UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

Transamination in Pseudomonas aeruginosa MacQuillan, Anthony Mullens 1958

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata


831-UBC_1958_A6_7 M24 T7.pdf [ 4.16MB ]
JSON: 831-1.0106173.json
JSON-LD: 831-1.0106173-ld.json
RDF/XML (Pretty): 831-1.0106173-rdf.xml
RDF/JSON: 831-1.0106173-rdf.json
Turtle: 831-1.0106173-turtle.txt
N-Triples: 831-1.0106173-rdf-ntriples.txt
Original Record: 831-1.0106173-source.json
Full Text

Full Text

TRANSAMINATION IN PSEUDOMONAS AERUGINOSA by Anthony M. MacQuillan A Thesis Submitted i n P a r t i a l F u l f i l l m e n t of the Requirements for the Degree of Master of Science i n A g r i c u l t u r a l Microbiology i n the Division of Animal Science We accept t h i s thesis as conforming to the standard required from candidates for the degree of Master of Science The University of B r i t i s h Columbia A p r i l 1958 ABSTRACT In attempting to study transamination i n Pseudomonas aeruginosa, with the object of determining the range of compounds concerned and whether or not more than one enzyme i s involved, an accurate, rapid, and generally applicable quantitative procedure f o r measuring amino acids was necessary. A method involving paper chromatography of reaction mixtures, spraying with ninhydrin and colorimetric measurement of the eluted spots was found and suitably modified. The reaction mixture was amino acid, keto acid, pyridoxal phosphate, water, phosphate buffer and enzyme. The range of a c t i v i t y of the crude c e l l - f r e e extract was i n v e s t i -gated by testing i t s a b i l i t y to transaminate from 23 amino compounds to glyoxylate, ^-ketoglutarate, oxalacetate and pyruvate. No transamination with pyruvate was observed and very l i t t l e oxalacetate. The range of trans-amination with glyoxylate and<<-ketoglutarate was extensive. In order to te s t whether or not these a c t i v i t i e s were due to one enzyme, p u r i f i c a t i o n was attempted. Isoleucine-glutamate was the system whose a c t i v i t y was followed. P a r t i a l p u r i f i c a t i o n of the enzyme catalyzing t h i s reaction was achieved by precip i t a t i n g the nucleic- acids with protamine sulphate and subsequently fractionating with ammonium sulphate. The i s o -leucine-glutamate a c t i v i t y was most concentrated i n the 50/60 f r a c t i o n . Further p u r i f i c a t i o n of t h i s enzyme system was attempted with the use of calcium phosphate gel adsorption and elution; ion-exchange r e s i n columns; paper electrophoresis i n phosphate buffers and ammonium sulphate e l u t i o n from a c e l i t e column - a l l without success. Having achieved some p u r i f i c a t i o n of the isoleucine-glutamate catalyzing system, the range and s p e c i f i c i t y of t h i s p a r t i a l l y p u r i f i e d f r a c t i o n was compared with that of crude c e l l - f r e e extract. The resu l t s showed that the p a r t i a l l y p u r i f i e d f r a c t i o n retained the broad range of glyoxylate and^-ketoglutarate a c t i v i t i e s while the range of oxalacetate a c t i v i t y was greatly increased. The p o s s i b i l i t y of chemical transamination under reaction conditions was examined and i t was observed that glyxoylate can be chemically aminated i n every case where i t was thought that enzymatic transamination might occur. The concentration of other transaminating a c t i v i t i e s i n a number of ammonium sulphate fractions was examined. The systems studied were i s o -leucine, methionine and phenylalanine, each with^-ketoglutarate, as w e l l as isoleucine phenylanaline and glutamate each with oxalacetate. Results indicated that the a c t i v i t i e s involving «<_-ketoglutarate were concentrating i n the 50/60 f r a c t i o n while those involving oxalacetate were concentrating i n the 60/70 f r a c t i o n . Specific a c t i v i t i e s corroborated these observations to a large extent. These results indicated at least two transaminases i n P. aeruginosa. The glyoxylate system was re-examined by quantitative comparison of chemical and enzymatic transamination and also by stopping the reactions with t r i c h l o r a c e t i c acid rather than by heat. Each of these procedures indicated that glutamate w i l l enzymatically transaminate with glyoxylate to form glycine. Other amino acids tested were inactive. The question of pyruvate p a r t i c i p a t i o n was investigated and the presence of a glutamate-alanine system was found i n fresh, crude preparations. This a c t i v i t y was not shown to occur i n the 50/60 f r a c t i o n . The observed facts therefore suggest the p o s s i b i l i t y of at least three transaminating systems i n P. aeruginosa. In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements fo r an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the Head of my Department or by h i s representative. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of The University of B r i t i s h Columbia, Vancouver 8\ Canada. Acknowledgements I wish to thank Dr. J.J.R. Campbell for his d i r e c t i o n and help i n t h i s work. Financial assistance given me by the B r i t i s h Columbia E l e c t r i c Railway Company and the National Research Council of Canada i s g r a t e f u l l y acknowledged. TABLE OF CONTENTS Page INTRODUCTION General Considerations and Transamination i n Metabolism 1 Chemical Transamination 3 Enzymatic Transamination 5 Mechanism 6 Scope and Occurrence 9 Evidence for the Existence of Separate Transaminases 15 MATERIALS AND METHODS Organism 25 Production of Crude Cell-Free Extract 25 Fractionation Procedures 25 Ammonium Sulphate, Protamine Sulphate Fractionation 25 Calcium Phosphate Gel Adsorption and El u t i o n 27 Column Chromatography 27 Paper Zone Electrophoresis 29 S t a b i l i t y of Transaminase to Heat and to A c i d i f i c a t i o n 30 Protein Determination Procedures 31 Specific A c t i v i t i e s 32 Transamination Reaction Procedure 32 Amino Acid Assay 32 Mouse Liver Homogenate Fractions 33 Reaction Optima 33 EXPERIMENTAL RESULTS 35 DISCUSSION 49 SUMMARY 55 BIBLIOGRAPHY 56 INTRODUCTION Transamination has been defined by Meister (87) as "a chemical reaction i n which an amino group i s transferred from one molecule to another without the intermediate p a r t i c i p a t i o n of ammonia" (130). I t should be stressed at the start that transamination may occur chemically or, i n b i o l o g i c a l systems, enzymatically. Chemical transamination was f i r s t observed by Herbst and Engel i n 1934 (44), who were able to show the transfer of thee£-amino group of amino acids to «*-keto acids i n b o i l i n g aqueous solutions. Braunstein and Kritsmann (12) working with pigeon breast muscle i n 1937, were the f i r s t to report enzymatic trans-amination between <K-amino acids and gt-keto acids. The importance of enzymatic transamination i n metabolism may be indicated by a b r i e f survey of i t s role i n many functional processes. A few examples of the types of reactions i n which transamination i s involved w i l l follow. However, f o r a more complete picture, reviews by Meister and others should be consulted (13, 46, 79, 84-87). The widespread occurrence and the broad s p e c i f i c i t y of transamination, to be discussed at greater length elsewhere, suggests the significance of the reaction i n metabolism. Transamination participates i n the oxidative deamination of L—amino acids and i s related also to the urea-forming mechanism i n mammals. The <X-keto acids have been shown to replacecK-amino acid analogues i n supporting growth of many organisms, which would indicate active transaminating systems. Attempts have been made to relate transamination d i r e c t l y to protein synthesis and to growth and development but without success. The degradation of tryptophan and that of kynurenine are thought to proceed by way of transamination reactions. - 2 -The r o l e of transamination i n the degradation and biosynthesis of amino acids, i n which i t i s f i r s t step i n the former and l a s t step i n the l a t t e r , i s most s i g n i f i c a n t . Tyrosine degradation, ornithine synthesis and h i s t i d i n e synthesis are but examples. An i n t e r e s t i n g mechanism wherein transamination plays a part i s that of hydrogen sulphide formation i n rat l i v e r (23, 8l). Cysteine i s . f i r s t observed to be transformed to/3-mercapto-pyruvate by transamination to^-ketoglutarate, desulphuration then occurs and the sulphur i s reduced to hydrogen sulphide. The removal of the amino group precedes the breakdown of the ^ s-acidic group, and there i s no formation of free ammonia p a r a l l e l with the appearance of hydrogen sulphide, therefore ind i c a t i n g occurrence of transamination. Hence the decrease i n hydrogen sulphide observed i n l i v e r s from vitamin B^-deficient rats i s due to reduced a c t i v i t y of the vitamin B^-requiring transaminase. Other randomly selected studies with rat tissues indicating the significance of transamination i n metabolism follow. Awapara (5) was able to show that the l i v e r s of rats on a protein-free d i e t l o s t f i f t y per cent of the alanine-glutamate transaminase i n about four days, so demonstrating that protein deficiency could ra p i d l y , markedly reduce the transaminating a c t i v i t i e s of the rat l i v e r . Weinhouse and Friedman (13&), i n 1951, studying the metabolism of l a b e l l e d two-carbon acids i n the i n t a c t r a t , demonstrated that glyoxylate could be converted to glycine, presumably by transamination. Sinitsyna (121+) found the transamination a c t i v i t i e s of l i v e r and kidney tissues from vitamin B 0 - d e f i c i e n t rats proceeded very slowly compared to controls. He also concluded that amination of-^-keto acids, other than»£-ketoglutarate, occurs p r i n c i p a l l y by transamination. In another important area of metabolism, Newburgh and Burris (94), studying the photosynthetic f i x a t i o n of carbon dioxide, found that transamination occurred very early i n the metabolic processes following f i x a t i o n . Pyruvate, - 3 -formed immediately after carbon dioxide fixation, was rapidly converted to alanine, which compound serves as a stable storage product for pyruvate. Schormuller and Gellrich (121) have noted transamination to be of some importance in the biochemistry of cheese ripening. Housewright and Thome (52), studying the capsular polypeptide of Bacillus anthracis, found that a considerable amount of the glutamate forming the polypeptide is synthesized via transamination. These workers subsequently noted the transamination of D-amino acids in this organism, so suggesting a possible use of D-amino acids in nature, transamination and racemization together forming a link between L- and D-amino acid utilization. Chemical Transamination Herbst, who, as previously noted, first observed chemical trans-amination, suggested the following mechanism to explain non-enzymatic trans-amination between the ethyl esters of =K-aminophenylacetic and pyruvic acids: I CH - COOCoHc I * NH0 + CH. I 3 C - 0 I cooc^ V CH-: C H - N = C - C O O C p H c I C00C2H5 H \ C - C00C2H5 0 + C H o I 3 C H - N H ' I C00C2H5 CH-: C _ N - CH - COOCgHj C O O C ^ - 4 -Since this f i r s t observation, a considerable quantity of work has been performed on chemical transamination involving keto acids and amino compounds. In particular, Snell's group has performed a voluminous amount of work, largely with the object of elucidating the mechanism of transamination. To mention some few instances of research i n this f i e l d , Metzler and Snell (88) found reversible transamination to occur at 100° C. between pyridoxal (an aldehyde) and most amino acids; moreover they observed the reaction at pH 4*5, to be catalyzed by copper, iron or aluminum salts. Meister et a l (77) i n 1952, found glutamine and asparagine to transaminate chemically with glyoxylate to a greater degree than glutamic and aspartic acids. Heyns and Walter (48) noted that chemical transamination would occur when^-keto acids and amino acids were melted together without solvent at temperatures below 150° C. Also, i n 1953, Nakada and Weinhouse (93), studying non-enzymatic transamination with glyoxylate and various amino acids, observed that glyoxylate was converted to glycine at low reaction temperatures (room temperature) i n the physiological pH range. Reactions were incubated for 2, 24 and 48 hours and after two hours only glutamate and glutamine produced glycine. These workers noted that the dicarboxylic amino acids and their amides were most active and at the same time confirmed Meister's observation that the amides were more active than their dicarboxylic analogues. Pyruvate and^-ketoglutarate were also tested by these same workers but no transamination was found under their experimental conditions. In 1954, Cennamo (21) reported the non-enzymatic transamination of several peptides, as well as amino acids, with pyridoxal i n the presence of alum. Reactions were incubated for t h i r t y minutes at 100° C. at pH 5.0. Meister and Fraser (83) observed the chemical transamination of asparagine with pyridoxal to form<*-ketosuccinamic acid and pyridoxamine; again the - 5 -r e a c t i o n temperature was 100° C. K a l y a n k a r and V a i d y a n a t h a n (58) found t h a t pyruvate p l u s amino a c i d s y i e l d e d no a l a n i n e when b o i l e d f o r two hours but when t h e r e a c t i o n m i x t u r e was f i r s t s p o t t e d on f i l t e r paper and d r i e d f o r h a l f a n hour a t 80° C. a l a n i n e was formed. S i m i l a r f i n d i n g s were made w i t h c ^ - k e t o g l u t a r a t e and amino a c i d s a t temperatures g r e a t e r t h a n 80° C. (37). That i s , t r a n s a m i n a t i o n o n l y o c c u r r e d i n t h e d r y s t a t e . T h i s o b s e r v a t i o n might f o l l o w f r o m the experiments o f Heyns and W a l t e r mentioned above. Enzymatic T r a n s a m i n a t i o n Subsequent-to t h e o r i g i n a l o b s e r v a t i o n s o f B r a u n s t e i n and K r i t s m a n n i t was thought t h a t o n l y t h r e e amino a c i d s p a r t i c i p a t e d i n enzymatic t r a n s -a m i n a t i o n : a l a n i n e + - k e t o g l u t a r a t e , g lutamate + o x a l a c e t a t e and a l a n i n e + o x a l a c e t a t e — t h o u g h t h i s l a s t r e a c t i o n was p o s s i b l y t h e sum o f the f i r s t t w o . L a t e r , t h e - i d e a h e l d sway t h a t glutamate o r ^ - k e t o g l u t a r a t e were n e c e s s a r y p a r t i c i p a n t s i n a l l enzymatic t r a n s a m i n a t i o n r e a c t i o n s . A c c o r d i n g t o M e i s t e r (87) the widespread occurrence o f glutamate-amino a c i d r e a c t i o n s made i t d i f f i c u l t t o prove t h e occurrence o f monocarboxyl ic amino-keto r e a c t i o n s . T h u s , he s a y s , " a p y r u v a t e - v a l i n e r e a c t i o n might o c c u r by d i r e c t amino group t r a n s f e r , o r a l s o by c o u p l e d g lutamate-pyruvate and v a l i n e - * < k e t o g l u t a r a t e r e a c t i o n s " . I t i s r e c o g n i z e d however, t h a t v i r t u a l l y a l l of t h e n a t u r a l amino a c i d s may p a r t i c i p a t e i n t r a n s a m i n a t i o n r e a c t i o n s and t h i s , w i t h o u t the n e c e s s a r y p a r t i c i p a t i o n o f g lutamate o r ^ - k e t o g l u t a r a t e . The r a p i d i n -c o r p o r a t i o n o f a d m i n i s t e r e d amino a c i d n i t r o g e n i n t o a lmost a l l the amino a c i d s i n animals (120) and the a b i l i t y of the©<-keto analogues o f c e r t a i n e s s e n t i a l amino a c i d s t o support growth o f animals and microorganisms made h i g h l y probable the occurence o f r e a c t i o n s i n v o l v i n g the exchange o f amino groups o f many o t h e r amino a c i d s . - 6 -Mechanism With regard to the mechanism of enzymatic transamination, Braunstein, following Herbst1s explanation of chemical transamination, proposed Schiff base formation thus: R R 1 R R 1 R R 1 R R 1 I I I I | I I I C - 0 + CHNHo — * C - N - C H — ^ H C - N = C — * CHNH9 + C = 0 I" I "~ I I I I COOH COOH COOH COOH COOH COOH COOH COOH Snell in 1944 (126) f i r s t proposed the involvement of the aldehyde and amine forms of vitamin B^ in transamination and later (127) he demonstrated reversible interconversion between pyridoxal and pyridoxamine by non-enzymatic transamination with amino acids and keto acids. In 1945 Schlenk and Fisher (118) observed the occurrence of a heat stable cofactor for transamination in pig-heart similar to and replaceable by synthesized pyridoxal phosphate (FLP). That same year, Snell (127) suggested that vitamin B^ played a role in enzymatic transamination. It is interesting to note, in this light, the occurrence in Streptococcus faecalis of pyridoxal kinase (40, 134), an enzyme catalyzing the conversion of pyridoxal to PLP. This enzyme was later studied in Esclerichia coli ( l l ) and in mouse brain tissue (103). Hurwitz in 1953 (53) attempted to purify the enzyme from yeast, and i t might therefore be assumed to be of general occurrence. A particularly interesting experiment indicating the participation of vitamin in enzymatic transamination was one performed by Holden, Wildman and Snell (51). S. faecalis cells were grown on media deficient in vitamin but which contained the necessary amino acids—hence growth resulted. But when the essential amino acids were replaced in the vitamin deficient media by theirX-keto acid analogues, growth did not occur. Growth on this latter medium was only observed when vitamin was added. That i s , vitamin provided the coenzyme for transamination. Studies on vitamin B^ in amino acid metabolism have shown that this vitamin and its derivatives are involved in many reactions which include, decarboxylation, transamination and racemization of amino acids and in addition, play a functional part in many more specific reactions such as the degradation of many amino acids and also of such compounds as kynurenine and cystathionine, also in the conversion of indole and serine to tryptophan. Braunstein and Shemyakin (14) in 1953 proposed a theory of amino acid metabolic processes catalyzed by pyridoxal-dependent enzymes and concluded that a l l reactions of amino acids mediated by PLP form an azomethine grouping: --- Protein COOH A redistribution of electron density is possible, so changing the character of the atom. The H atom at dissociates, allowing an increased electron density as indicated by the arrows. Metzler et a l (89) in 1954 proposed much the same mechanism—a Schiff base is fromed involving pyridoxal, amino acid and a metal ion and suggest the following mechanism for transamination: RCHCOO" I + H - 8 - I RC - C = 0 CHO M + + + H0H2C H0H2C H0H 2C—. C H 3 0 II RC c o o -CHoNH RC - COO-2wn3 HOHgC H+ N l + + H O H 2 C -C H , H W h e r e i n t h e f o r m y l g r o u p f u n c t i o n s i n S c h i f f b a s e f o r m a t i o n w i t h t h e a m i n o a c i d . C h e l a t i o n t h e n s t a b i l i z e s t h i s S c h i f f b a s e v i a t h e n i t r o g e n o f t h e a z o m e t h i n e l i n k a g e , t h e p h e n o l i c g r o u p a n d p r o b a b l y a l s o t h e c a r b o x y l o f t h e amino a c i d . The r e s u l t i n g p l a n a r s y s t e m o f c o n j u g a t e d d o u b l e bonds p r o v i d e s a m e c h a n i s m f o r d i s p l a c e m e n t o f a n e l e c t r o n p a i r f r o m a n y o f t h e bonds o f t h e ^ - c a r b o n o f t h e amino a c i d t o w a r d t h e s t r o n g l y e l e c t r i p h i l i c h e t e r o c y c l i c n i t r o g e n . A g r e a t vo lume o f w o r k h a s b e e n p e r f o r m e d t o d e m o n s t r a t e S c h i f f b a s e f o r m a t i o n , p a r t i c i p a t i o n o f m e t a l i o n s v i a c h e l a t i o n , i n p a r t i c u l a r b y S n e l l e t a l . t o show t h e l a b i l i t y o f t h e o<-hydrogen a t o m , e t c . R e c e n t l y L o n g e n e c k e r a n d S n e l l (69) compared t h e a c t i v i t i e s o f 17 m e t a l s i n p r o m o t i n g p y r i d o x a l - c a t a l y z e d r e a c t i o n s o f amino a c i d s a n d f o u n d t h a t s t a b i l i t y o f c h e l a t e s b e t w e e n t h e s e m e t a l i o n s a n d t h e l i g a n d s p a r a l l e l s t h e o r d e r o f t h e i r c a t a l y t i c a c t i v i t y i n t r a n s a m i n a t i o n . M a t s u o (73) e l a b o r a t e s f u r t h e r u p o n t h e p r o b a b l e e f f e c t s o f c h e l a t i o n upon n o n - e n z y m a t i c t r a n s a m i n a t i o n a n d - 9 -presents evidence f o r chelate formation with S c h i f f bases of pyridoxal and amino acid with Cu + + or A l + + + ions. S n e l l et a l have suggested that metal chelation s t a b i l i z e s the Schiff base i n aqueous solutions and also enhances tautomerism of the S c h i f f base by increasing electron displacement at the ^-carbon. I t should be noted that Nakada and Weinhouse (93) were able to demonstrate non-enzymatic transamination between glyoxylate and amino acids' without added metal ions. Matsuo (74) points out however, that i n the enzymatic reaction the carboxyl group of the amino or keto acid, also the phosphate and other groups of the coenzyme may each react with some s i t e on the apoenzyme molecule. Such bondings might, i n e f f e c t , s t a b i l i z e the S c h i f f base i n t e r -mediate and may maintain suitable p l a n a r i t y without the help of metal chelation. However, Happold (43) claims that magnesium ions stimulate glutamate-oxalacetate transaminase from sheep-heart muscle and Patwardham (98) has shown ferrous ion to reactivate aspartate-glutamate transaminase a f t e r prolonged d i a l y s i s against 8-hydroxyquinoline. He points out though, that Merck PLP may contain F e + * i r o n which fact would c e r t a i n l y complicate such experiments. Vitamin then, i s probably involved i n a l l transamination reactions. That the coenzyme i s f i r m l y bound to the enzyme might explain the observation that certain transaminases do not require addition of coenzyme f o r a c t i v i t y . I t i s conceivable that transamination reactions involving aldehydes, f o r example, glyoxylate, may not involve vitamin B D. Although t h i s reaction has been shown non-enzymatically, as yet there i s no positive evidence f o r a vitamin B^-free transaminase. Scope and Occurrence The scope and spectrum of the transamination reaction i s by the nature of the reaction a r e p e t i t i v e point to discuss. Since the days when - 10 -the three transamination reactions, previously mentioned, were considered to be the only important systems, a great deal of work has been performed by numbers of investigators, and today i t is considered that the scope of trans-amination is very broad. Virtually a l l of the natural amino acids are active in enzymatic transamination. The various "types1 of transamination reactions have been quite fully discussed, by Meister in particular (85, 87). To avoid the mere repetition of his discussion, only more recent work, such trans-amination reactions as: might be considered unusual and certain reactions which pertain to conclusions based upon the experimental work contained in this thesis, wi l l be dealt with. In these discussions, nomenclature employed by the various authors concerned wi l l be used, although Meister (85, 87) has proposed a system of nomenclature which might be adopted for the present time. The transamination reaction as we know i t today consists largely in the transfer of the amino group of an^-amino acid to an<?<-keto acid yielding new^-amLno and keto acids. The keto acids usually employed to study transamination are^-ketoglutarate, oxalacetate, pyruvate and glyoxylate. To indicate the relative degree to which two of these keto acids participate, Snell and Rannefield (128) noted that twenty-two amino acids transaminated with^-ketoglutarate and seven with pyruvate. In 1943 Herbst and Shemin (45) reported the participation of peptides in transamination, observing that pyruvylalanine was formed from alanylalanine. However, Meister (86) states that transamination of ©^-peptides, though shown in non-enzymatic systems, has not been shown enzymatically. The participation of pyruvate in transamination is of interest owing to the difficulty in demonstrating this reaction in Pseudomonas aeruginosa (ATCC 9027) • The glutamate-alanine and alanine-aspratate reactions were - 11 -considered to be two of the three primary transamination reactions, yet, as wi l l be seen later, we could demonstrate the participation of pyruvate in only one case with P. aeruginosa. A brief survey of the occurrence of pyruvate transaminases might serve to elucidate its participation in metabolism. Barron and Tahmisian (7) reported glutamate-alanine transaminase in cockroach muscle. Quastel and Witty (100) reported an ornithine-pyruvate transamination reaction in mammalian and in avian liver tissue and in mammalian kidney, but found l i t t l e or none in muscle or brain. Rowsell (105) observed transamination to pyruvate, in rat liver particles and supernatant, from several amino acids; he also demonstrated reversal of these reactions. In 1953 Fowden and Done (34) reported transamination involving ^-methylene glutamic acid with oxalacetate, ^-ketoglutarate and with pyruvate in groundnut plants. This reaction is interesting both from the point of view of the participation of pyruvate and the unusual amino acid. The participation of pyruvate in transamination in microorganisms does not appear to be as readily demonstrable. But Kakimoto and Kanazawa (57) report aspartate-alanine and glutamate-alanine in Bacillus natto. Barban (6) reports several transamination reactions forming alanine from pyruvate in studies with a spirochaete. However, Yonemura and Iisuka (142) state that they found that only glutamate would transaminate with pyruvate in a strain of bovine tubercle bac i l l i . Hicks (49) reports aspartate-alanine reactions in Clostridium welchii. One of the more interesting aspects of pyruvate parti-cipation in transamination may be concerned with the use of D-amino acids in bacterial metabolism. That i s , L-amino acids transaminate pyruvate and the alanine formed is racemized by alanine racemase, the D-alanine resulting is transaminated back to thee^-keto acid to form a new D-amino acid. Molnar and Thome (92, 132) reported on this possibility and Thome, Gomez and - 12 -Housewright (131) investigated t h i s occurrence i n B a c i l l u s anthracis and B a c i l l u s s u b t i l i s respectively. Jordan (56) observed the presence of both D-aspartate-alahirie, transaminase and alanine racemase i n Rhizobium m e l i l o t i . Thome (133) states that alanine racemase i s the only racemase found i n B a c i l l u s and Rhizobium species. Saito and Tanaka (109) report D-amino acid-pyruvate reactions and also the presence of glutamate and alanine racemases i n Mycobacterium avium. There i s no evidence yet of D-amino aci d transamination i n animal t i s s u e . Saxena (113) reports that pyruvate was not an acceptor i n reactions involving a large number of amino acids i n Pasteurella p e s t i s . Yonatt (143), investigating transaminases i n Mycobacterium tuberculosis (B.C.G.) found that glutamate only would transaminate with pyruvate. Rowsell (107) found that rat l i v e r transaminated pyruvate with a number of amino acids and he reaffirmed the f a c t that such reactions are independent of glutamate and <^-ketoglutarate. He also states that l i v e r i s the only tissue able to catalyze transamination with pyruvate. K i l b y and N e v i l l e (6l) reported the presence of both glutamate-alanine and leucine-alanine transaminases i n the tissues of the desert locust. Schales and Schales (114) report that plant extracts catalyze transamination involving alanine. The p a r t i c i p a t i o n of glyoxylate i n transamination i s of interest because of the f a c t that t h i s compound i s an aldehyde and may or may not require PLP and, or,metal c a t a l y s i s i n the enzymatic reaction. The following workers have reported involvement of glyoxylate: Barban (6 ) , i n a spirochaete, Wright (141) i n Neurospora, Meister (82), Wilson et a l (140 i n tobacco leaves, Kolesnikov (63) i n plant leaves, Marshadev and Pavlova (7l) and Campbell (18) i n Pseudomonas. This reaction ( p a r t i c i p a t i o n of glyoxylate) i s d i f f i c u l t to evaluate when one considers the case of non-enzymatic transamination with - 13 -glyoxylate investigated by Nakada and Weinhouse (93)• Our own work has indicated that i n the absence of good quantitative data, transamination i n v o l -ving glyoxylate, especially i n those cases where the reaction has been stopped by b o i l i n g , should be suspect. Several transamination reactions involving wj-amiiio acids and aldehydes are now known. A unique case i n which an aldehyde i s both a reactant and a product i s the glycine-ornithine transamination reaction (82). Transamination between ornithine and pyruvate and ornithine and cK-ketoglutarate has been shown i n l i v e r preparations (15, 82, 100) and i n Neurospora crassa (32). Such transamination reactions y i e l d a c i d i c semialdehydes and might therefore be considered analogous to reactions involving glyoxylate. I t would be i n t e r e s t i n g , i n t h i s l i g h t , to investigate and compare the requirement f o r metallic ions and fo r PLP i n these reactions with that of glyoxylate. In 1955 Scher and Vogel (117) explored the occurrence of ornithine ^ -transaminase i n various microorganisms. This enzyme catalyzes the reversible transamination of the S-araLno groups of ornithine to ^-ketoglutarate. They found t h i s enzyme i n extracts of Neurospora  s i t o p h i l a . Torulopsis u t i l i s and i n commercial baker's yeast, also i n B. s u b t i l i s and B a c i l l u s pumilus. No appreciable amounts of t h i s enzyme were found i n the enterobacteriaceae, E. c o l i . Aerobacter aerogenes. Erwinia carotovora. S e r r a t i a  marcescens and Proteus v u l g a r i s . Lately, Otani and Meister (97) have worked on the p a r t i c i p a t i o n of<£*-amides and thec£.-amino acid derivatives of ^-keto-glutarate and oxalacetate i n transamination. Glutamine and asparagine have been found to participate i n trans-amination. For example, Campbell (18) found that they were more active than t h e i r respective dicarboxylic amino acid analogues i n transamination with glyoxylate. Meister (86, 87) states that we can conclude that transamination of glutamine and asparagine with«K-keto acids occurs i n two steps: f i r s t , - 1 4 -transamination to y i e l d the«K-keto analogues of the amino acid (A-amides and second, the enzymatic deamidation of t h e ^ - k e t o acid-*o-amide by an enzyme system which has no a c t i v i t y toward the amino acid-*0-amides. However, Kinney and Werkman (62) state that, i n experiments with Propionibacterium .iensenii. neither asparagine nor glutamine participated i n transamination. Douglas and San Clemente (28) studying the metabolism of Streptomvces scabies, concluded that glutamine and asparagine were deamidated p r i o r to transamination. Youatt (143) found that M. tuberculosis transaminated asparagine with <?<-ketoglutarate. Experiments i n our laboratory show that where glutamine and asparagine were involved i n transamination, using crude and p a r t i a l l y p u r i f i e d c e l l extracts, paper chromatograms show markedly dimnished amino acid amide spots but strong glutamate or aspartate spots i n addition to the amino acid formed i n the reaction. This observation suggests that i n b a c t e r i a l extracts at l e a s t , a strong deamidase nay be present which produces the dicarboxylic amino acids which then take part i n transamination. Such a consideration cannot be ignored, therefore, when investigating the p a r t i c i p a t i o n of these two amino ac i d amides i n transamination. The formation of kynurenic acid from kynurenine and ^-ketoglutarate i n Pseudomonas which form glutamate and 2-aminobenzoylpyruvic a c i d which compound spontaneously cy c l i z e s has been reported by M i l l e r et a l (91). Hog l i v e r kynureninase w i l l form kynurenic a c i d i n the presence of pyruvate or ^-ketoglutarate due to contamination with transaminase (95) • Meister (86) states that there i s no d e f i n i t i v e enzymatic work on the transamination of l y s i n e . There i s evidence, he notes, that=£-keto-^-amino caproic acid i s formed from l y s i n e during i t s conversion to pipecolic acid i n the rat (104); such a conversion could occur by oxidative deamination or by transamination. - 15 -In 1952 Gunsalus and Tonzetich (41) reported the formation of glutamate f rom ^ C-ketoglutaric acid plus PLP and adenine, guanine or cytosine as amino donors in extracts of E. co l i . Marshadev and Pavlova (7l) reported that rat liver slices could catalyze the formation of glycine from glyoxylate and guanine, adenosine, guanosine and adenylic acid. Meister claims that such reactions must be considered a typical transamination. However, Schein and Brown (115) were unable to repeat the observations of Gunsalus and Tonzetich either with E. co l i or with rat l iver and, moreover, point out that such trans-amination, i f i t does occur, is "difficult to reconcile . . . with present mech-anisms proposed for transamination". It w i l l be remembered that in the brief discussion on mechanism above, dissociation of the^-hydrogen in the amino donor was prerequisite in order that the electron density might be shifted favourably to allow transfer of the amino group. With adenine or cytosine neither of the tautomeric structures has a proton at carbon-6, thus intra-molecular oxidation reduction cannot take place as in typical transamination. Evidence for the Existence of Separate Transaminases "With the rapidly widening scope of transamination, i t is not particularly remarkable that many new transaminase enzymes should come to light. It is of interest that those enzymes which have been thus far recog-nized appear to exhibit.fairly specific substrate requirements. Although i t might appear that virtually any amino acid might transaminate with any other in an intact tissue, the intermediate stages in such a transformation might well require catalysis by several separate enzymes" (84)• This statement presupposes the existence of separate transaminases for each transamination reaction, or each few transamination reactions. However, as Meister (8?) later points out, "the separation and purification of these enzymes has not - 16 -kept pace with the discovery of new transamination reactions, possibly because of the difficulties inherent in enzyme purification, and perhaps also because of the preoccupation of the investigator with the large number of active and potential substrates". Early reports on the separation and purification of transaminases were made in 1945 (66), concerning a bacterial transajninase and later by Lichstein, Gunsalus and Umbreit (67) who obtained transamination activity in cell-free antolysates of S. faecalis. That same year, 1945, Green, Leloir and Nocito (39) isolated and purified the two classical transaminases from pig-heart, alanine-glutamate and aspratate-glutamate, verifying that these two reactions were indeed catalyzed by two different enzymes. The latter enzyme was highly specific while with the former, alanine could be replaced to some extent by^-amino-butyric acid. These workers, using what are now well known procedures for enzyme fractionation and purification, claimed that their purified preparations were very nearly a l l enzyme and this assumption was corroborated by Tiselius electrophoresis. A number of other investigators worked with these purified enzymes from pig-heart establishing their properties. O'Kane and Gunsalus (96) produced evidence that the aspartic-alanine trans-aminase was an artifact and was indeed the net effect of two other enzymes. This finding was later verified by Heyns and Koch (47)• Schlenk and Fisher (119) showed glutamate-aspartate transaminase to be stable to 60° C. for as long as one hour; in fact, many of these workers used a heat purification step in their studies. Umbreit et a l (135) demonstrated that this enzyme was activated by PLP but not by pyridoxamine phosphate (PMP), though this observ-ation was later found to erroneous (78, 80). Later, in 1951, Cammarata and Cohen (16) prepared and purified an unresolved glutamate-aspartate transaminase from pig-heart by means of alcohol fractionation rather than by ammonium sulphate - 17 -fractionation. They claimed that this unresolved enzyme was very much more active than the apoenzyme plus PLP. Considering the possibility of soluble and particulate enzymes, Hird and Rowsell (50) found that a glutamate-phenylpyruvate transaminase was located solely in the particulate fraction of rat-liver homogenates; tyrosine, alanine and aspartate were also formed from glutamate and their respective <X-keto acid analogues. Differential centrifugation enabled these workers to show that mitochondrial preparations possessed these activities and that they therefore might possibly be the site for these reactions. Dialyzed supernatant from these same homogenates possessed only the glutamate-aspartate and glutamate-alanine transaminases. Later (105) i t was noted that particles from rat-liver homogenates would transaminate pyruvate from L-leucine, phenylalanine, methionine, histidine and tyrosine, but while the supernatant was similarly active with pyruvate, transamination toc^-ketoglutarate from L-leucine, phenylalanine, methionine and valine would not occur. Such data therefore seem to indicate enzymatic differentiation (localization) within the c e l l . Rowsell notes that i t is therefore obvious that there are differences between different animal species and between different animal tissues in transaminating abil i ty. In later studies (106) on localization of transaminases in rat kidney and liver preparations, Rowsell concludes that the formation of phenylalanine, tyrosine and leucine from their correspondinge^-keto acids and L-glutamate is associated with the particulate fraction. Also, he states that transamination between L-amino acids, except alanine and aspartate, and L-glutamate is associated with the sedimentable particles of kidney and liver homogenates. Emmelot and Brombacker (30) state that transamination from valine to iK-ketoglutarate is associated with the mitochondria, of tumour tissue. Quastel and Witty (IOO) found that in the presence of rat l iver and pyruvate an additive effect on - 18 -alanine formation was given by mixtures of ornithine and glutamate, hence indicat ing the presence of separate ornithine-alanine and glutamate-alanine transaminases. And in fact i n 1952 Meister (77) states, "The catalysis of a number of transamination reactions by l i v e r and by other b io logica l materials suggests the existence of at least several separate transaminases different from the highly speci f ic glutamate-alanine and glutamate-aspartate systems." In this paper he presented' data indicating that glutamine and asparagine were degraded by an associated transamination deamidation reaction and that the transaminases involved were di f ferent . Awapara (5), studying the effect of protein depletion on the transaminating act iv i t ies of rat l i v e r s , found that the l i ve rs lost 50 per cent of their or ig ina l alanine-glutamate transaminase within about four days but there was no change in the ac t iv i ty of the aspartate-glutamate transaminase — such data suggest the existence of two quite separate transaminases. Darling (24, 25) achieved the par t ia l separation and pur i f icat ion of three transaminases from ox-heart, u t i l i z i n g a heat pur i f icat ion step and cutting the supernatant with ammonium sulphate into 0/50, 50/65 and 65/8O f r a c -t ions. Examining each f ract ion for alanine-glutamate, aspartate-glutamate and cysteic acid-glutamate ac t iv i ty , he found that the f i r s t reaction was catalyzed by the 0/50 but not by the 50/65 f ract ion . The cysteic acid reaction was catalyzed by the 50/65 and 65/8O f ract ions, therefore suggesting two enzymes. Then, on the basis of the qualitative comparison of glutamate chromatogram spots, he compared the ratios of the aspartate-glutamate and the cysteic ac id -glutamate reactions i n the 50/65 and 65/80 f ract ions. He found that the 50/65 f ract ion was better for the former reaction while the 65/8O was better for the l a t t e r . Hence he postulated the existence of three enzymes i n ox-heart. Smith and Williams (125), attempting to relate transaminases to growth and protein synthesis, found that the amounts of glutamate-alanine and - 19 -glutamate-aspartate transaminases in the embryos of germinating plants( increased™ increased at different rates in the same plant species, suggesting therefore that again these are two separate enzymes in plants. Turning to transaminases in microorganisms, Feldman and Gunsalus (31) appeared to stimulate research in this direction with their study noting that a large number of amino acids would transaminate^-ketoglutarate in extracts of E. co l i . B. subtilis and Pseudomonas fluorescens. They concluded from the number of reactions that a wide variety of transaminases must occur in bacteria. In 1951 Meister, Sober and Tice (76) found that C. welchii would catalyze transamination between PMP and pyruvate to form alanine and PLP. Kakimoto and Kanazawa (57) postulated at least two transaminases in B. natto. aspartate-alanine and aspartate-glutamate, on the basis of different pH optima. Halpern and Grossowicz (42) also postulate two transaminases in mycobacteria on the basis of pH optima. An interesting point with regard to alanine synthesis in bacteria was noted by Marr et al (70) who present N^^ ammonia evidence to show that alanine formed during glutamate oxidation contains more than could be accounted for by exchange of the glutamate amino group with the medium, and much less N 15 than the ammonia. They presume from this evidence that two mechanisms for alanine synthesis exist in Brucella abortus, transamination and direct amination. Wlame and Pierard (139) have since shown the occurrence of an alanine de-hydrogenase in B. subtilis and suggest the following reaction as an alternative forming system in bacteria: L-anine + DPN* + H20 » pyruvate" f DPNH 4- H + + NH^+ In connection with B. abortus. Altenbern and Housewright (3) had already shown the existence of glutamate-alanine transamination. These latter workers also - 20 -found that while leucine-alanine and glutamate-alanine transaminase activities were precipitated by 30 per cent ammonium sulphate from B. abortus cell-free extracts, the former would transaminate directly but the latter required PLP to react, hence indicating two enzymes. Rudraan and Meister (108) obtained evidence for the existence to two major transaminase systems in B. col l extracts. This evidence is discussed to some degree by Meister (84, 87, 108). These enzymes were separated by elution, at different pH levels, from calcium phosphate gel. The f irst , eluted at pH 4*5, catalyzed reactions between^-ketoglutarate and phenylalanine, tryptophan, tyrosine and aspartate; the second, eluted at pH 6.0 catalyzed reactions between^-ketoglutarate and isoleucine, leucine, valine, norleucine and norvaline. A third transaminase catalyzing valine-alanine transamination was eluted at pH 6.5 and found not to be identical with the former two enzymes. These workers then state that this evidence suggests that a single transaminase may catalyze reactions between any of several amino acids and their«K-keto analogues, for, further attempts to separate the above activities proved futile. Fincham (32) demonstrated the occurrence of an ornithine-glutamate transaminase in Neurospora which transfers the £-amino group of L-ornithine to«s<-ketoglutarate, forming glutamic acid-^f-semialdehyde. One might conclude therefore the likelihood of a specific <^ -amino transaminase (136). Fumio and Tadaaki (35) claim to have separated transaminases of avian tubercle bac i l l i by treatment of ce l l extracts with 30 per cent ammonium sulphate. Thome et a l (92, 131, 132, 133), in their studies on D-amino acid transamination in bacillus species, claim the separation, by ammonium sulphate fractionation, of L- and D-amino acid transaminases. Moreover, they state that the active D-amino acids and L-glutamate w i l l transaminate with pyruvate while the active L-amino acids and D-alanine wi l l transaminate with<^y-ketoglutarate. - 21 -These data provide evidence of a rather elegant l i n k between D- and L-amino acid metabolism. In addition, these investigators continually state the importance of a quantitative transamination balance to prove that true trans-amination occurs i n a reaction. They used manometric methods and f i l t e r paper chromatography i n t h e i r work; however t h e i r procedures for the l a t t e r were overly long. The apparent adaptive nature of some transaminases i s shown by the work of Adelberg and Umbarger ( l ) who found that valine-alanine transaminase of E. c o l i was increased i n a c t i v i t y by growth on valine-deficient media. This adaptive behaviour would therefore appear to denote the presence of a s p e c i f i c enzyme. Fincham and Boulter (33) observed that the addition of c e r t a i n amino acids to the growth medium increased the concentration of certain transaminases i n mycelial extracts of N. crassa. On the basis of the effects they observed, they postulated the existence of at l e a s t four transaminases. Three transaminases, alanine-glutamate, aspartate-glutamate and ornithine-glutamate, each of which i s apparently s p e c i f i c , behave d i f f e r e n t l y to the influence of exogenous amino acids. In addition, the behaviour of nine other transaminase a c t i v i t i e s appeared to be correlated and these workers suggest that they may a l l be properties of a single non-sp e c i f i c enzyme. This l a t t e r enzyme appeared to be increased by the addition of certain exogenous amino acids to the medium. Differences i n the dependence of these four transaminases on PLP were also observed. Ames and Horecker (4) have p u r i f i e d the h i s t i d i n o l phosphate-glutamate transaminase of N. crassa to some extent by conventional methods and have found i t to be PLP-requiring. They observed that neither h i s t i d i n o l nor imidazolacetol w i l l replace t h e i r phosphate esters i n transamination, but that several other a c t i v i t i e s are contained i n the p u r i f i e d preparation. - 22 -The reactants were noted to be structurally related, hence__it was postulated that a l l these reactions were carried out by one enzyme. Meister (87) points out that the phosphate ester reaction is analogous to the reaction of PMP with<^-keto acids. Jakoby and Bonner (54) have partially purified kynurenine transaminase from Neurospora and have separated i t from kynureninase. They note that a variety of^-keto acids w i l l act as amino group acceptors. Gasior (36) found the aspartate-glutamate transaminase of Mycobacterium phlei to belong to the -globulin fraction, employing electrophoresis with a partially purified fraction. Shiio (122, 123), working with a halophilic bacterium, a Pseudomonas. has had some success with starch zone electrophoresis of water-lysed extracts and has observed two components, which move towards the cathode, possessing separate activities, phenylalanine-Q<-ketoglutarate and aspartate-^-ketoglutarate. Youatt (143), studying the sensitivity of different transaminase reactions in M. tuberculosis (B.C.G.) to isonizaid inhibition, presents data which are consistent with the presence of at least two transaminases catalyzing the formation of glutamate from alanine, aspartic and phenylalanine in the one case, and from leucine, isoleucine, valine and methionine in the other. Beechey and Happold (9) have lately produced evidence for the existence of a specific transaminase which catalyzes the reversible inter-conversion of PMP and PLP in E . co l i . Whether such an enzyme vrould be a necessary participant in transamination reactions is difficult to say. Such a mechanism might be difficult to reconcile with different pH optima of transaminases, and, in addition, the separation of transaminases would therefore be rendered difficult to prove. The fact that many workers have claimed separation of transaminases indicates that such an enzyme, i f wide-spread, is not a necessary participant in transamination. - 23 -It is apparent that in l iver tissue, separate enzymes exist which catalyze glutamine-^-keto acid (75), asparagine-«£-keto acid (77), serine-alanine (110, 112) and glutamate-hydroxyaspartate ( i l l ) transamination reactions. The transamination-deamidation of glutamine in guinea-pig kidney has been studied more recently (38, 102) and two such systems have been found which differ in their resistance to heat and acid. Chatagner et a l (22) have also shown, in rat l iver, the existence of a cysteinesulphinic acid (CSA)-c<-ketoglutarate transaminase, which, as well as glutamate-alanine transaminase, is unaffected by vitamin deficiency, whereas CSA-alanine transamination activity is strongly diminished. Hence two enzymes must be postulated here. A one hundred-fold purification of a tyrosine-v<-ketoglutarate transaminase from dog liver has been claimed by Canellakis and Cohen (19, 20). They note a sharp pH optimum at 7.7 and that neither pyruvate nor oxalacetate would substitute for^-ketoglutarate. Mason (72) has partially purified kynurenine transaminase from rat kidney and claims a six-fold purification of the apoenzyme. In this connection, he states that keto acids prevent dis-sociation of the apoenzyme-PLP complex at pH 6.3. Lastly, Jenkins and Sizer (55) state that they have purified glutamate-aspartate transaminase from pig heart to the point where their preparation is 70 per cent pure, obtaining a specific activity of 380 uM of glutamate formed per minute per milligram of protein. The problem of transamination is therefore of interest from the point of view of the mechanism of the reaction, the scope and variety of the reaction and the numbers and specificity of the enzymes catalyzing these reactions. Transamination i s , however, of interest not merely from an academic aspect, but is already proving to be of use in the study and the care of illness and disease and also in vitamin B^ deficiency studies (2, 8, 26, 27, 59, 64, 65). - 24 -The objects therefore in this study of transamination in P. aeruginosa (ATCC 9027) were to determine the range of compounds concerned and whether or not more than one enzyme was involved. MATERIALS AND METHODS Organism A l l work was performed with extracts from a strongly pigmenting strain of Pseudomonas aeruginosa (ATCC 9027). Cultures were grown for 20 hours at 30° C. at pH 7.2 in Roux flasks. The medium consisted of ammonium dihydrogen phosphate, 0.3$; dipotassium hydrogen phosphate, 0.2$; yeast extract, 0.1$; iron as FeSO^, 0.5 p.p.m.; glucose, 0.3$; and magnesium sulphate heptahydrate, 0.3*. Production of Crude Cell-Free Extract (CFX). Cells, obtained by centrifugation, were washed once and resuspended in M/30 phosphate buffer pH 7.0. In later experiments cells, similarly washed, were resuspended in 0.2% potassium chloride solution. Suspensions containing about 200 mgm. wet weight of cells per ml. were subjected to sonic oscillation for twelve minutes in a 10 kc. Raytheon oscillator. At f irst a l i t t l e grinding alumina was included with the suspension but this material did not seem to improve the ce l l breakage, hence in later experiments i t was excluded. Cel l sonicates were then centrifuged at 18,000 g. in the cold. Cell-free extracts (CFX), so prepared, were stored at -20 ° C. Extracts contained on an average run about 16 mgm. of protein per ml. Fractionation Procedures Ammonium Sulphate-Protamine Sulphate Fractionation Fresh CFX, prepared as above, was subjected to conventional ammonium- sulphate-protamine sulphate fractionation (68). Detailed procedure was as follows: extracts were placed in an ice bath on a magnetic stirrer and solid ammonium sulphate crystals slowly added t i l l 25$ saturation had been - 26 -reached. The mixture was allowed to stir slowly for a further ten minutes when the precipitated material, designated 0/25, was centrifuged out at 18,000 g. for 15 minutes in the cold. The supernatant was then replaced in the ice bath and more ammonium sulphate crystals added to make a total of 90$ saturation. The precipitate, designated 25/90, which formed here was again spun down at 18,000 g. in the cold. Precipitates were redissolved in chilled M/30 phosphate buffer pH 7.0, later, 0.2$ potassium chloride solution was used instead, then, the neutralized solutions were dialyzed against M/200 phosphate buffer at pH 7.4« Materials were always dialyzed against at least 20 times their volume of buffer. Dialysis was carried out either overnight, i . e . for a period of about twelve hours, or for a period of five hours, util izing a stirring motor to rotate the sacs and including one complete change of buffer. After dialysis, fractions were frozen at - 2 0 ° C. or subjected to further fractionation. The dialyzed 25/90 fraction was placed in an ice bath as before and its pH lowered to 6.0 with acetate buffer. A solution of protamine sulphate containing 200 mgm. per ml. at pH 5*4 was then slowly added from a fine t ip pipette to the 25/90 fraction, with constant stirring, to precipitate the nucleoprotein material. Periodically one ml. of the material was removed, centrifuged for 5 minutes on the Misco microcentrifuge and the 280/260 mu absorption ratio of the supernatant checked on the spectro-photometer. When a ratio of between 0.6 and 1.0 (usually close to 1.0) was reached, addition of protamine sulphate was stopped. The material was then centrifuged at 18,000 g. for 15 minutes, in the cold, the precipitate discarded and the supernatant dialyzed as before. Any precipitation occurring during dialysis was centrifuged out and the dialyzed material then cut into various fractions with solid ammonium sulphate. Fractions resulting were designated thus, e.g. 5O/6O, this being the fraction of protein precipitating between - 27 -50 and 60 per cent ammonium sulphate saturation. Fractions were centrifuged, the precipitate redissolved, neutralized and dialyzed as above. A l l fractions were stored at -20° C. Calcium Phosphate Gel Adsorption and Elution Fractionation of the 50/60 ammonium sulphate fraction was attempted by adsorption on, and elution from, calcium phosphate gel (60), a stock laboratory preparation being used. Two tubes, each containing 0.3 ml. of the 50/60 fraction, were treated as follows: (l) calcium phosphate gel, 0.1 ml. ; dist i l led water, 0.4 ml.; (2) calcium phosphate gel, 0.2 ml.; dist i l led water, 013 ml. These amounts constitute 12.5 % adsorbant respectively. The tubes were incubated in ice for ten minutes, then centrifuged for five minutes on the Misco microcentrifuge. The supematants were neutralized, then tested for activity and protein content. The precipitate from (2) was drained and then eluted with 1.0 ml. of M/30 phosphate buffer pH 7.4 for 10 minutes in an ice bath. This material (3) was centrifuged as above and the eluate neutralized and tested for activity and protein content. Three more tubes were prepared exactly as ( l ) , i .e* for 12.5$ adsorption, then 0.6 ml. of each supernatant from centrifugation was treated with a further calcium phosphate gel adsorption, 0.12, 0.15 and 0.18 ml. of gel being added respectively, constituting 15, 18.7 and 22.5$ adsorbant. After 10 minutes adsorption time, these materials were centrifuged and the supernatants (4), (5) and (6) tested for protein and activity. The drained precipitates were then eluted for 10 minutes, each with one ml. of M/30 phosphate buffer, pH 7.4. These eluates, (7), (8) and (9), were tested for activity and protein after centrifugation. Column Chromatography I. Purification of the 50/60 fraction by ion-iexchange chromatography on Amberlite IRC-50, XE 64 resin was attempted, after the manner of - 28 -Privat de Garilhe and Laskowski (99)• Phosphate buffers were used for elution rather than acetate, for i t was felt that the lower pH values employed by these workers might inactivate the transaminase. The resin, washed clear of fines, was poured to give a 5x1 cm. column which was than washed with M/5 phosphate buffer at pH 6.64. The column was attached to a Gilson fraction collector and the whole apparatus set up in a cold room. Five ml. of the 50/60 fraction, containing 4.65 mgm. protein per ml. , was adjusted to pH 6.64 and added to the column. Elution was then begun with 100 ml. of chilled M/5 phosphate buffer at pH 6.64, which was followed by 200 ml. of M/l phosphate buffer at 7-17. Between 3 and 4 ml. were collected per tube. The rate of flow, averaged over 24 hours, was 8.0 ml. per hour. These columns seemed to expand and slow the rate of flow as time progressed. The protein content of the eluates was determined by 280 mu readings on the spectrophotometer and the contents of selected tubes were f irst dialyzed against distil led water for 2 hours in the cold, then lyophilized to a powder. The lyophilized materials were then redissolved in 0.5 ml. of disti l led water and tested for activity. II . A celite column was also used in an effort to effect a separation of proteins. A quantity of crude celite was f irst boiled three times in 4.0 N. hydrocholric acid, decanting the acid each time. The acid treated celite was then washed with dist i l led water t i l l the washings gave no precipitation upon the addition of a drop of 2$ silver nitrate solution. Following this treatment, the washed celite was rinsed and resuspended in 70% saturated ammonium sulphate solution and a 12x1 cm. column poured. The column was then washed once with a small amount of 70% saturated ammonium sulphate solution. At this point a suspension of protein in more of this same solution was applied to the top of the column. A protein solution was - 29 -used which, having gone through the preliminary 25/90 ammonium sulphate and protamine sulphate purification, had a concentration of 5 mgm. protein per ml. Finally, the protein used was that fraction which precipitated between 35 and 10% ammonium sulphate saturation. The whole column was enclosed in a crude crushed ice jacket and attached to the Gilson fraction collector. Gradient elution was then begun, employing two connected flasks, one containing 500 ml. of 10% saturated ammonium sulphate solution and the other, 500 ml. of dist i l led water. The ammonium sulphate flask was placed on a magnetic stirrer which mixed the fluids as they intermingled (cf. diagram, 17). A nitrogen pressure of 1 lb . p . s . i . was used to drive the fluid over into the column and thence into the fraction collector. The whole apparatus was at room temperature with the exception of the cooled column. Fractions were 5 ml. in volume and the rate of flow was approximately 50 ml. per hour. The 280 mu adsorption readings of fractions were taken on the spectrophotometer to estimate their protein content. Owing to the lack of elution peaks, every ten fractions were combined and their protein precipitated with the addition of ammonium sulphate to saturation point. Precipitates, so obtained, were opalescent and colloidal in nature and hence proved difficult to centrifuge out even at 18,000 g. in the cold. These precipitates were then redissolved or resuspended (solubility limited) in 0.2% potassium chloride solution and each dialyzed against M/200 phosphate buffer at pH 7.4 in the cold, overnight. The activity of these dialyzed fractions was then tested and protein estimations made on those which proved to catalyze enzymatic transamination. Paper Zone Electrophoresis Preliminary experiments, employing M/10 phosphate buffers at various pH levels, indicated that separation of the 50/60 fraction into at least two fractions might occur at pH 7-7 with paper zone electrophoresis. Six strips - 30 -of N°3MM Whatman f i l ter paper were placed on the electrophoresis apparatus, and on each, at the origin, 20 ml. of the 50/60 fraction was deposited. The paper strips were f irst dampened with buffer solution, the excess buffer being blotted up before the 50/60 material was applied. This procedure was calculated to reduce denaturation of protein due to drying, however, excess moisture must be avoided to prevent irregular diffusion of the protein from the origin. Two runs were made: the f irs t , employing an i n i t i a l current of 6.0 milliamps and a final of 11.5 ma. at a voltage of 100 v . , was run for eighteen hours; the second, using only two strips of paper at 700 v. with in i t i a l and f inal currents of 4«75 ma. and 19 ma. respectively, was run for two hours. Protein bands were located by staining control strips for 15 minutes with 10$ mercuric chloride contained in 100 ml. of 95$ ethanol to which 0.1 gm. of bromphenol blue had been added (29). Employing an innovation in the procedure, the strips were decolourized with 0.5$ acetic acid, rather than with water and blotted dry. Protein appeared as blue bands along the strips. Stability of Transaminase to Heat and to Acidification Experiments to test the effects of heating and acidification on the crude CFX were performed in the following manner: three separate mis. of crude CFX, at pH 7.0, were heated in a water bath at 60° C. for 5, 10 and 15 minutes respectively; the activity of each was tested ((l) , (2), and (3)). A small quantity of the crude CFX was taken to pH 5.5 with M/l KHgPO^ and the pH immediately restored to 7.0 with N/ l NaOH, and the activity tested (4). The remainder of this material was heated at 60° C. for 10 minutes and its activity also tested (5)• Another quantity of crude CFX was adjusted to pH 5.5 with 0.4 M acetate buffer, which formed a precipitate immediately. The pH of this material was then brought back to 7.0 and the activity tested (6). - 31 -Similarly acidified material was first centrifuged for 5 minutes on the microcentrifuge and the supernatant neutralized and tested for activity (7); the precipitate was redissolved in M/lOO phosphate buffer at pH 8.0, neutrali-zed and similarly tested (8) . Recombined, neutralized supernatant and preci-pitate were also tested for activity (9). Three separate mis. of crude CFX at pH 5.5 (adjusted with the acetate buffer) were heated at 60° C. in a water bath for 5, 10 and 15 minutes respectively, then neutralized and tested for activity ((10), ( l l ) and (12)). Untreated CFX and boiled CFX were run as controls ((13), (14)). Protein Determination Procedures The protein content of the various fractions was estimated in two ways: (1) A modification of the method employed by Sutherland et al (130) was used to determine the protein content of the crude CFX and fractions thereof before protamine sulphate treatment. To 0.5 ml. of the diluted protein solution, 3.0 ml. of the dilute copper sulphate, sodium tartarate, sodium carbonate solution was added and the mixture incubated as required. Then 0.3 ml. of the 1:3 Folin Ciocalteau solution was added and further incubation at room temperature was allowed for 15 minutes. Readings were made on the Fisher electrophotometer using a 65O mu f i l t e r . A standard curve was prepared using purified egg-albumen as protein source. (2) For fractions which had been treated with protamine sulphate the optical method of Warburg and Christian (138) was employed, wherein the 28O/26O mu absorption ratio of fractions was determined on the Beckman DU spectrophotometer. - 32 -S p e c i f i c A c t i v i t i e s The sp e c i f i c a c t i v i t i e s of c e l l f ractions were calculated on the basis of the above protein determinations and, denoted the uM of amino ac i d formed by transamination i n one hour per mgm. of protein i n the f r a c t i o n s . Transamination Reaction Procedure Test tubes (1x12 cm.) containing reactants, previously ice cooled, were placed i n a water bath at the' required temperature, usually 35° C., allowed a minute to equilibrate then incubated, without shaking, f o r 15 minutes. Reactions were stopped by plunging the t e s t tubes into a b o i l i n g water bath f o r f i v e minutes, thence to crushed i c e . Where excessive heating was not desired, reactions were stopped by the addition of 0.1 ml. of 90$ t r i c h l o r a c e t i c acid (TCA) to each tube. The reaction mixture usually contained M/lOO phosphate buffer at pH 8.04, 0.4 ml.j X-amino a c i d , 10 uM; <?<-keto aci d , 10 uM; pyridoxal phosphate (PLP) where required, 2.5 ug; c e l l f r a c t i o n s and d i s t i l l e d water to a t o t a l volume of 1.0 ml. When TCA was used to stop the reaction, the t o t a l reaction volume was 0.9 ml. to which 0.1 ml. TCA was added. Af t e r stopping the reactions, the tube contents were centrifuged f o r 5 minutes on the microcentrifuge and the supernatants used f o r analysis. Amino Acid Assay Descending f i l t e r paper chromatography at 30° C. was used to assay reaction mixtures f o r amino acids formed. A l l chromatograms were run i n the butanol, g l a c i a l acetic acid, water, 40s10:50 solvent system. For q u a l i t a t i v e work chromatograms were run f o r approximately eight hours on Whatman N° 4, or sixteen hours on Whatman M° \ f i l t e r paper, then dried at room temperature, sprayed with ninhydrin solution and heated at 100° C. f o r 5 minutes. At f i r s t , 0.1$ solutions of ninhydrin i n butanol were used, but i n l a t e r work a solution - 33 -of 0.5$ ninhydrin in acetone containing 10$ galcial acetic acid v/v was used. For quantitative determinations the supernatants from reactions described above were chfomatographed in 10 u l . amounts on Whatman N° 4 f i l ter paper. Chromatograms were run for periods varying from five to seventeen hours, depending upon the reaction pair of amino acids. After running, chromatograms were dried at room temperature, then sprayed with 0.5$ ninhydrin solution in acetone containing 10$ glacial acetic acid v/V !. Sheets were then heated in the oven at 65° C. for 10 minutes. Developed spots were then excised on approximately equal areas of paper which were placed in test tubes and the colour eluted in 7.0 ml. of 75$ ethanol. Tubes were shaken for 15 minutes (101). Colour concentrations were read at 560 mu on the Beckman DU spectrophotometer and the amino acid values read off on standard curves. The accuracy of the method was about 1.5 - 8$. Standard curves, employing solutions of amino acids containing concentrations varying between 0.20 and 0.025 uM of amino acid per 10 u l . were similarly prepared. Mouse Liver Homogenate Fractions To test the reaction procedure with pyruvate, fractions from mouse l iver homogenate were obtained from the Department of Biochemistry. (1) Homogenized liver of male mice was centrifuged at 600xg for 15 minutes, yielding a supernatant labelled S-^ . (2) The S-^  fraction was then centrifuged at 6000xg for 30 minutes, yielding a supernatant labelled S2. These two fractions were suspended in Krebs phosphate buffer at pH 7.4« Reaction Optima Curves plotting reaction conditions for the isoleucine-glutamate system in the 50/60 fraction were made. Reaction times of zero, 1, 2, 3, 4, - 34 -5, 10 and 15 minutes were plotted. Isoleucine concentrations of 1, 2, 3, 5, 8 and 10 uM per reaction; and 50/60 amounts varying from zero, 0.01, 0.02, 0.05, 0.1 and 0.2 ml. were plotted. Reaction temperatures of 0, 25, 30, 35, 40, 50, 60, 65, 70, 75 and 80° C. and reaction pH through 5-6, 5.9, 6.46, 6.81, 7.17, 7.38, 7.73, 8.04 and 8.4 were determined. EXPERIMENTAL RESULTS Preliminaries The reaction procedure outlined in methods was in fact the result of a multitude of elementary but necessary experiments. Initial ly the re-action involved a total volume of 3 ml. and was carried out with crude CFX, at 3 0 ° C , for 60 minutes. Prior to incubation, the mixture was flushed with nitrogen to establish anaerobiosis. The reaction was stopped by boiling. After being cooled, the tubes with their contents were centrifuged. It was then felt that the supernatant, which was used for analysis for amino acids, should be concentrated. With the small volumes employed, and the numbers of reactions involved, evaporation in a fat-bath at 1 3 0 ° C. proved to be the most efficient, routine method. Interest was at f irst centred on the involvement of glyoxylate in transamination. It was soon discovered that chemical transamination could occur with the alanine-glycine system under study. The chemical amination of glyoxylate by alanine proved to be a function of temperature and therefore both the method of stopping the reaction and the method of evaporation had to be changed. It was found that by stopping the reaction with TCA and then evaporating at 3 5 ° C. on a flash evaporator, no chemical transamination occurred and the enzymatic reaction could be measured. It was assumed at this point that the keto acids would also take part in chemical transamin-ation and that boiling temperatures could not be used in the analytical procedure. It was found that transamination occurred equally well aerobically or anaerobically, hence the flushing with nitrogen was eliminated from the procedure. Fractionation of the CFX was begun with the object of purifying the alanine-glycine transaminase, but the activity of the fractions was disappointingly - 36 -low. In view of this fact, the abil ity of the crude CFX to transaminate each of glyoxylate,K-ketoglutarate, pyruvate and oxalacetate from each of fourteen amino acids was determined. The object here was to find another more active transaminating system which could be used in following the isolation and purification of a transaminase. A number of transaminating systems were found in the crude CFX and a particularly active one (judged qualitatively), isoleucine-glutamate, was chosen for further study. In addition to the pronounced activity of this system, the fact that the Rf. values of glutamate and isoleucine differ markedly in the solvent system used for chromatography, lent itself to speeding up the analytical procedure, a point which proved to be important in view of the volume of work involved. Another factor which helped to confuse the issue was that the crude CFX apparently contained a number of free amino acids or small peptides whose presence on chromatograms made the detection of amino acids, formed in trans-amination, a l i t t l e diff icult . It was also observed that such untreated crude CFX would aminate added^-keto acids in the absence of added amino acids. Such contamination of CFX appeared to be a function of the age of the preparation. Dialysis of the crude CFX eliminated much of this trouble. Subsequently, fractionation was found to completely eliminate this difficulty. At this stage of the procedure i t was noted that the alanine-glycine activity of weakly active ammonium sulphate fractions was not stimulated by addition of pyridoxal, adenosine triphosphate (ATP) and magnesium, indicating that no active pyridoxal kinase was present in the fractions, and that PLP would have to be added routinely to fractionated extracts. Preliminary ammonium sulphate fractionation of the CFX to purify and isolate the isoleucine-glutamate transamination did not yield completely isolated activity, i . e . a l l fractions possessed some activity. It therefore became - 37 -obvious that a quantitative procedure for' amino acid determination would have to be used to determine the specific activity of the fractions. Hence, an accurate quantitative micro method was needed, but as well as accuracy, rapidity of method too was important. Good spectrophotometric methods are known for a few amino acids but these are not generally applicable and a general method was desirable. Paper chromatographic procedures were felt to be the most rapid, general methods available. After trying a number without success, the procedure outlined in the methods was found and modified accordingly. Running chromato-grams on Whatman N° 4 f i l ter paper, rather than on Whatman N° 1, was found to double the speed of flow and separation of amino acids (9a). Having obtained this quantitative procedure, i t was possible to experiment with reaction time and on this basis the time was cut to 15 minutes at 35°. The TCA precipitation to stop the reaction and neutralization of the supernatants was not desirable from a quantitative point of view, and, in addition, i t was found that the Rf. value of isoleucine was considerably increased by the TCA treatment. Experimentation proved, at this point, that boiling isoleucine with ^ -ketoglutarate in solution would not produce glutamate, i . e . chemical transamination would not occur under these conditions and therefore enzymatic reactions involving this system could be stopped by boiling. Evaporation of the reaction supernatants was not easily performed routinely on large numbers of samples. Reducing the total reaction volume to one ml. did nothing detrimental to the reaction and eliminated the need for evaporation in the quantitative procedure. Having thus developed the reaction and quantitative assay procedures and, in addition, established the existence of an active isoleucine-glutamate transaminase, i t was then possible to embark upon the problem of its isolation and purification. - 38 -Before proceeding with the results on isoleucine-glutamate trans-aminase purification the amount of isoleucine remaining, and glutamate formed during the reaction was accurately determined. This experiment was run in accordance with Thorne's statement that quantitative estimation of at least two of the participants in the transamination reaction must be made in order to conclusively state that such a reaction does occur (Table I) . These results indicated that a reaction time of 15 minutes was sufficient. It may also be seen that, with longer reaction times, amino acids disappear, i . e . the crude CFX does possess the ability to diminish the amount of amino acid present. TABLE I Amounts of glutamate formed, and isoleucine remaining in transamination reactions. uM/lO y of reaction supernatant Reaction time (mins.) glutamate isoleucine total amino acid 15 .020 .135 .155 30 .023 .127 .150 45 .026 .121 .147 60 .027 .113 .140 90 .028 .107 .135 120 .032 .103 .135 Reaction tubes contained: M/30 phosphate buffer pH 8.04, 0.4 ml; _-ketoglutarate, 10 uM; DL-isoleucine, 15 uM; crude CFX, 0.4 ml.; disti l led water to a total volume of 1 ml. Reaction temperature was 30° C. Fractionation and Purification Studies After a number of preliminary investigations, separation of the crude - 39 -CFX into 0/25, 25/90, 25/50, 50/60 and 60/70 fractions was achieved, employing the solid ammonium sulphate-protamine sulphate procedure outlined. Each of these fractions was tested for isoleucine-glutamate transaminase activity. Their specific activities are listed in Table II. TABLE II Specific activities of various CFX fractions obtained by ammonium sulphate and protamine sulphate treatment. Fraction Protein uM glutamate Specific activity mgm./ml. per 10 >; uM/mgm. protein/hr. Crude CFX 0/25 25/90 Protamined 25/90 25/50 50/60 60/70 50/60 + 60/70 14.55 2.95 15.27 5.8 34.33 6.56 4.97 5.76 0.0185 0.016 0.014 0.011 0.031 0.010 0.025 1.7 2.78 3.22 1.29 19.0 8.05 17.3 In addition, the effect of PLP upon the reaction was tested with the two most active fractions, the 50/60 and 60/70 (Table III). On the basis of these experiments PLP stimulates the activity of the 50/60 fraction 30$, and the 60/70 fraction 90$. -RO-TABLE III Stimulation of isoleucine-glutamate transaminase by the addition of Pyridoxal phosphate (PLP) Fraction Protein mgm./ml. uM glutamate per 10 >. Specific Activity 50/60 6.56 0.31 19.0 50/60 + PLP 6.56 0.416 25.6 60/70 4.97 0.10 8.05 60/70 + PLP 4.97 0.0178 14.32 It was therefore decided to include PLP in the reaction mixture of subsequent experiments. The results of fractionation, shown in Tables II and III, thus demonstrated the fact that the isoleucine-glutamate transaminating system was concentrated in the 50/60 fraction. Further fractionation of this 50/60 fraction was then attempted with the use of the calcium phosphate gel procedure. Preliminary work to determine suitable proportions of gel, pH of adsorption, pH and molarity of elution buffers etc., led to the procedure outlined in methods. Fractions produced by this procedure were tested for activity which is shown in terms of specific activity of fractions numbered as in the procedure (Table IV). - 41 -TABLE IV Specific a c t i v i t y of f r a c t i o n s obtained by calcium phosphate gel treatment of 50/60 Fraction Protein mgm./ml. uM glutamate per 10 A Specific A c t i v i t y 50/60 5.125 0.0413 32.2 1 12.5$ 1.34 0.0225 22.8 2 25$ 0.73 - -3 Eluate from 25$ 1.14 0.0088 10.2 4 12.5$ then 15$ 0.618 — — 5 12.5$ then 18.7$ 0.545 — — 6 12.5$ then 22.5$ 0.726 - -7 Eluate from 4 1.3 0.0044 4-5 8 Eluate from 5 0.86 — — 9 Eluate from 6 0.8 Fractionation of the 50/60 ammonium sulphate f r a c t i o n with calcium phosphate gel produced either f r a c t i o n s with markedly reduced s p e c i f i c a c t i v i t y or completely inactive preparations. Adjustments of pH did not improve the picture. I t was f e l t therefore that some other p u r i f i c a t i o n procedure should be investigated. Ion-exchange chromatography of the 50/60 f r a c t i o n on Amberlite IRC-50 resin was attempted. The separation of protein fractions was very poor and only one broad peak resulted. The fractions i n tubes 3, 6, 10, 15, 18, 21 and 25 were l y o p h i l i z e d and tested f o r a c t i v i t y . None of these f r a c t i o n s showed isoleucine-glutamate transaminase a c t i v i t y . Paper zone electrophoresis employing M/lOO phosphate buffer at pH 7.17 under the conditions stated f a i l e d to effect the separation of proteins of the - 42 -50/60 fraction, as evidenced by examination of stained paper strips. In fact movement of the protein was very poor indeed, much of i t being adsorbed at the origin. Adsorption and desiccation on the strip would appear to effect rapid protein denaturation. That movement of protein which did occur was towards the positive pole. During the higher voltage run considerable condensation occurred on the supporting plates, a condition which could allow the protein to streak unevenly on the paper. This fact suggests that longer runs at lower voltages might give clean separation. At this point i t became evident that further purification of the isoleucine-glutamate transaminase from the 50/60 fraction might prove to be diff icult . Hence i t was decided to determine the range or specificity of the 50/60 fraction per se and to compare i t with the range and specificity of the crude CFX. In view of the possible contaminating amino acids and oxidizing enzymes in the crude CFX i t was also decided to include the specificity and range of the 25/90 fraction in the comparison. Thus each of these three enzyme sources was tested for its ability to transaminate from the 23 amino compounds listed to each of pyruvate, glyoxylate, oxal-acetate and ^ -ketoglutarate. PLP was included in a l l reaction mixtures. Qualitative paper chromatographic analysis, util izing the 0.5$ ninhydrin spray, yielded the results listed in Table V. Controls were run to check possible chemical transamination on a l l positive reactions. - 43 -TABLE V Transamination by Pseudomonas aeruginosa Crude CFX 25/90 50/60 Non-enzymatic <a> u a bO O - P V 0> -p rt -p © o rt H A o Q> & rH bO 0) 18 a) t O • P 0) •s -p o rt H 8 0) •p 0) -p rt rH •a crj 0) - P - P <D 3 rt - P H - P Q> a) bO 0) - P r-4 O O flj rS •P rt > K <1> rH pf O I. K l>» H •^1 O O, bO 2 a •p a o M - P o rt rH O 0) 1 a, a> H bO A l a n i n e A r g i n i n e Asparagine A s p a r t i c Cysteine Glutamic Glutamine G l y c i n e G l y c y l g l y c i n e H i s t i d i n e Hydroxyproline I s o l e u c i n e Leucine L y s i n e Methionine N o r l e u c i n e O r n i t h i n e Phenylalanine P r o l i n e Serine Threonine Tryptophan V a l i n e + + + + 4-+ 4- + + + + + + + - -+ + + + + + + + + + + - 4 f f + + + + + -+ -+ + -4- -4- -4-4-4-+ + + + 4-+ + + r + -4- 4. 4- -- 4-+ 4-- 4-4-+ 4-+ 4-4-+ 4-- 44 -I t w i l l be observed here that no transamination to pyruvate occurred. Chemical transamination with glyoxylate took place with a l l positive reactions, hence there i s no conclusive evidence here of enzymatic transamination involving glyoxylate. Another point of considerable interest i s that the p a r t i c i p a t i o n of oxalacetate i s increased, i . e . widened, i n the 25/90 and 50/60 frac t i o n s over that of the crude CFX. The main point of interest however, i s the broad range of transamination reactions catalyzed by the p a r t i a l l y p u r i f i e d protein, the 50/60 f r a c t i o n . One more attempt to pur i f y the isoleucine-glutamate transaminase further was made, using the c e l i t e column technique, wherein gradient elution of the precipitated protein i n ammonium sulphate solution was ca r r i e d out with the hope of more s e l e c t i v e l y d i s s o l v i n g and separating f r a c t i o n s . Absorption readings at 280 mu, on the spectrophotometer, of the frac t i o n s produced by t h i s procedure revealed no protein peaks. The protein from combined fractions was then tested f o r a c t i v i t y . The fractions from tubes 62 - 71, 72 - 81 and 82 - 91 showed some a c t i v i t y . The glutamate spots produced by the f r a c t i o n from tubes 62 - 71 proved t o be too weak to measure. The s p e c i f i c a c t i v i t y of the other two fractions i s compared with that of the 50/60 f r a c t i o n i n Table VI. TABLE VI Specific a c t i v i t y of protein fractions eluted from a c e l i t e column with gradient ammonium sulphate solution Fraction Protein mgm./ml. uM glutamate per 10 X Speci f i c a c t i v i t y 50/60 3.72 0.0213 22.8 Tubes 62 - 71 0.216 - — Tubes 72 - 81 0.786 0.0013 3.98 Tubes 82 - 91 0.955 0.0013 3.12 - 45 -A l l efforts to purify the isoleucine-glutamate transaminase beyond the 50/60 fraction had thus far been defeated. Recalling that many of the transaminases investigated by other workers had proved to be stable to heating, and, i n addition, i t had been observed i n this laboratory that the crude CFX retained much of the isoleucine-glutamate transaminating a b i l i t y for at least eight months, i t was therefore decided to investigate the s t a b i l i t y of this enzyme to heat and a c i d i -f i ca t ion . It was hoped that such an investigation might provide means to another puri f icat ion step which would be of use i n future work. Specific ac t iv i t i e s on these variously treated preparations were not run; however, the glutamate formed i n reactions i s l i s t e d i n Table VII . These results indicate some degree of heat s t ab i l i ty , which point might possibly be of use i n fractionation. The enzyme appears to be less sensitive to phosphate than to acetate ac id i f i ca t ion . I t would appear that ac idi f icat ion effects resolution, for PLP becomes essential i n reactions after the pH has been lowered; neutralization alone, does not appear to effect recombination to holoenzyme. These observations have not as yet been incorporated into any puri f icat ion procedure. - 46 -TABLE VII Stability of isoleucine-glutamate transaminase in crude CFX to heat and acidification uM glutamate/ml. Fraction - PLP + PLP 1 CFX 60° 51 4.0 2 CFX 60° 10' 5.0 3 CFX 60° 15' 5.3 4 Phosphate pH 5.5 0 8.0 5 Phosphate pH 5.5 60° 10' 0 8.5 6 Acetate pH 5.5 0 6.25 7 Acetate pH 5.5 supernatant 2.5 7.5 8 Acetate pH 5.5 precipitate 0 4.0 9 Acetate pH 5.5 recombined 0 9-0 10 Acetate pH 5.5 60° 5' 0 11 Acetate pH 5-5 60° 10' 0 12 Acetate pH 5-5 60° 15' 0 13 Untreated CFX 15.0 18.0 14 Boiled CFX 0 0 Concentration of Activity Since efforts to achieve a clean separation of enzymes, or further purification of the 50/60 fraction, had not proved fruitful i t was decided to see i f several activities were concentrated in the same fraction, indicating concentration of the same enzyme. Before beginning this procedure determinations of certain reaction optima were made for the isoleucine-glutamate transaminase. Optimum pH, temperature, time, substrate and enzyme were determined and are plotted in Figs. 1, 2, 3> 4 and 5- PIP was included in these reactions, with the exception of the time curve, a l l were run with a reaction time * ' • • • • • • I I . . 0 25 30 35 40 q 50 60 65 70 Fig-1. Influence of temperature on 9he rate of iso leucine-glutamate transamination : reaction time 15 min. PH Fig. 2. Influence of pH on the rate of isoleucine-glutamate transamination; reaction time 15 min. 3 1 2 3 4 5 10 15 M i n u t e s Fig-3- Rate of glutamate formation from isoleucine S u b s t r a t e (uM i so leuc ine /m l . ) Fig.4 . Influence of substrate concentration on rate of isoleucine-glutamate transamination*, reaction time 15 min. ugm of p r o t e i n / m l . F i g - 5 . Ra te of i s o l e u c i n e - g l u t a m a t e t r a n s a m i n a t i o n as a f unc t i on of enzyme concent ra t ion . - 47 -of 15 minutes. To examine the concentration of activities with degree of purification, the systems studied were: isoleucine, methionine and phenyl-alanine, each witho(-ketoglutarate, as well as isoleucine, phenylalanine and glutamate,each with oxalacetate. The specific activities of each of these systems in crude CFX, 25/90, 25/50, 50/60 and 60/70 fractions were compared (Table VIII). The glyoxylate system has been re-examined, f irst by comparison of chemical and enzymatic transamination and second by stopping the reactions with TCA rather than by heat. Systems studied were glutamate, phenylalanine and ornithine, each with glyoxylate. Quantitative comparison of enzymatic with chemical transamination proved that as much glycine was formed chemically as enzymatically with the phenylalanine and ornithine systems. However, glycine formed from glutamate by the 25/50, 50/60 and 60/70 fractions is compared in Table IX. These results were borne out when the reaction was stopped with TABLE VIII Relative transaminase activity in several enzyme fractions. uM of amino acid formed/mgm. protein/hr. Fraction isoleucine phenylalanine methionine isoleucine phenylalanine glutamate -ketoglutarate oxalacetate CFX • 44 .074 .185 0 .22 .22 25/90 2.71 1.08 .840 1.90 6.77 4.36 25/50 3.06 1.95 1.30 1.53 1.95 3.66 50/60 15.50 6.86 6.80 5.15 10.75 16.4 60/70 10.57 3-52 7.05 15.00 33.48 18.1 TCA, i .e . no glycine spots were visible with phenylalanine or ornithine but glutamate had formed glycine in the enzymatic reaction. Unfortunately the - 48 -chromatograms with TCA were somewhat smudged and separation of glutamate and glycine was not sufficient for quantitative determinations. TABLE IX Glycine formed in enzymatic transamination from glutamate. Fraction Glycine formed uM/lO X. 25/50 .0225 50/60 .032 60/70 .018 Chemical .019 The question of pyruvate participation was investigated again. F i r s t , to indicate whether or not the reaction conditions would permit formation of alanine from pyruvate, a known source of glutamate-alanine transaminase was tested. Mouse l i v e r homogenate fractions were used as enzyme source under usual reaction conditions, PLP being added. Qualit-ative paper chromatography revealed a very active enzyme, large amounts of alanine being formed. Evidently then the conditions for transamination to pyruvate were suitable. Thus fresh crude CFX, 0/25 and 25/90 fractions were re-examined for pyruvate transamination with each of glutamate, aspartate and isoleucine as amino group donors. Again PLP was included. Reactions were carried out i n duplicate at pH 8.04 and at pH 5*9. Neither aspartate nor isoleucine was found to aminate pyruvate under these conditions. However, with glutamate, both the crude CFX and the 25/90 fraction formed alanine, and moreover, at both pH levels . An experiment was run to show that alanine was not oxidized by the three fractions used above and quantitative data proved this to be fact . DISCUSSION The above data indicate a number of elementary facts concerning the study of transamination in microorganisms, i f not in a l l tissues. To begin with, the fact that crude extracts may contain free amino acids, which has been observed by others (10, 116), indicates the need for quite thorough dialysis before determinations are made. The amination of keto acids by crude, untreated preparations, in the absence of added amino acids, suggests that observed results of transamination reactions should be treated with reservation. In Table I, i t may be observed that the crude CFX possesses the ability to remove amino acids to some small extent. Then, in Table V, dealing with the range of reactions, i t was pointed out that while the ability of the crude CFX to transaminate to oxalacetate was small, the range of the 25/90 and 50/60 fractions was very much greater. This increase in oxalacetate participation may be a result of eliminating aspartate oxid-izing, or oxalacetate inactivating systems, or may merely be a function of increased specific activity. Therefore negative data with crude preparations may be quite misleading. A fraction equivalent to the 25/90 might better be employed in spectrum studies. Reaction time is an important point to observe as may be seen from Table I, wherein the amount of glutamate formed in f i f -teen minutes was almost as much as that formed in one hour. The possibility of chemical transamination cannot be ignored, in particular with glyoxylate. As may be seen from Table V, in every case where i t was thought enzymatic transamination occurred, glyoxylate was chemically aminated. Hence glyoxylate involvement in enzymatic transamination cannot be determined on a purely qualitative basis where any heating is involved in the procedure. The fractionation studies merely contribute to the probability, a l -ready observed in a large number of cases, that the isolation and purification - 50 -of transaminases is difficult, and in a large measure has not been successful. The ammonium sulphate-protamine sulphate fractionation procedure wi l l be observed to produce isoleucine-glutamate transaminase, in the 50/60 fraction, of variable specific activity. The specific activities listed in Table VIII indicate a 35-fold purification of this enzyme which is the best obtained to date. It is interesting to note that purification of transaminases with ammonium sulphate-protamine sulphate is the limit of purification achieved by a number of workers. However, those workers who claim to have produced highly purified transaminase preparations, have used conventional procedures such as have been employed in this work, without success. The failure to succeed in purification of the 50/60 fraction with calcium phosphate gel treatment was disappointing in view of the i n i t i a l results which, qualitatively speaking, looked promising; other workers have employed this procedure successfully to purify transaminases (108). As was noted before, this procedure completely inactivated the preparations or markedly reduced specific activity. Such results are not likely to be due to the low adsorption pH levels nor to the pH of elution, for pH studies (Fig. 2) indicate that considerable activity is retained at pH 5.6 and even lower. Neither are these results l ikely to be a function of resolution to apoenzyme and coenzyme and their consequent separation, for though i t has been shown that resolution does probably occur at low pH levels, addition of PLP restores activity (Table VII) and PLP was included in these activity tests. It is only possible therefore to conclude that the actual processes of ad-sorption and elution denatured the enzyme sufficiently, by some rearrangement might possibly have covered up reactive sites, or possible binding sites for PLP, in such a way that the postulated reaction chelate could no longer form. - 51 -Similarly one might explain the failure of ion-exchange chroma-tography to produce active fractions. The length of this procedure might contribute to inactivation, though the operation was carried out in the cold. However, a l l indications are that the isoleucine-glutamate transaminase is remarkably stable both to heat and to aging with frequent freezing and thawing. The poor separation of protein in this procedure indicates that other eluting buffers might be used - possibly those used by Privat de Garilhe and Laskowski might prove to be of use in view of the stability of this transaminase to low pH levels. Zone electrophoresis might possibly achieve some separation of proteins i f the correct conditions could be found. Time did not permit experimenting with other buffers and with agar or starch electrophoresis beds. Shiio (123) has had some success in this connection. Inactivation by the adsorption process cannot be invoked to explain the failure of the gradient elution from a celite column, for the celite merely provides surface for solution to take place. Here, possibly the length of time in ammonium sulphate solution and the warmer temperatures were responsible for inactivation. The fraction collector was not working too well in the cold and was therefore used in the laboratory at normal temperatures. Therefore, despite cooling the column, fractions were warmed to room temperature for some time before cooling. The precipitation and removal of protein from these fractions was unsatisfactory in view of its colloidal nature, a fact which suggests that denaturation had already occurred. The- majority of fractions produced by this procedure should have had some activity unless separation of protein was very selective. Denaturation then, seems to provide a probable explanation. The spectrum of transamination in P. aeruginosa preparations (Table V) - 52 -i s quite broad. This fact i s better shown by the p a r t i a l l y p u r i f i e d f r a c t i o n s than by the crude CFX. In t h i s experiment pyruvate was not shown to participate i n transamination. The lack of s p e c i f i c i t y shown by the most p u r i f i e d f r a c t i o n , the 50/60, indicated that possibly a l l these reactions were catalyzed by a single enzyme or a number of very s i m i l a r enzymes. I f one can assume that formation of alanine can occur by the process of transamination to pyruvate, perhaps i n addition to those path-ways mentioned i n the introduction, then the data i n Table V constitute negative evidence f o r at least two transaminases, the one catalyzing those positive reactions shown, the other transaminating to pyruvate. Re-examination of pyruvate involvement i n transamination has subsequently shown that alanine can indeed be formed by transamination from glutamate to pyruvate. This reaction however, has only been shown i n the less p u r i f i e d preparations and not i n the 50/60 f r a c t i o n . Such observations suggest that the enzyme which transaminates to pyruvate i s not contained i n the 50/60 f r a c t i o n . E a r l i e r preliminary investigations had suggested that a glutamate-alanine transaminase was contained i n ammonium sulphate fractions of the crude CFX up to 40 per cent saturation. Owing to the d i f f i c u l t y of separating alanine and glutamate by paper chromatography, t h i s observation was not examined further at that time. The lack of success i n separating transaminases by p u r i f i c a t i o n suggested that some other route to conclusive evidence f o r more than one enzyme should be sought. Having shown some concentration of the isoleucine-glutamate transaminase a c t i v i t y i n the 50/60 f r a c t i o n , the p o s s i b i l i t y that other reactions were s i m i l a r l y concentrated was examined. A cursory comparison of results indicated that the a c t i v i t i e s involving^-ketoglutarate were concentrating i n the 50/60 f r a c t i o n while those involving oxalacetate - 53 -were c o n c e n t r a t i n g i n the 60/70 f r a c t i o n . The c a l c u l a t e d s p e c i f i c a c t i v i t i e s (Table V I I I ) i n d i c a t e t h a t such i s the f a c t . The isol e u c i n e - g l u t a m a t e and phenylalanine-glutamate a c t i v i t i e s are more concentrated i n the 50/60 c u t w h i l e the i s o l e u c i n e - a s p a r t a t e and phenylalanine-aspartate a c t i v i t i e s are more concentrated i n the 60/70 c u t . These observations suggest some degree of«£-keto a c i d s p e c i f i c i t y . The glutamate-aspartate a c t i v i t y appears t o be e q u a l l y w e l l concentrated i n both f r a c t i o n s ; which f a c t , e q u i l i b r i u m p e r m i t t i n g , might be expected i n view of the r e a c t i o n p a i r . The methionine-glutamate r e a c t i o n a l s o appears t o be c a t a l y z e d e q u a l l y w e l l by both o f these f r a c t i o n s . I t i s f e l t t h a t these r e s u l t s i n d i c a t e p o s i t i v e evidence f o r the e x i s t e n c e of at l e a s t two transaminases. The s p e c i f i c i t y of these two enzymes may not be narrow, f o r a l a r g e number of r e a c t i o n s have been shown t o occur, and each c a t a l y z e s methionine-glutamate transamination t o the same degree. The enzymatic formation of g l y c i n e from glutamate (Table LX), f i r s t , i n d i c a t e s t h a t the 50/60 f r a c t i o n a l s o contains t h i s a c t i v i t y , and second, corroborates the evidence f o r separate enzymes i n the 50/60 and 60/70 f r a c t i o n s . Both of these f r a c t i o n s have been shown t o possess t r a n s -aminase a c t i v i t y yet o n l y the former has been shown, q u a n t i t a t i v e l y , t o form g l y c i n e . The f a c t t h a t both pyruvate and g l y o x y l a t e have thus f a r been shown t o transaminate w i t h glutamate i n t h i s s t r a i n of P. aeruginosa i s i n keeping w i t h the i d e a t h a t glutamate, w h i l e not n e c e s s a r i l y i n v o l v e d , s t i l l may f i l l a key p o s i t i o n i n the i n t e r c o n v e r s i o n of amino a c i d s . The o v e r a l l o b s e r v a t i o n , then, i n these s t u d i e s suggests the p o s s i b i l i t y of at l e a s t three transaminases i n t h i s b a c t e r i a l s t r a i n . F u r t h e r i n v e s t i g a t i o n might e l u c i d a t e the p i c t u r e . Perhaps heat p u r i f i c a t i o n - 54 -might be included. It may be important to determine the specificity of reactions for a period of time shorter than fifteen minutes and possibly at temperatures higher than 35° C. It would be interesting to observe whether the specificity of the 50/60 fraction would s t i l l remain as wide i f reactions were carried out for ten minutes at 60° C. Other possibilities for investigation would include the transamination of D-amino acids, and other purification procedures such as fractionation with organic solvents. These studies have indicated the need for quantitative deter-minations in connection with enzyme purification; such determinations can, as has been shown, indicate existence of two enzymes where their activities have not been cleanly separated. SUMMARY 1. A quantitative procedure was developed for the assay of amino acids formed during transamination by cel l- free extracts of P. aeruginosa (ATCC 9027). 2. Par t i a l purif icat ion of an isoleucine-glutamate transaminase was achieved employing ammonium sulphate and protamine sulphate precipitat ion. The enzyme was concentrated i n the 50/60 ammonium sulphate fract ion. 3. Further purif icat ion of this enzyme was attempted with the use of calcium phosphate gel adsorption, ion-exchange chromatography, paper zone electrophoresis i n phosphate buffers and ammonium sulphate elution from a cel i te column, a l l without success. A considerable decrease i n specific ac t iv i ty resulted. 4. The range and spec i f ic i ty of transamination reactions catalyzed by the par t ia l ly puri f ied fraction was compared with that of the crude c e l l -free extract and 25/90 f ract ions . Broad speci f ic i ty was indicated. 5. The s t ab i l i ty of the isoleucine-glutamate transaminase to heat and ac idi f icat ion was examined. 6. The concentration of several transamination ac t iv i t i e s was examined. Reactions involving ^<-ketoglutarate appear to be more concentrated i n the 50/60 ammonium sulphate fraction while those involving oxalacetate are more concentrated i n the 60/70 f ract ion. These results suggest the existence of two separate transaminases. 7. Transamination to glyoxylate and to pyruvate has only been shown to occur from glutamate. 8. I t i s concluded that the data provide evidence for at least three transaminases i n P. aeruginosa. BIBLIOGRAPHY 1. Adelberg, E.A. and Umbarger, H.E. Isoleucine and valine metabolism i n Escherichia c o l i . V ^ - k e t o i s o v a l e r i c acid accumulation. J . B i o l . Chem. 205., 475, 1953-2. Agress, CM., Jacobs, H.I., Glassner, H.S., Lederer, M.A., Clark, W.G., Wroblewski, F., Karmen, A. and La Due, J.S. Serum transaminase le v e l s i n experimental myocardial i n f a r c t i o n . C i r c u l a t i o n 9_, 711, 1955. 3. Altenbern, R.A. and Housewright, R.D. Transaminases i n Smooth Brucella Abortus St r a i n 19. J . B i o l . Chem. 204,, 159, 1953. 4. Ames, B.N. and Horecker, B.L. The Biosynthesis of H i s t i d i n e : Imid-azoleacetol Phosphate Transaminase. J . B i o l . Chem. 220, 113, 1956. 5. Awapara, J . Effect of protein depletion on the transaminating a c t i v i t i e s of some rat organs. J . B i o l . Chem. 200, 537, 1953. 6. Barban, S. Metabolism of h i s t i d i n e and transamination reactions of the Treponemata. Bact. Proc. 123, 1955-7. Barron, E.S.G. and Tahmisian, T.N. The metabolism of cockroach muscle (Periplaneta Americana). J . C e l l . Comp. Physiol. 32, 57, 1948. 8. Beaton, J.R., Beare, J.L., Beaton, G.H., Caldwell, E.F., Ozawa, G. and McHenry, E.W. Vitamin B^. V Chronological sequence of biochemical defects i n the vitamin B^-deprived r a t . J . B i o l . Chem. 202, 385, 1954-9. Beechey, R.B. and Happold, F.C. Pyridoxamine Phosphate Transaminase. Biochem. J . 66, 520, 1957. - 57 -9a. Bentley, H.R. and Whitehead, J.K. Water-miscible solvents i n the Separation of Amino acids by Paper Chromatography. Biochem. J. 2*6, 341, 1950. 10. Bigger-Gehring, L. Transamination Reactions i n Saccharomyces f r a g i l i s . J . Gen. Microbiol. 1 ,^ 45, 1955-11. Binkley, F. and Christensen, G.M. Pyridoxal Phosphate, the Coenzyme of Thioether Cleavage. J . Am. Chem. Soc. 7J3, 3535, 1951. 12. Braunstein, A.E. and Kritsmann, M.G. Uber den Ab-und Aufbau von Aminosauren durch Umaminierung. Enzymologia 2, 129, 1937. 13- Braunstein, A.E. Transamination and the I n t e g r a t i v e Functions of the Dicarboxylic Acids i n Nitrogen Metabolism. Advances i n Protein Chem. 1, 1947-14. Braunstein, A.E. and Shemyakin, M.M. A theory of amino acid metabolic processes catalyzed by pyridoxal-dependent enzymes. Biokhimiya 18, 393, 1953-15. Cammarata, P.S. and Cohen, P.P. The Scope of the Transamination Reaction i n Animal Tissues. J . B i o l . Chem. 182, 439, 1950. 16. Cammarata, P.S. and Cohen, P.P. Fractionation and Properties of Glutamic-Oxalacetic Transaminase. J. B i o l . Chem. 193. 53, 1951. 17. Campbell, J.N. A Study of the Incorporation of Inorganic Phosphate by Pseudomonas aeruginosa. M.Sc. Thesis, p. 47, 1957. Dept. of Dairying, U.B.C. 18. Campbell, L.L. Transamination of Amino Acids with Glyoxylic Acid i n B a c t e r i a l Extracts. J . B a c t e r i o l . 2 i , 81, 1956. 19. Canellakis, Z.N. and Cohen, P.P. P u r i f i c a t i o n studies of tyrosine-^ - k e t o g l u t a r i c acid transaminase. J . B i o l . Chem. 222, 53, 1956. - 58 -20. Canellakis, Z.N. and Cohen, P.P. Kinetic and su b s t r a t e - s p e c i f i c i t y study of tyrosine-o<-ketoglutaric acid transaminase. J. B i o l . Chem. 222, 63, 1956. 21. Cennamo, C. Non-enzymatic transamination between peptides and pyridoxal. Naturwissenschaften 41, 39, 1954-22. Chatagner, F., Bergeret, B. and Fromageot, C. Transaminations taking part i n the metabolism of cy s t e i n e s u l f i n i c acid i n mammals. Ann. Acad. S c i . Fennicae Ser. A I I No. 60, 693, 1955. 23. Chatagner, F. and Sauret-Ignazi, G. Role of transamination and of pyridoxal phosphate i n the enzymic formation of hydrogen sulphide from cysteine by the rat l i v e r i n aerobiosis. B u l l , soc. chim. b i o l . ^8, 415, 1956. 24. Darling, S. The transamination between cysteic acid and«<-keto-glu t a r i c acid, catalyzed by a sp e c i f i c transaminase. Cong, intern, biochim., Resumes communs., 2 e Congr. Paris 304, 1952. 25. Darling, S. Cysteic acid Transaminase. Nature, 17J), 749, 1952. 26. DeRitis, F., C o l t o r t i , M. and G i u s t i , G. Transaminase a c t i v i t y i n human serums i n v i r a l h e p a t i t i s . Minerva med. I , 1207, 1955. 27. DeRitis, F., C o l t o r t i , M. and G i u s t i , G. An enzymic test f o r the diagnosis of v i r a l h e p a t i t i s : the serum transaminase a c t i v i t i e s . C l i n . Chim. Acta 2, 70, 1957-28. Douglas, R.J. and San Clemente, C L . Some Observations on the Metabolism of Streptomvces scabies. Can. J . Microbiol. _2, 905, 1957. 29. Durrum, E.L. A Microelectrophoretic and Microionophoretic Technique. J . Am. Chem. Soc. 72, 2943, 1950. - 59 -30. Emmelot, P. and Brorabacker, P.J. Enzymic a c t i v i t i e s of tumour mitochondria. Pyridine nucleosidases and amination processes. Biochem. et Biophys. Acta 22, 487, 1956. 31. Feldman, L.I. and Gunsalus, I.C. The Occurrence of a Wide Variety of Transaminases i n Bacteria. J . B i o l . Chem. 182, 821, 1950. 32. Fincham, J.R.S. Ornithine Transaminase i n Neurospora and i t s Relation to the Biosynthesis of Proline. Biochem. J . j>3_, 313, 1953. 33. Fincham, J.R.S. and Boulter, A.B. Effects of Amino Acids on Trans-aminase Production i n Neurospora crassa: Evidence f o r Four Different Enzymes. Biochem. J . 62, 72, 1956. 34- Fowden, L. and Done, J. A New Transamination Reaction. Nature 171. 1068, 1953-35. Fumio, I . and Tadaaki, S. Transaminases of avian tubercle b a c i l l i . Kekkaku 2£, 368, 1954-36. Gasior, E. Aspartic-glutamic transaminase i n Mycobacterium p h l e i . Ann. Univ. Mariae Curie - Sklodowska, Lublin-Polonia Sect. D 11, 161, 1957. 37. G i r i , K.V. and Kalyankar, G.D. Non-enzymic transamination reaction between"(.-amino acids and keto acids. Naturwissenschaften 40. 224, 1953. 38. Goldstein, L., Richterich-van Baerle R. and Dearborn, E.H. Kidney glutaminases I I . The glutamine-o<-keto acid transamination-deamidation system of the guinea pig. Enzymologia 18, 26l, 1957-39. Green, D.E., L e l o i r , L.F. and Nocito, V. Transaminases. J . B i o l . Chem. 161, 559, 1945-40. Gunsalus, I . C , Bellamy, W.D. and Umbreit, W.W. A Phosphorylated Derivative of Pyridoxal as the Coenzyme of Tyrosine Decarboxylase. J . B i o l . Chem. 15J>, 685, 1944. - 60 -41. G u n s a l u s , C F . and T o n z e t i c h , J . T r a n s a m i n a s e s f o r P y r i d o x a m i n e and P u r i n e s . N a t u r e 122, l 6 2 , 1952. 42. H a l p e r n , Y . S . a n d G r o s s o w i c z , N . T r a n s a m i n a t i o n r e a c t i o n s i n M y c o -b a c t e r i a c e a e . B u l l . R e s e a r c h C o u n c i l I s r a e l 6E, 21, 1956. 43- H a p p o l d , F . C and T u r n e r , J . M . E f f e c t o f magnes ium i o n s on h e a r t m u s c l e t r a n s a m i n a s e . N a t u r e 179. 155, 1957. 44. H e r b s t , R . M . and E n g e l A R e a c t i o n B e t w e e n a £ - k e t o n i c A c i d s a n d <*-amino A c i d s . J . B i o l . Chem. 102, 505, 1934-45- H e r b s t , R . M . a n d S h e m i n , D . The S y n t h e s i s o f P e p t i d e s b y T r a n s -a m i n a t i o n . J . B i o l . Chem. 11^2, 541,. 1943. 46. H e r b s t , R . M . The T r a n s a m i n a t i o n R e a c t i o n . A d v a n c e s i n E n z y m o l . £ I n t e r s c i e n c e , N . Y . 1946, p . 75. 47- H e y n s , K . a n d K o c h , ¥ . T r a n s a m i n a t i o n . The E n z y m i c f o r m a t i o n o f a l a n i n e f r o m a s p a r t i c a c i d . H o p p e - S e y l e r ' s Z . p h y s i o l . Chem. 288, 272, 1951. 48. H e y n s , K . and W a l t e r , W. The r e a c t i o n o f k e t o a c i d s w i t h amino a c i d s a n d ammonium s a l t s . N a t u r w i s s e n s c h a f t e n ZjO, 362, 1953. 49. H i c k s , R . M . T r a n s a m i n a s e A c t i v i t y i n C l o s t r i d i u m w e l c h i i . B i o c h e m . J . £0 i i i , 1955. 50. H i r d , F . J . R . and R o w s e l l , E . V . A d d i t i o n a l T r a n s a m i n a t i o n s b y I n -s o l u b l e P a r t i c l e P r e p a r a t i o n s o f R a t L i v e r . N a t u r e 166. 517, 1950. 51. H o l d e n , J . T . , W i l d m a n , R . B . a n d S n e l l , E . E . G r o w t h P r o m o t i o n b y K e t o and H y d r o x y A c i d s and i t s R e l a t i o n t o V i t a m i n B ^ . J . B i o l . Chem. 121, 559, 1951. 52. H o u s e w r i g h t , R . D . a n d T h o r n e , C B . S y n t h e s i s o f G l u t a m i c A c i d a n d G l u t a m y l P o l y p e p t i d e b y B a c i l l u s a n t h r a c i s . J . B a c t e r i d . 60, 39, 1950. - 61 -53. Hurwitz, J . The Enzymatic Phosphorylation of Pyridoxal. J . B i o l . Chem. 205., 935, 1953-54. Jakoby, W.B. and Bonner, D.M. Kynurenine Transaminase from Neurospora. J . B i o l . Chem. 221, 689, 1956. 55. Jenkins, W.T. and Sizer, I.W. Glutamic-aspratic transaminase. J . Am. Chem. Soc. 2 2 , 2655, 1957-56. Jordan, D.C. Observations on the Enzymatic Degradation and Conversion of Certain L - and D-Amino Acids by an Effective Strain of Rhizobium m e l i l o t i . Can. J . Microbiol . 1, 743, 1955. 57. Kakimoto, D. and Kanazawa, A. Transamination i n Baci l lus natto No. 8, Kagoshima Daigaku Suisan Gakuba Kiyo 2, 121, 1953-58. Kalyankar, C D . and Vaidyanathan, C.S. The reaction of keto acids with amino acids. Naturwissenschaften 41, 14, 1954. 59. Karmen, A . , Wroblewski, F . and La Due, J.S. Transaminase Act iv i ty i n Human Blood. J . C l i n Invest. 2kt 126, 1955. 60. K e i l i n , D. and Hartree, E . F . On the Mechanism of the Decomposition of Hydrogen Peroxide by Catalase. Proc. Roy. Soc. BI24. 397, 1938. 61. K i lby , B.A. and Nevi l le , E . Amino Acid Metabolism i n Locust Tissues. Biochim. et Biophys. Acta 1£, 389, 1956. 62. Kinney, R.W. and Werkman, C H . Transamination i n propionic acid bacteria. Bact. Proc. 118, 1957. 63. Kolesnikov, P.A. Formation of glycine from glyoxylic acid in extracts from green leaves. Doklady Akad. Nank. S.S.S.R. j)6, 125, 1954-64. I»a Due, J .S. Wroblewski, F . and Karmen, A. Serum glutaraic-oxalacetic transaminase ac t iv i ty i n human acute transmural myocardial infarct ion. Science 120, 497, 1954-- 62 -65. l a Due, J.S. and Wroblewski, F. The Significance of Serum Glutamic-oxalacetic Transaminase a c t i v i t y following Acute Myocardial Inf-a r c t i o n . C i r c u l a t i o n 2, 871, 1955-66. L i c h s t e i n , H.C. and Cohen, P.P. Transamination i n Bacteria.. J . B i o l . Chem. 15X 85, 1945-67. L i c h s t e i n , H.C.,Gunsalus, I.C. and Umbreit, W.W. Function of the Vitamin B^ Group: Pyridoxal Phosphate (Codecarboxylase) i n Transamination. J . B i o l . Chem. l 6 l , 311, 1945-68. Lindstrom, E.S. Thec^-ketoglutaric oxidase System of Azotobacter. J . B a c t e r i o l . 6j>, 565, 1953-69. Longenecker, J.B. and S n e l l , E.E. The comparative a c t i v i t i e s of metal ions i n promoting pyridoxal-catalyzed reactions of amino acids. J . Am. Chem. Soc. 22, 142, 1957. 70. Marr, A.G., Olsen, C.B., Unger, H.S. and Wilson, J.B. The oxidation of glutamic acid by Brucella abortus. J . B a c t e r i o l . 66, 606, 1953. 71. Marshadev, S.R. and Pavlova, N.A. Doklady Akad. Nauk. S.S.S.R. 101, 135, 1955. 72. Mason, M. Kynurenine Transaminase of Rat Kidney: A Study of Coenzyme Dissociation. J . B i o l . Chem. 222, 6 l , 1957-73. Matsuo, Y. Formation of Sc h i f f Bases of Pyridoxal Phosphate Reactions with Metal Ions. J . Am. Chem. Soc. 22, 2011, 1957-74. Matsuo, Y. Pyridoxal Catalysis of Non-enzymatic Transamination i n Ethanol Solution. J . Am. Chem. Soc. 22, 2016, 1957-75. Meister, A. and Tice, S.V. Transamination from Glutamine tocxS-keto Acids. J . B i o l . Chem. 182, !73, 1950. 76. Meister, A., Sober, H.A. and Tice, S.V. Enzymatic Decarboxylation of Aspartic Acid to<x-Alanine. J . B i o l . Chem. 182, 577, 1951. - 63 -77- Meister, A., Sober, H.A., Tice, S.V. and Fraser, P.E. Transamination and Associated Deamidation of Asparagine and Glutamine. J. Biol. Chem. 122, 319, 1952. 78. Meister, A., Sober, H.A. and Peterson, E.A. Activation of Purified Glutamic-Aspartic Apotransaminase by Crystalline Pyridoxamine Phosphate. J. Am. Chem. Soc. Jk> 2385, 1952. 79. Meister, A. Enzymatic Transfer of Alpha-Amino Groups. Science 120. 43, 1954. 80. Meister, A., Sober, H.A. and Peterson, E.A. Studies on the Coenzyme Activation of Glutamic-Aspartic Apotransaminase. J. Biol. Chem. 206. 89, 1954. 81. Meister, A., Fraser, P.E. and Tice, S.V. Enzymic desulfuration of ^-mercaptopyruvate to pyruvate. J. Biol. Chem. 206, 56l, 1954-82. Meister, A. Enzymic transamination reactions involving arginine and ornithine. J. Biol. Chem. 206, 587, 1954. 83. Meister, A. and Fraser, P.E. Enzymic formation of L-asparagine by transamination. J. Biol. Chem. 210. 37, 1954-84. Meister, A. General Reactions of Amino Acids. A Symposium on Amino Acid Metabolism. W.D. McElroy and H.B. Glass, The Johns Hopkins Press, Baltimore, 1955, p. 3-85. Meister, A. Transamination. Advances in Enzymol. 16, 185, 1955-86. Meister, A. Nonoxidative and Nonproteolytic Enzymes. (Amino Acid Transaminases and Racemases) . Ann. Rev. of Biochem. 2j>, 29, 1956. 87- Meister, A. Biochemistry of the Amino Acids. A.P. Inc. N.Y. 1957-88. Metzler, D.E. and Snell, E.E. Some Transamination Reactions Involving Vitamin B^. J. Am. Chem. Soc. Jk> 979, 1952. - 64 -89. Metzler, D.E., Ikawa, M. and S n e l l , E.E. A general mechanism f o r vitamin B D-catalyzed reactions. J . Am. Chem. Soc. 7.6, 648, 1954-90. Milch, L.J. and Albaum, H.G. Serum transaminase a c t i v i t y i n x-irradia t e d rabbits. Proc. Soc. E x p t l . B i o l . Med. <j£, 595, 1956. 91. M i l l e r , I.L., Tsuchida, M. and Adelberg, E.A. The Transamination of kynurenine. J . B i o l . Chem. 2pJJ, 205, 1953-92. Molnar, D.M. and Thome, C.B. Transamination of D-amino acids by Ba c i l l u s anthracis. Bact. Proc. 123, 1955. 93- Nakada, H.I. and Weinhouse, S. Non-enzymatic transamination with glyoxylic a c i d and various amino acids. J . B i o l . Chem. 204. 831, 1953. 94. Newburgh, R.W. and B u r r i s , R.H. Effe c t of i n h i b i t o r s on the photo-synthetic f i x a t i o n of carbon dioxide. Arch. Biochem. and Biophys. 4i, 98, 1954-95• Ohashi, I . , T a t s u j i , N., Kiyoshi, M. and Makio, U. Kynurenine Trans-aminase. Symposium on Enzyme Chem. (Japan) 2> 84, 1954-96. O'Kane, D.E. and Gunsalus, I.C. Aspartic-Alanine Transaminase, an A r t i f a c t . J . B i o l . Chem. 122, 433, 1947. 97- Otani, T.T. and Meister, A. w_-Amide and o>amino acid derivatives of o^-ketoglutaric a c i d and oxalacetic acid. J . B i o l . Chem. 224. 137, 1957. 98. Patwardham, M.U. Role of Ferrous Iron i n Enzymatic Transamination. Nature 181, 187, 1958. 99. Privat de Garilhe, M. and Laskowski, M. Studies of the Phosphodies-terase from Rattlesnake Venom. Biochim. et Biophys. Acta 18. 370, 1955. - 65 -100. Quastel, J.H. and Witty, R. Ornithine Transaminase. Nature 167. 556, 1951. 101. Reusser, F., Spencer, J.F.T. and Sallans, H.R. E s s e n t i a l Amino Acids i n Microorganisms. Can. J . Microbiol, _3_, 721, 1957. 102. Richterich^van Baerle, R., Goldstein, L. and Dearborn, E.H. Kidney glutaminases I . Glutaminase I i n the guinea pig kidney. Enzymologia 18, 190, 1957-103. Roberts, E. and Frankel, S. Further Studies of Glutamic Acid De-carboxylase i n Brain. J . B i o l . Chem. 190. 505, 1951. 104. Rothstein, M. and M i l l e r , L.L. The Conversion of Lysine to Pipecqtic Acid i n the Rat. J . B i o l . Chem. 211, 851, 1954-105. Rowsell, E.V. Transamination to Pyruvate and Some other*£-keto Acids. Nature 168, 104, 1951. 106. Rowsell, E.V. Transaminations with L-Glutamate and K-Oxoglutarate i n Fresh Extracts of Animal Tissues. Biochem. J . 64. 235, 1956. 107. Rowsell, E.V. Transaminations with Pyruvate and Other<=^-keto Acids. Biochem. J . 6^, 246, 1956. 108. Rudman, D. and Meister, A. Transamination i n Escherichia c o l i . J . B i o l . Chem. 200, 591, 1953-109- Saito, M. and Tanaka, S. Transamination from D-amino acids. Kekkaku 31, 329, 1956. 110. Sallach, H.J. Evidence f o r a s p e c i f i c alanine-hydroxypyruvic trans-aminase. A Symposium on Amino Acid Metabolism. W.D. McElroy and H.B. Glass, The Johns Hopkins Press, Baltimore, 1955, p. 782. 111. Sallach, H.J. and Peterson, T.H. The formation of hydroxyaspartic ac i d from dihydroxy-fumaric acid and L-glutamic acid. J . B i o l . Chem. 223_, 629, 1956. - 66 -112. Sallach, H.J. Formation of serine from hydroxy-pyruvate and L-alanine. J. B i o l . Chem. 223, 1101, 1956. 113. Saxena, K.C., Sagar, P., Agarwala, S.C. and Shrivastava, D.L. Enzyme make-up of Pasteurella pestis. IV Transamination reactions i n vir u l e n t and avirulent strai n s . Indian J. Med. Research 45, 161, 1957-114. Schales, 0. and Schales, S.S. Decarboxylations and transaminations i n extracts of higher plants. Arch. Biochim. and Biophys. 69, 378, 1957. 115. Schein, A.H. and Brown, E.M. Purine and Pyrimidine Transaminases i n Escherichia c o l i . Biochem. J. 67, 594, 1957. 116. Schepartz, B. Transamination as a step i n tyrosine metabolism. J. B i o l . Chem. 1£3, 293, 1951. 117. Scher, W.I. J r . and Vogel, H.J. On the occurrence of ornithine ^-transaminase i n various microorganisms. Bact. Proc. 123, 1955. 118. Schlenk, F. and Fisher, A. Note on the P u r i f i c a t i o n and Properties of Glutamic-Aspartic Transaminase. Arch. Biochem. 8, 337, 1945-119. Schlenk, F. and Fisher, A. Studies on Glutamic-Aspartic Acid Trans-aminase. Arch. Biochem. 12, 69, 1947. 120. Schoenheimer, R. The Dynamic State of Body Constituents. Harvard Univ. Press, Cambridge, 1949. 121. Schormuller, J. and G e l l r i c h , W. Biochemistry of cheese ripening. XI. Transaminase reactions i n ripening sour milk cheese, with regard to glutamate-aspartate system. Z. Lebensm.-Untersuch. u- Forsch. 100, 200, 1955. - 67 -122. Shiio, I . , Maruo, B. and Akabori, S. Transaminases i n a ha l o p h i l i c bacterium. J. Biochem. (Japan) 43_, 779, 1956. 123. Sh i i o , I. Transaminases i n a ha l o p h i l i c bacterium. 'N° 101. I I . Phenylalanine-glutamic acid transaminase. J. Biochem. (Japan) 44_, 175, 1957. 124. Sinitsy.na, A.L. The Effect of B^-avitaminosis on the deamination of L-amino acids and on the synthesis of t h e i r -=£-keto and ^-hydroxy acids and ammonia i n the l i v e r and kidneys of the r a t . Biokhimiya 19, 80, 1954-125. Smith, B.P. and Williams, H.H. Transaminase Studies i n Germinating Seeds. Arch Biochem. and Biophys. ^1, 366, 1951. 126. S n e l l , E.E. The Vitamin A c t i v i t i e s of "Pyridoxal" and "Pyridoxamine". J. B i o l . Chem. 154, 313, 1944-127. S n e l l , E.E. The Vitamin B^ Group. V. The Reversible Interconversion of Pyridoxal and Pyridoxamine by Transamination Reactions. J. Am. Chem. Soc. 67_, 194, 1945. 128. S n e l l , E.E. and Rannefield, A.N. The Vitamin Bg, Group. I I I . The Vitamin A c t i v i t y of Pyridoxal and Pyridoxamine for Various Organisms. J. B i o l . Chem. 15j7_, 475, 1945-129. Sutherland, E.W., Cori , C.F., Haynes, R. and Olsen, N.S. P u r i f i c a t i o n of the Hyperglycemic-Glycogenolytic Factor from Ins u l i n and from Gastric Mucosa. J. B i o l . Chem. 180, 825, 1949-130. Tanenbaum, S.W. A Study of the Transamination Reaction by Use of Isotopic Nitrogen. J. B i o l . Chem. 218, 733, 1956. 131. Thorne, C.B., Gomez, C.G. and Housewright, R.D. Transamination of D-Amino Acids by B a c i l l u s S u b t i l i s . J . B a c t e r i o l . 69, 357, 1955. - 68 -132. Thorne, C.B. and Molnar, D.M. D-amino Acid Transamination i n B a c i l l u s Anthracis. J. B a c t e r i o l . 70, 420, 1955. 133' Thome, C.B. Metabolism of Nitrogenous Compounds. Ann. Rev. Microbiol. 10, 329, 1956. 134. Umbreit, W.W., Bellamy, W.D. and Gunsalus, I.C. The Function of Pyridoxin Derivatives: A Comparison of Natural and Synthetic Codecarboxylase. Arch. Biochem. 7, 185, 1945* 135. Umbreit, W.W., O'Kane, D.J. and Gunsalus, I.C. Function of the Vitamin B Q Group: Mechanism of Transamination. J. B i o l . Chem. 176, 629, 1948. 136. Vogel, H.J. On the Glutamate-Proline-Ornithine I n t e r r e l a t i o n i n Various Microorganisms. A Symposium on Amino Acid Metabolism. W.D. McElroy and H.B. Glass, The Johns Hopkins Press, Baltimore, 1955, p. 335. 137. Warburg, 0. and Christian, ¥. Isolierung and K r y s t a l l i s a t i o n des Garungsferments Enolase. Biochem. Z. 310, 384, 1941. 138. Weinhouse, S. and Friedman, B. Metabolism of l a b e l l e d 2-carbon acids i n the intact rat. J. B i o l . Chem. 191, 707, 1951. 139. Wiame, J.M. and Pierard, A. Occurrence of an L (+) - Alanine De-. hydrogenase i n B a c i l l u s s u b t i l i s . Nature 176, 1073, 1955. 140. Wilson, D.G., King, K.W. and B u r r i s , R.H. Transamination reactions i n plants. J. B i o l . Chem. 208, 863, 1954. 141. Wright, B.E. U t i l i z a t i o n of Glyoxylic and Gl y c o l i c Acids by a Neurospora Mutant Requiring Glycine or Serine. Arch. Biochem. and Biophys. 31, 332, 1951. - 69 -142. Yonemura, T. and Iisuka, M. Jap. J. Tuberc. 3_, 22, 1955-143. Youatt, J. The Action of Isoniazid on the Transaminases of Myco-bacterium tuberculosis (BCG). Biochem. J. 68, 193, 1958. 


Citation Scheme:


Citations by CSL (citeproc-js)

Usage Statistics



Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            async >
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:


Related Items