Crystalline Urease Research Articles

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

One-step Metabolomics: Carbohydrates, Organic and Amino Acids Quantified in a Single Procedure

Every infant born in the US is now screened for up to 42 rare genetic disorders called "inborn errors of metabolism". The screening method is based on tandem mass spectrometry and quantifies acylcarnitines as a screen for organic acidemias and also measures amino acids. All states also perform enzymatic testing for carbohydrate disorders such as galactosemia. Because the results can be non-specific, follow-up testing of positive results is required using a more definitive method. The present report describes the "urease" method of sample preparation for inborn error screening. Crystalline urease enzyme is used to remove urea from body fluids which permits most other water-soluble metabolites to be dehydrated and derivatized for gas chromatography in a single procedure. Dehydration by evaporation in a nitrogen stream is facilitated by adding acetonitrile and methylene chloride. Then, trimethylsilylation takes place in the presence of a unique catalyst, triethylammonium trifluoroacetate. Automated injection and chromatography is followed by macro-driven custom quantification of 192 metabolites and semi-quantification of every major component using specialized libraries of mass spectra of TMS derivatized biological compounds. The analysis may be performed on the widely-used Chemstation platform using the macros and libraries available from the author. In our laboratory, over 16,000 patient samples have been analyzed using the method with a diagnostic yield of about 17%--that is, 17% of the samples results reveal findings that should be acted upon by the ordering physician. Included in these are over 180 confirmed inborn errors, of which about 38% could not have been diagnosed using previous methods.

ChemInform Abstract: 70 Years of Crystalline Urease: What Have We Learned?

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70 Years of Crystalline Urease: What Have We Learned?

70 Years of Crystalline Urease: What Have We Learned?

Magnesium metabolism in the hyperammonemic rat.

SummaryHyperammonemia was induced by injection of crystalline urease intraperitoneally in normal and Mg-deficient male rats. In hyperammonemic rats, plasma Mg tended to increase and plasma phosphate fell to about 50% of control values. Total Mg and the uptake of 28Mg by the liver, brain, heart, colon, or muscle were not changed. Mg concentrations in the kidney were decreased following acute ammonia toxicity, and release of 28Mg by liver slices in vitro was not changed by previous hyperammonemia. Hyperammonemia did not alter the rate of disappearance of 28Mg from blood. Serum Mg of rats fed a Mg-deficient diet for 7 days was 50% of control, but the biochemical and toxicological responses to hyperammonemia were not markedly altered. Serum lactate, pyruvate, and α-ketoglutarate were increased in all urease-injected animals. Serum succinate was significantly lower in Mg-deficient rats. Significant changes in Mg metabolism during hyperammonemia were not observed.The assistance of Mrs. Mabel Goetchius, Mr. John...

Quantitative studies on the inhibition of crystalline urease by rabbit anti-urease

1. 1. Quantitative precipitin studies with crystalline urease and antiurease have been performed. The resulting data provide information concerning the combining proportions of the enzyme and antibody on a weight basis. 2. 2. Urease is inhibited by rabbit anti-urease. 3. 3. The extent of inhibition of the enzyme by specific antiserum is dependent upon the ratio of antibody to antigen employed. The greatest inhibition, about 80 %, was achieved with a reaction mixture containing a large excess of antibody. 4. 4. Under the conditions of this study, complement was not found to play a role in the inhibition of urease by anti-urease.

THE ACTIVATION OF UREASE

1. It has been shown that the activity of solutions of twice recrystallized urease is reversibly increased by moderate heating and reversibly decreased by storage in the cold, even in the frozen state. 2. Crude extracts of jack bean meal containing potent urease undergo this same type of reversible activation by heating and inactivation by cooling. Dilution has the same potentiating effect on the activity as moderate heating. As much as a fivefold increase in activity can be obtained when a sample previously inactivated by storage for 24 hours at -10 degrees C. is heated for 5 minutes at 60 degrees C. 3. Solutions of crystalline urease protected by serum albumin and preserved in the cold give a constant "potential" activity over a period of more than 30 days if heated 5 minutes at 60 degrees C. before assay. 4. The data presented have been interpreted to mean that an association between urease molecules (or between urease and other proteins) might occur, resulting in inactivation of the enzyme which would be reversed on dissociation. 5. It has been postulated that the same forces are responsible for the reversible inactivation brought about by standing at temperatures above or below the freezing point.

Effects of Soft X-rays on Urease and Catalase

Summary1. The effects of soft X-rays on crude urease, crystalline urease, crude catalase, and crystalline catalase are described. 2. The sensitivity of the enzymes to the radiation is dependent upon purity and concentration. 3. Results are given which are in accordance with the observations of Dale on the effects of hard X-rays on polyphenol oxidase and carboxypolypeptidase.

TEMPERATURE ACTIVATION OF THE UREASE-UREA SYSTEM USING CRUDE AND CRYSTALLINE UREASE

1. The hydrolysis of urea catalyzed by jack bean meal has been followed by determining colorimetrically after Nesslerization the ammonia nitrogen, and volumetrically the carbon dioxide liberated at successive intervals during the reaction. During the early part of hydrolysis the rate of ammonia or carbon dioxide liberation is constant for all the urease solutions which were used. 2. When log rate of NH(3) or CO(2) formation was plotted against 1/T, the points fell along a straight line, the slope of which corresponded to an activation energy of either 8,700 or 11,700 calories per gram mol. Frequently urease, when dissolved in sulfite solution, was characterized by an activation energy of 11,700 below and 8,700 above the critical temperature of about 23 degrees C. At high temperatures the plotted points fell off from the curve due to temperature inactivation. 3. Essentially the same results on temperature activation were obtained with crude jack bean meal, Arlco urease, crystalline urease not recrystallized, and crystalline urease once recrystallized. The temperature characteristic which was obtained depended in part upon the composition of the medium. When dissolved in water, or aqueous solutions of glycerine, KCN, Na(2)S(2)O(2), cystine, Na(2)SO(4), and K(4)Fe(CN)(6), the temperature characteristic or micro of urease is 8,700. On the other hand, when urease is dissolved in solutions of K(3)Fe(CN)(6) or H(2)O(2) the micro value is 11,700. When dissolved in a solution containing Na(2)SO(3) and NaHSO(3) the micro value may be either 8,700 or 11,700 over the whole temperature range, or 11,700 below and 8,700 above 23 degrees C. 4. When crystalline urease is dissolved in varying mixtures of K(4)Fe(CN)(6) and K(3)Fe(CN)(6), the temperature characteristic depends upon the oxidation-reduction potential of the digest. When E(h) is greater than +0.46 volt micro = 11,700, when less than +0.42 volt micro = 8,700, when between +0.42 - +0.46 micro = 11,700 below and 8,700 above the critical temperature. 5. It is suggested that in reducing or in indifferent solutions the configuration of the urease molecule (as determined especially by SH groups present) is such that the activation energy is 8,700 calories. In oxidizing solutions the urease molecule has been so altered (perhaps by the oxidation of the SH groups) as to be partly inactivated and now has an activation energy of 11,700. Such changes in the urease molecule are reversible (unless oxidation has proceeded too far) and are accompanied by a corresponding change in the activation energy.

The Non-Identity of Jack Bean Agglutinin with Crystalline Urease

Abstract Hotchkiss and Tauber (1) have stated that crystalline urease agglutinates washed rabbit and mouse erythrocytes, but not those of sheep. As little as 0.00014 mgm. of urease is said to have caused a perceptible effect. This work is quite erroneous. We find that washed rabbit and dog erythrocytes are not agglutinated in the slightest when incubated for two hours at 37°C. with as much as 3 mgm. of recrystallized urease. It is true that the jack bean, like many other beans, contains a substance which agglutinates red cells. This is thermolabile and non-dialyzable. We have obtained it in solution in such concentration that 1 cc. will cause an almost complete agglutination of 5 cc. of 2 per cent erythrocyte suspension in five minutes at room temperature. Diluted 100 times the material will produce an agglutination detectable by microscope within thirty minutes at 37°C. This 100-fold diluted solution contained only about 0.00015 mgm. of urease per cubic centimeter.

X-Ray Patterns of Crystalline Urease and Pepsin

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTx-Ray Patterns of Crystalline Urease and PepsinIsador FankuchenCite this: J. Am. Chem. Soc. 1934, 56, 11, 2398Publication Date (Print):November 1, 1934Publication History Published online1 May 2002Published inissue 1 November 1934https://doi.org/10.1021/ja01326a502RIGHTS & PERMISSIONSArticle Views34Altmetric-Citations8LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (125 KB) Get e-Alerts Get e-Alerts

The Reaction between Crystalline Urease and Antiurease

Abstract In the study of immunologic reactions the advantage of employing a substance which is both enzyme and antigen is manifest, since one can readily estimate the concentration of the enzyme by determination of its activity and since by means of activity measurements it is possible to follow the reaction between enzyme and antibody. In the past numerous attempts to establish the production of antienzymes after injecting enzymes into animals have been made, but the results have been questioned, partly because the specificity in many cases appeared to be related in some manner to the accompanying protein, and partly because the antienzymatic activity has been so weak as to cause doubt as to the actual existence of any antienzyme. Much work has been done in recent years to clear up the chemical nature of enzymes. Four enzymes, namely, urease (1), pepsin (2), trypsin (3), and pancreatic amylase (4) have been prepared in crystalline and presumably pure form.

Urease Activity as Influenced by Oxidation and Reduction

During recent years there have appeared numerous descriptions of the effects of hydrocyanic acid, of hydrogen sulphide and especially of organic sulphydryl compounds in activating certain hydrolytic enzymes, for example, intracellular proteinases (cathepsins),' arginase2 and pancreatic amylase.3 Indeed, reduced glutathione has been called by WaldschmidtLeitz the natural activator of animal cathepsin and of arginase. Recently Perlzweig4 suggested that is associated with a sulphhydryl activator, possibly glutathione. This suggestion was based, in part, upon the old observation that cyanides and hydrogen sulphide reactivate crude preparations which have been poisoned by ions of heavy metals. Perlzweig assumes that the poisoning consists of the catalysis of the activator's oxidation and that the activator can be regenerated by the reducing action of cyanides and sulphides. Perlzweig also reported a weakening of the nitroprusside test (for sulphhydryl) accompanying the partial inactivation of by aeration. Sumner5 has given evidence that the protein-urease molecule itself contains a group which gives a nitroprusside test. Such groups are probably sulphhydryl. Furthermore Sumner's work indicates that highly active, recrystallized preparations of possess characteristic activities in the absence of supplementary material such as glutathione. It has appeared to us that some of the phenomena in this field might be ascribed not to changes in a supplementary activator but to oxidation of and regeneration of the sulphhydryl groups of the enzyme itself. Accordingly, we have put this working hypothesis to a test with preparations and we shall report in this preliminary communication only sufficient of our experiments to indicate typical results. It is a pleasure to acknowledge the cooperation of Dr. J. B. Sumner and Dr. D. B. Hand who enabled us to obtain highly active jackbean meal and advised us in the preparation of urease. Crystalline was used throughout the investigation except when it was occasionally desired to compare its behavior with that of the usual crude preparations. The reactions with the various reagents studied were carried out at room temperature. Conductivity water was used throughout this work. For the determination of activity a mixture corresponding to 2.5 ml. of freshly prepared crystalline urease solution diluted with water or other reagents to 5.0 ml., was added to 25.0 ml.

Sulfhydryl Compounds and Crystalline Urease

Perlzweig1 states that both crystalline urease and extracts of jack bean meal give a positive sulfhydryl test with nitroprusside and that the more active preparations give stronger tests than the weaker ones. He estimates one of his samples of jack bean meal to contain the equivalent of 70 mg. of glutathione per 100 gm. and believes the substance in the meal to be glutathione, rather than cysteine, since the material is negative to the Sullivan reaction until after hydrolysis. He suggests that urease activity is due in part to the sulfhydryl compound and that it may be possible to separate a sulfhydryl activator from the urease proper.We have independently observed that urease, prepared from jack bean meal by the Sumner procedure and twice recrystallized from 30% alcohol, produces a moderate amount of red color when treated with nitroprusside, ammonium sulfate and ammonia, and had ascribed this reaction to the presence of sulfhydryl groups attached to the urease molecule. It was recently shown that no co-...

The heat-inactivation of crystalline pepsin; the critical increment of the process

IN a recent series of publications Northrop [1930, 1, 2; 1931] has advanced very strong evidence that pepsin is a protein. Starting from commercial pepsin, by suitably regulating the pH and precipitating with the aid of magnesium or ammonium sulphate a crystalline product was obtained which had the general properties of a protein. In particular, solutions of this crystalline pepsin were coagulated by heat, and Northrop states that the inactivation of the enzyme either by heat or by alkali is quantitatively proportional to the denaturation of the protein. A successful effort was made [1931] to reactivate denatured inactivated pepsin along the lines used by Anson and Mirsky [1929; 1931, 1, 2] for the reversal of the denaturation of certain proteins, and the product obtained was identical with the original freshly prepared material, both in general properties and in its specific proteolytic activity. It seems safe to conclude therefore that the crystalline pepsin is in fact a protein. More recently Northrop [1932, 2] has shown that trypsin can similarly be obtained in a crystalline form and has the properties of a protein, whilst Sumner [1926] claims that the crystalline urease prepared by him is also a protein. Work has been proceeding in this Department for a number of years with a view to determining the mechanism of the process of the denaturation of proteins. In this connection the conclusions mentioned above are of great importance, for if the processes of inactivation and denaturation of the crystalline enzymes do go hand in hand, then an investigation of the heat-inactivation of the enzymes might yield valuable information regarding the mechanism of the process of denaturation, especially as the rate of inactivation can be determined experimentally with great accuracy by a number of different methods. Consequently an investigation of the heat-inactivation of crystalline pepsin as prepared by Northrop was undertaken, and the results obtained are given in the present communication. EXPERIMENTAL.

Crystalline Urease - Preparation of Meal from Jack Beans

Crystalline Urease - Preparation of Meal from Jack Beans

Immunological Identity of Soy and Jack Bean Urease

Since proteins are usually species specific it might be expected a priori that soy urease would be chemically distinct from jack bean urease. In order to decide this question we have treated clear solutions of soy urease with jack bean antiurease. The antiurease was obtained by injecting crystalline urease into rabbits, followed by purification of the antiurease according to our method.1 In all cases a precipitin reaction was obtained even when the soy urease contained as little as 1 unit per cc. Furthermore, we have found that soy urease is inactivated by the addition of jack bean antiurease. This inactivation is not so great as the inactivation of jack bean urease by jack bean antiurease. However, the difference is probably due to the presence of colloidal impurities in the soy urease. Table I shows some of our results.As further confirmation of the inhibitory effect of jack bean antiurease on soy bean urease we have employed animal tests. Soy urease is as toxic to rabbits as is jack bean urease. If soy...

STUDIES ON CRYSTALLINE UREASE

1. Crystalline urease is not inactivated by trypsin in the absence of a gum. In fact, the presence of trypsin alone in aqueous solutions of urease has an action similar to that of gum, that is, it acts as a "protective colloid" for urease. 2. Crystalline urease is inactivated by trypsin in the presence of a gum. This occurs with great rapidity if purified (crystalline) trypsin is used. 3. If trypsin is added to urease a considerable time before the addition of gum no inactivation of the urease takes place. 4. The suggested explanation is that an inhibitive group of the urease molecule ("antitrypsin") may combine with trypsin to form an irreversible inactive trypsin compound, which cannot attack the urease but has unchanged ureolytic power. This compound cannot be formed if gum is present because the gum has united with a portion of the urease molecule; the tryptic power is thus unimpaired and urease is then digested. This speaks for the protein nature of urease.

Studies on Crystalline Urease

Summary Crystalline urease in solution produced agglutination of the erythrocytes of the rabbit and of the mouse, but not of the sheep. In the tests where high concentrations of urease were used hemolysis occurred, complicating the results. This was subsequently controlled by the use of a buffer solution, when agglutination could be readily seen. After agglutination the urease was found to be active. Agglutination could not be demonstrated in the whole blood of rabbits dead after urease injection. Crystalline urease represents a highly purified form of toxic hemagglutinin (10).