hemizygotes and heterozygotes for Fabrys disease can be confirmed by the deficient activity of ceramide trihexosidase in plasma, urine, leukocytes, cultured fibroblasts, or biopsied tissue. The chapter also discusses clinical characteristics of Fabrys disease. The clinical manifestations of the disease result from the generalized visceral deposition of the Fabry glycosphingolipid, particularly in the cardiovascular-renal system. Three separate approaches to enzyme replacement have been utilized in Fabrys disease. These are: (1) the plasma infusion, (2) renal transplantation, and (3) direct enzyme infusion.