N u m e r o u s reports [see review b y N a s o n ( 1 7 5 ) ] h a v e implicated v a n a d i u m in nitrogen fixation. T h e s e studies indicated t h a t t h e ad-dition of v a n a d i u m (or m o l y b d e n u m ) to soils or to the growth m e d i u m stimulated nitrogen fixation a n d g r o w t h w h e r e nitrogen was n o t added in combined form. F o r example, H o r n e r et al. ( 1 0 8 ) reported that m o l y b d e n u m or v a n a d i u m w a s essential for nitrogen fixation b y a n u m b e r of Azotobacter species w i t h similar concentration ranges for both metals. T h e m a x i m u m effect b y v a n a d i u m was 5 0 - 8 0 % of t h a t produced b y m o l y b d e n u m . T h e responses to tungsten w e r e due to a m o l y b d e n u m i m p u r i t y . A l t h o u g h t h e above tends to indicate that v a n a d i u m can replace m o l y b d e n u m as a catalyst in nitrogen fixation b y a n u m b e r of soil bacteria, t h e r e is no evidence t h a t v a n a d i u m is required i n t h e presence of m o l y b d e n u m . O n t h e other h a n d , it has been shown b y two groups of investigators t h a t although tungstate is a competitive inhibitor of m o l y b d a t e in nitrogen fixation a n d nitrate assimilation b y Azotobacter, v a n a d a t e did not compete w i t h tungstate.
T h i s would suggest t h a t v a n a d i u m cannot replace the m o l y b d e n u m requirement.
Additional a r g u m e n t s against a role of v a n a d i u m in nitrogen fixation are the report b y Esposito a n d Wilson ( 6 5 ) t h a t v a n a d i u m cannot re-place m o l y b d e n u m in Azotobacter vinelandii O, a n d t h e review b y Allen ( 6 ) declaring t h a t the m o l y b d e n u m r e q u i r e m e n t of t h e blue-green alga Anabaena cylindrica for nitrogen fixation a n d n i t r a t e re-duction cannot be replaced b y v a n a d i u m . I n view of the controversial evidence it would appear t h a t a role for v a n a d i u m in nitrogen fixation has as yet not been established.
504 A . N A S O N A N D W . D . M C E L R O Y
3. Possible Role of Vanadium in Animais
T h u s far v a n a d i u m has not been established as a n essential element for animals. T h e v a n a d i u m in t h e blood of tunicates is in the form of vanadium-protein complex in special blood cells called vanadocytes.
T h e r e is some question w h e t h e r v a n a d i u m is indeed a p a r t of a n organic compound w i t h i n t h e vanadocyte or bound as t h e inorganic ion to the cell m e m b r a n e or some protein. T h e function of the v a n a d i u m chromogen of t h e blood of tunicates is u n k n o w n . T h e r e is n o clear evidence t h a t the m e t a l chromogen serves as a respiratory pigment, the combination of oxygen w i t h t h e p i g m e n t not being comparable with that of oxygen w i t h hemoglobin. T h e possibility has not been eliminated that t h e p i g m e n t serves in reduction processes, perhaps in the reduction of carbon dioxide for the production of t h e celluloselike substance of the tunic. T h e r e are a n u m b e r of reports i n t h e literature concerning the effects of v a n a d i u m salts on t h e development of cells, on hemo-globin formation a n d other processes, a n d as a n inhibitor of certain e n z y m e s [see review b y N a s o n ( 1 7 5 ) ] . T h e r e seems to be a relation-ship between v a n a d i u m a n d lipid metabolism. Small concentrations of sodium m e t a v a n a d a t e or v a n a d i u m acetate m a r k e d l y increased oxida-tion of phospholipid b y washed r a t or guinea pig liver suspensions ( 2 8 ) . T h e fact t h a t m a n g a n e s e nullified both t h e stimulatory effect of v a n a d i u m on phospholipid oxidation as well as t h e depressant action on cholesterol synthesis suggests a possible link between the metabolism of cholesterol a n d liver phospholipids. T h e r e is also some evidence that v a n a d i u m inhibits cholesterol synthesis a n d accelerates cholesterol breakdown. T h e r e also are indications t h a t v a n a d i u m m a y be function-ing in teeth b y increasfunction-ing the hardness of t h e apatite structure as well as serving as a binding substance between t h e organic a n d inorganic m a t t e r in e n a m e l a n d dentine.
H . C O B A L T , S E L E N I U M , A N D I O D I N E
Although these three elements h a v e been established or implicated as necessary n u t r i e n t s for h i g h e r animals, t h e r e is little or no evidence for their essentiality in p l a n t nutrition.
Cobalt appears to be required b y r u m i n a n t animals a n d certain micro-organisms solely because it is a constituent of v i t a m i n B1 2 ( 4 8 , 1 1 3 , 151 ) . I n r u m i n a n t s t h e m e c h a n i s m of action of cobalt is concerned w i t h the formation of v i t a m i n B1 2 a n d of related v i t a m i n B1 2-like substances b y microorganisms in t h e digestive tract. V i t a m i n B1 2 is required b y most a n i m a l species. It is a p p a r e n t l y synthesized only b y microorganisms a n d is concerned w i t h (a) protein metabolism, (b) the synthesis of m e t h y l
groups i n animals, a n d (c) v e r y probably t h e utilization of other single carbon compounds [see review b y D i n n i n g ( 5 6 ) ] . Barker a n d co-workers ( 2 1 , 22, 284, 285) h a v e isolated a v i t a m i n B1 2 coenzyme from Clostridium tetanomorphum w h i c h serves as a n essential cofactor for the e n z y m a t i c conversion of g l u t a m a t e to ß-methylaspartate. T h e en-z y m e h a s been shown to be present in several bacteria a n d i n rabbit liver (248, 2 8 5 ) . Barker's v i t a m i n B1 2 coenzyme is also essential for the isomerization of succinyl coenzyme A to m e t h y l m a l o n y l coenzyme A (59, 2 4 7 ) . T h e suggestion has been m a d e t h a t t h e coenzyme m a y p l a y a general role i n one-carbon metabolism of a large n u m b e r of bacteria ( 2 4 8 ) .
H i g h e r plants n o r m a l l y h a v e no v i t a m i n Bi 2 i n their tissues. W h a t little v i t a m i n B1 2-like activity has been found i n p l a n t extracts was shown to be due to deoxyribosides or possibly related to t h e occurrence of associated organisms containing v i t a m i n B1 2. T h e deoxyribosides of adenine, of h y p o x a n t h i n e a n d of cytosine, deoxyribonucleic acid, a n d t h y m i d i n e can substitute for v i t a m i n B12 i n t h e nutrition of certain bacteria, p r e s u m a b l y because these compounds are able to provide a n essential metabolic substance w h i c h t h e cells cannot produce in t h e absence of v i t a m i n Bi 2 ( 1 5 1 ) . V i t a m i n B1 2 is required, however, for the nutrition of t h e chlorophyll-containing flagellate Euglena gracilis and cannot be replaced b y t h y m i d i n e . T h e r e are also a n u m b e r of reports on the response to, or t h e occurrence of, v i t a m i n Bi 2 factors in certain algae (78, 2 0 3 ) . A l t h o u g h t h e r e is n o experimental evidence t h a t cobalt is an essential n u t r i e n t for green plants, a n u m b e r of interesting effects of co-balt h a v e been reported. Miller (161) a n d T h i m a n n (264) observed a n e n h a n c e d elongation of etiolated pea stems i n a u x i n a n d sugar solutions.
T h e expansion of slices of etiolated b e a n leaves w a s also favored b y C o+ +
( 1 6 1 ) . T h e possibility has been suggested t h a t cobalt acts specifically on the properties of t h e cell m e m b r a n e or i n some m a n n e r makes m o r e e n e r g y available for growth. Evidence h a s also been presented w h i c h in-dicates t h a t cobalt m a y also be concerned i n the depression of peroxide formation or i n facilitation of peroxide decomposition in p l a n t tissues.
Although added cobalt resulted i n significant increases in d r y weight, p l a n t height, a n d stem girth i n tomato a n d r u b b e r plants supplied w i t h purified n u t r i e n t solutions, t h e lack of cobalt failed to produce visual symptoms of deficiency ( 3 2 ) . Cobalt w a s therefore n o t claimed to be a n essential element for h i g h e r plants. Relatively high concentrations of cobalt (greater t h a n 0.1 m g Co p e r milliliter) i n w a t e r cultures are toxic to plants w h i c h m a y b e offset b y further addition of m o l y b d e n u m
( 1 5 5 ) .
Most recently, however, t h e r e h a v e been a n u m b e r of independent
506 A . N A S O N A N D W . D. M C E L R O Y
findings w h i c h implicate cobalt as a n essential n u t r i e n t in t h e sym-biotic fixation of nitrogen b y leguminous plants. T h e first of these, b y S h a u k a t - A h m e d a n d E v a n s ( 2 2 9 ) , shows t h a t added cobalt produced a m a r k e d increase in the d r y weight of soybean shoots of the Rhizobiumsoybean system as well as prevented nitrogendeficiency s y m p -toms; this was followed, almost simultaneously, b y similar or related reports b y Reisenauer ( 5 3 , 211) a n d H a l l s w o r t h et al. (91) using dif-ferent leguminous plants. T h e cobalt-containing v i t a m i n Bi 2 h a d already been shown to be present in t h e roots of nonleguminous species (46) a n d in the nodules of leguminous plants w i t h its concentration in pink nodules being four times as great as in w h i t e ones ( 1 3 8 ) . I n the former case t h e vitamin was attributed to soil microorganisms a n d in t h e latter case to the nitrogen-fixing bacteria in the nodules. Of special interest is the early report b y H o l m - H a n s e n et al. (107) w h i c h demonstrated that certain blue-green algae a r e dependent u p o n either cobalt or v i t a m i n B1 2 for n o r m a l growth a n d t h a t those species t h a t fix nitrogen display a greater response t h a n those w h i c h r e q u i r e a source of fixed nitrogen.
F u r t h e r experiments h a v e indicated t h a t cobalt is a n essential ele-m e n t for the growth of soybean plants u n d e r syele-mbiotic conditions, but no response could be demonstrated in experiments w h e r e adequate fixed nitrogen w a s supplied ( 2 3 0 ) . A m o r e recent report indicates t h a t co-balt is also a n i m p o r t a n t growth factor for t h e bacterium Rhizobium japonicum in p u r e culture, a symbiotic p a r t n e r in leguminous nitrogen fixation ( 1 4 2 ) . It therefore appears t h a t cobalt h a s a n essential role in the bacteria regardless of w h e t h e r it is g r o w n w i t h or without the le-guminous plant. It seems quite possible t h a t t h e r e q u i r e m e n t of sym-biotically grown legumes for cobalt can be accounted for simply in terms of the r e q u i r e m e n t of t h e bacteria for t h e metal. I t m a y b e t h a t cobalt functions as p a r t of the v i t a m i n Bi 2 molecule which in t u r n m a y also be m o r e directly involved in nitrogen fixation.
At the e n z y m a t i c level it has been found t h a t the inhibition of in-corporation of acetate into fatty acids in homogenates of Saccharomyces cerevisiae b y ethylenediaminetetracetic acid was completely a n d spe-cifically removed b y C o+ + ( 1 2 7 ) . T h e glycylglycine dipeptidase from bakers' yeast has been reported to be specifically activated b y C o+ +
( 1 9 1 ) , while carboxypeptidase A experiences a 1 0 0 % increase in pep-tidase activity w i t h no a p p a r e n t increase in esterase activity b y specific incubation w i t h cobaltous ions ( 7 1 ) .
Selenium has recently been implicated as a n essential element in a n i m a l nutrition. T h e r e h a v e been a n u m b e r of i n d e p e n d e n t reports t h a t selenium i n v e r y low concentrations is effective in t h e prevention of liver necrosis in rats a n d of exudative diathesis in chicks w h e n t h e y
are m a i n t a i n e d on a special diet containing Torula yeast ( 2 7 4 ) . T h e t e r m "factor 3 " or "active s e l e n i u m " has been used to designate the biologically active selenium-containing component (s) in n u t r i e n t s and other biological m a t e r i a l ( 2 2 7 ) . It w a s originally observed as a n u n -identified substance t h a t prevented necrotic liver degeneration of rats fed a diet containing protein supplied b y Torula yeast ( 2 2 6 ) . T h e u n -classified complexity of t h e biological relationship between factor 3 a n d v i t a m i n Ε in preventing or alleviating exudative diathesis in the chick, m u s c u l a r dystrophy in laboratory animals, dialuric acid-induced hemolysis, resorption gestation i n rats, a n d depigmentation of r a t in-cisor teeth has recently been s u m m a r i z e d b y Vasington et al. ( 2 7 3 ) .
I n higher plants it has been demonstrated t h a t t h e growth of cer-tain species is stimulated b y selenium (cf. Chapter 2 ) . These particular plants, w h i c h are classified in t h e genera Stanleya, Oonopsis ( = Hap-lopappus), Xylorrhiza (= Machaeranthera), a n d Astragalus, appar-e n t l y grow on soils w h i c h contain sappar-elappar-enium. Thappar-esappar-e plants h a v appar-e sappar-ervappar-ed as valuable indicators of seleniferous soils a n d seleniferous soil areas ( 1 6 5 , 2 6 6 ) , a n d selenium has been referred to as a n essential element for these "indicator" plants. T h e element occurs in plants in concentra-tions as high as 3 5 0 0 p p m in organic a n d inorganic forms, the latter being present for the most p a r t as selenate. A n u m b e r of species h a v e been reported to h a v e t h e selenium present only in the organic form.
A m i n o acids of seleniferous w h e a t protein hydrolyzates showed m u c h of its selenium b y p a p e r chromatographic analysis to be in t h e same areas as m e t h i o n i n e a n d cystine. T h e toxicity of selenium in t h e selenium analog of cystine is comparable to the toxicity of selenium in n a t u r a l l y seleniferous grains a n d in sodium selenite. According to a recent report ( 1 3 5 ) selenate interferes competitively w i t h the absorption of sulfate b y plants. Shrift ( 2 3 4 ) h a s recently reviewed the chemical properties of selenium a n d the biological activities of selenium compounds w i t h par-ticular emphasis on its effects a n d possible roles in plants. H e has in-dicated t h a t sulfate is the one substance w h i c h has been consistently found to counteract selenate toxicity in microorganisms a n d higher plants. T h e antagonism is competitive a n d seems to be best explained on t h e basis of t h e structural similarity of t h e two ions. T h e ratio dependence of metabolite to antimetabolite has been reported in a n u m -ber of growth studies w i t h Chlorella vulgaris ( 2 3 2 ) , Saccharomyces cerevisiae ( 7 0 ) , Aspergillus niger ( 2 8 8 ) , a n d Desulfovibrio desul-furicans ( 2 0 6 ) . T h e assumption is often m a d e t h a t unlike most anti-metabolites selenate can be converted b y plants into organic forms by the same enzymes t h a t convert sulfate. T h e s e organic selenium com-pounds in t u r n are believed to be competitive. T h e recent work w i t h
508 A . N A S O N A N D W . D . M C E L R O Y
the sulfate " a c t i v a t i n g " system ( A T P -f sulfate -> A M P — sulfate + pyrophosphate) indicates t h a t t h e e n z y m e designated as ATP-sulfury-lase p r e p a r e d from a n i m a l a n d Neurospora tissues is inhibited b y sele-n a t e a sele-n d sele-not reversed b y sulfate ( 1 0 2 ) . A similar system from yeast yielded only trace a m o u n t s of t h e corresponding A M P s e l e n a t e i m p l y -ing a hydrolysis of t h e adenosine phosphoselenate, b u t no experiments w e r e reported to demonstrate a n actual competition between sulfate a n d selenate ( 2 9 3 ) .
A t t h e organic selenium level, experiments w i t h a n i m a l e n z y m e s h a v e shown t h a t t h e selenium analog of m e t h i o n i n e is converted to
"active selenomethionine" (Se-adenosylselenomethionine) at a r a t e w h i c h is similar to t h a t at w h i c h "active m e t h i o n i n e " is formed u n d e r identical conditions ( 1 6 7 ) . T h e Se-adenosylselenomethionine can i n t u r n serve as a m e t h y l donor for t h e biosynthesis of creatine (167) a n d choline (35) i n e n z y m a t i c t r a n s m e t h y l a t i o n reactions using cell-free liver preparations. T h e m a n n e r in w h i c h selenium is built into protein molecules a n d to w h a t extent sulfur can be replaced b y selenium with-out i m p a i r m e n t of protein function a r e v e r y m u c h i n need of clarification.
T h e w o r k of P i n s e n t (202) represents t h e o n l y report of a specific e n z y m e r e q u i r e m e n t for selenite. She observed a specific need for sel-enite ( a n d molybdate) in t h e formation of formic dehydrogenase b y m e m b e r s of t h e coli-aerogenes group of bacteria. I n t h e absence of these ions from t h e n u t r i e n t m e d i u m no e n z y m e activity could be de-tected even though growth was n o r m a l .
Iodine, w h i c h is a n essential element for a n i m a l s a n d functions as a component of the t h y r o x i n e molecule, has thus far not been shown to be a necessary n u t r i e n t for plants.