Joseph Roberts’s research while affiliated with University of Washington and other places

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Publications (5)


Therapeutic Properties of a New Glutaminase-Asparaginase Preparation and the Influence of the Lactate Dehydrogenaseelevating Virus
  • Article

March 1974

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17 Reads

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23 Citations

Cancer Research

Vernon Riley

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Darrel Spackman

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[...]

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William C. Dolowy

A new enzyme termed GA:1.2, possessing approximately equal amounts of glutaminase and asparaginase activity, has antitumor activities against cancers other than leukemia, thus enlarging the potential for cancer therapy by amino acid deprivation. When tested against the asparagine dependent EARAD 1 leukemia in the presence of the lactate dehydrogenase elevating virus (LDH virus), striking tumor regression was obtained. This asparagine dependent mouse tumor was used as a model to study the basic mechanism of the therapeutic action of the enzyme and its influence upon various plasma amino acids in the presence and absence of the LDH virus. At doses of 150 IU/kg and higher, both plasma glutamine and asparagine were depleted to undetectable levels (less than 1 nmole/ml) when the virus was present, but not in its absence. Tumor regression was correlated with asparagine and glutamine depletion in the plasma. Although there were no measurable differences in the plasma glutamine and asparagine depletion below 1 nmole/ml as a function of increasing enzyme dose, both glutamic and aspartic acids increased systematically with dose. The plasma half life of GA:1.2 is 1 to 2 hr in normal mice, but was increased to 12 to 18 hr when mice were infected with the LDH virus. Thus, the presence of the LDH virus in the host is necessary for the expression of the therapeutic capabilities of the enzyme preparation on this and presumably other mouse tumor systems.


Physical Properties of Acinetobacter Glutaminase-Asparaginase with Antitumor Activity
  • Article
  • Full-text available

January 1973

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21 Reads

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21 Citations

Journal of Biological Chemistry

Acinetobacter glutaminase-asparaginase has been shown to consist of 4 subunits (molecular weight 33,000) by sedimentation equilibrium in 5.5 m guanidine HCl and electrophoresis in sodium dodecyl sulfate on polyacrylamide gels after cross-linking the protein with dimethyl suberimidate. Moving boundary velocity experiments showed that most of the native enzyme sediments as the tetramer (s20,w = 7.42 ± 0.03 S). On the other hand, equivalent boundary calculations always showed a smaller s20,w. Analytic sedimentation equilibrium experiments revealed a tetramerdimer dissociation with a dimer molecular weight of 69,000 ± 3000. The molecular weight on calibrated Sephadex G-200 and Bio-Gel P-200 was 97,000 and 93,000, respectively, which may indicate reversible dissociation. The hydrolysis of 5-diazo-4-oxonorvaline was used to determine the sedimentation coefficient of the active species. Sedimentation of the enzyme in 5-diazo-4-oxonorvaline showed complex patterns with ultraviolet optics due to protein absorbance. A new double sector cell was devised which allows layering of enzyme into both sectors simultaneously, cancelling the absorbance of the enzyme. The s20,w value for the species which degraded 5-diazo-4-oxonorvaline was 7.6 ± 0.2 S. By matching zone sedimentation and active enzyme experiments, enzyme species smaller than tetramer were shown to have 4% or less of the activity of the tetramer.

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Isolation, Crystallization, and Properties of Achromobacteraceae Glutaminase-Asparaginase with Antitumor Activity

February 1972

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34 Reads

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129 Citations

Journal of Biological Chemistry

Crystalline glutaminase-asparaginase with antitumor activity was prepared from an Achromobacteraceae soil isolate organism. This enzyme has l-glutaminase and l-asparaginase activity in a ratio of 1.2:1. The purification procedure provides an over-all yield of 40 to 60% from crude cell-free extract to homogeneous glutaminase-asparaginase and is adaptable to large scale isolation of the enzyme. Glutaminase-asparaginase is crystallizable from aqueous alcohol solutions in the absence of heavy metal cations. The highest yields of enzyme were obtained when cells were grown aerobically in a basal synthetic medium composed of l-glutamic acid, ammonium sulfate, trace minerals, and phosphate buffer. Glutaminase-asparaginase content remained relatively constant when the organism was at temperatures between 15 and 25°. Above 25° the enzyme content decreased with increasing temperature. The isoelectric point by isoelectric focusing of glutaminase-asparaginase on ampholytes is 8.43. The specific activity of homogeneous enzyme is 190 ± 20 i.u. per mg of protein and the E1%280 is 10.2. No carbohydrate or phospholipid was detected in the enzyme. No disulfide or sulfhydryl groups appear to be present on the enzyme. The Km values for l-glutamine and l-asparagine are 5.8 ± 1.5 x 10⁻⁶ and 4.8 ± 1.4 x 10⁻⁶m, respectively. Glutaminase-asparaginase catalyzes the hydrolysis of the d isomers of glutamine and asparagine at about one-third the rate of the l isomers. The enzyme is not inhibited by ethyl-enediaminetetraacetate (0.1 mm), ammonia (10 mm), l-glutamate (30 mm), or l-aspartate (30 mm). 6-Diazo-5-oxo-l-norleucine which is not a substrate for the enzyme irreversibly inactivates glutaminase-asparaginase at very low concentrations. In contrast the l and d isomers of 5-diazo-4-oxonorvaline are attacked by the enzyme and are considerably poorer inhibitors.


In vitro Cytocidal Effect of L-Glutaminase on Leukaemic Lymphocytes

August 1971

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23 Reads

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17 Citations

Nature

MOUSE lymphomas, such as 6C3HED, undergo complete regression after one or more injections of L-asparaginase1, and remissions have been reported in some patients with acute leukaemia after treatment with L-asparaginase2-4. Roberts et al.5 found that purified bacterial L-glutaminase inhibited the growth of Ehrlich mouse carcinoma. Before considering this enzyme for therapeutic studies in man, it is necessary to study in vitro effects of the enzyme on human blood cells. Previous work6 demonstrated that the cells of 6C3HED lymphoma do not survive incubation with very low concentrations of L-asparaginase (1.7 mIU/ml.), while the cells of the in vivo resistant variant of 6C3HED were not killed by 170 mIU of enzyme/ml. The blood lymphocytes from patients with chronic lymphocytic leukaemia were found to be more sensitive than normal lymphocytes to incubation with 170 mIU of L-asparaginase/ml. but not as sensitive as 6C3HED mouse lymphoma which responds in vivo to the enzyme. In this study, the slide chamber method7 was used to compare the sensitivity of normal and leukaemic lymphocytes to glutaminase.


Antineoplastic Activity of Highly Purified Bacterial Glutaminases

October 1970

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32 Reads

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123 Citations

Nature

SEVERAL lines of evidence motivated the treatment of neoplasms by glutaminase to cause glutamine deprivation. Certain tumour cells grown in tissue culture require glutamine at a level which is tenfold or greater than that of any other amino-acid1,2. Glutamine participates in a wide variety of metabolic reactions in mammalian cells3. It has been suggested that one of the important functions of glutamine in the metabolism of certain tumours may be as a direct precursor of glutamic acid, which can then furnish the carbon for the partial operation of the tricarboxylic acid cycle from α-ketoglutarate to oxaloacetate4. Compared with other tissues, certain tumour cells seem to operate at a marginal level of glutamine availability because of slow synthesis5 and rapid utilization4. The glutamine antagonists, azaserine and 6-diazo-5-oxonorleucine (DON) have been shown to possess moderate antineoplastic activity, which may be enhanced by L-asparaginase6-8. Greenberg et al.9 reported that a glutaminase-asparaginase preparation with a relatively high glutamine Km (7 × 10-3M) decreased the initial rate of growth of a number of tumours, including an Ehrlich ascites carcinoma, but caused no significant increase in the survival time of tumour-bearing animals. In this study, more intensive therapy with three extensively purified glutaminase preparations with considerably lower Km values resulted in marked inhibition of an Ehrlich ascites carcinoma and significant increases in the survival time of tumour-bearing animals.

Citations (5)


... having dual specificity towards L-glutamine and L-asparagine, and which proved to have substantial effectiveness against a variety of tumors that have abnormally high requirements for L-glutamine and L-asparagine. These organisms are different pseudomonads , such as Pseudomonas species45, Pseudomonas fluorescens678, Pseudomonas aeruginosa [9] , Pseudomonas acidovorans [10], Pseudomonas 7A1112131415 , Pseudomonas boreopolis 526 [7,1617 and Pseudomonas aurantiaca BKMB 54818192021 , but also Acinetobacter gluta- minasificans2223242526, Achromobacter272829, and one yeast characterized as Cryptococcus nodaensis has also been suggested to contain this enzyme [30]. According to Ortlund et al. [15] , the most studied representatives of this class of enzymes are those of Pseudomonas 7A and Acinetobacter glutaminasificans. ...

Reference:

© by PSP Volume 33 – No 2. 2011 Advances in Food Sciences STUDIES AND SOME KINETIC PROPERTIES OF L-ASPARAGINASE FROM Penicillium politans NRC 510
Physical Properties of Acinetobacter Glutaminase-Asparaginase with Antitumor Activity

Journal of Biological Chemistry

... In C3H/HE mice carrying the 6C3HED Gardner tumor the half-life of the AG enzyme was approximately 27 h, whereas in Swiss mice carrying the Ehrlich lymphoma it was 6.5 h. The markedly slower clearance rate from the tumor-bearing C3H mice can most likely be attributed to the presence of lactic dehydrogenase elevating virus in such animals (22,23). DISCUSSION In this paper we describe the isolation of two asparaginases from P. geniculata using a relatively straightforward purification procedure. ...

Therapeutic Properties of a New Glutaminase-Asparaginase Preparation and the Influence of the Lactate Dehydrogenaseelevating Virus
  • Citing Article
  • March 1974

Cancer Research

... The hydrolysis of L-glutamine to L-glutamic acid and ammonia is catalyzed by Lglutaminase (L-glutamine amidohydrolase EC 3.5.1.2) [24]. Due to its potential use as an anti-leukemic agent [25][26][27][28], in addition to its application in the c-glutamyl transfer processes that yield specialized compounds like theanine, and as a potent antiretroviral drug [29], it has drawn a lot of attention. By selectively denying glutamine-dependent tumor cells of their glutamine, amidases cause neoplasms to lose vital nutrients and ultimately die [30,31]. ...

Antineoplastic Activity of Highly Purified Bacterial Glutaminases
  • Citing Article
  • October 1970

Nature

... Discovery of L-glutaminase was observed while working with L-asparaginase by Wade and Philips in 1971 [13]. Shreck et al. in 1971 worked on in vitro studies on leukemic lymphocytes 6C3HED [14]. Ehrlich mouse carcinoma treatment was identified by Roberts et al. [15]. ...

In vitro Cytocidal Effect of L-Glutaminase on Leukaemic Lymphocytes
  • Citing Article
  • August 1971

Nature

... 3.5.1.38) [20,43]. The clinical application of GLNase-ASNase enzymes has been poorly studied, but earlier studies performed In vitro [44] and In vivo with humans [45,46] showed anti-leukemia effects. ...

Isolation, Crystallization, and Properties of Achromobacteraceae Glutaminase-Asparaginase with Antitumor Activity

Journal of Biological Chemistry