Cloning of cDNA Sequences Encoding the Calcium-Binding Protein, Calmodulin, from Barley (Hordeum vulgare L.)

Full- and partial-length cDNAs encoding calmodulin mRNA have been cloned and sequenced from barley (Hordeum vulgare L.). Barley leaf mRNA, size-fractionated in sucrose density gradients, was used to synthesize double-stranded cDNA. The cDNA was cloned in λgt10 and screened with a synthetic, 14-nucleotide oligonucleotide probe, which was designed using the predicted coding sequences of the carboxy termini of spinach and wheat calmodulin proteins. The primary structure of barley calmodulin, predicted from DNA sequencing experiments, consists of 148 amino acids and differs from that of wheat calmodulin in only three positions. In two of the three positions, the amino acid changes are conservative, while the third change consists of an apparent deletion/insertion. The overall nucleotide sequence similarity between the amino acid coding regions of barley and vertebrate calmodulin mRNAs is approximately 77%. However, a region encoding 11 amino acids of the second Ca²⁺-binding domain is very highly conserved at the nucleotide level compared with the rest of the coding sequences (94% sequence identity between barley and chicken calmodulin mRNAs). Genomic Southern blots reveal that barley calmodulin is encoded by a single copy gene. This gene is expressed as a single size class of mRNA in all tissues of 7-day-old barley seedlings. In addition, these analyses indicate that a barley calmodulin cDNA coding region subclone is suitable as a probe for isolating calmodulin genes from other plants.     

Sidedness studies of thylakoid phosphatidylglycerol in higher plants.

The transmembrane distribution of phosphatidylglycerol was determined in thylakoids from barley (Hordeum vulgare), lettuce (Lactuca sativa) and pea (Pisum sativum) chloroplasts. Phospholipase A2 and phospholipase D digestion and chemical-labelling methods were used. Phosphatidylglycerol was preferentially localized in the outer (stromal) leaflet. The proportion of the phospholipid in this leaflet ranged from about 66% in pea to about 75% for barley and lettuce thylakoids. One of the main fatty acids, trans-delta 3-hexadecenoic acid, was exclusively located in the outer leaflet in all three plant types. The data are discussed in relation to suggested roles for phosphatidylglycerol in thylakoid function.     

Nonhost resistance of barley is successfully manifested against Magnaporthe grisea and a closely related Pennisetum-infecting lineage but is overcome by Magnaporthe oryzae

Magnaporthe oryzae is a major pathogen of rice (Oryza sativa L.) but is also able to infect other grasses, including barley (Hordeum vulgare L.). Here, we report a study using Magnaporthe isolates collected from other host plant species to evaluate their capacity to infect barley. A nonhost type of resistance was detected in barley against isolates derived from genera Pennisetum (fontaingrass) or Digitaria (crabgrass), but no resistance occurred in response to isolates from rice, genus Eleusine (goosegrass), wheat (Triticum aestivum L.), or maize (Zea mays L.), respectively. Restriction of pathogen growth in the nonhost interaction was investigated microscopically and compared with compatible interactions. Real-time polymerase chain reaction was used to quantify fungal biomass in both types of interaction. The phylogenetic relationship among the Magnaporthe isolates used in this study was investigated by inferring gene trees for fragments of three genes, actin, calmodulin, and beta-tubulin. Based on phylogenetic analysis, we could distinguish different species that were strictly correlated with the ability of the isolates to infect barley. We demonstrated that investigating specific host interaction phenotypes for a range of pathogen isolates can accurately highlight genetic diversity within a pathogen population.     

Calmodulin isoform-specific activation of a rice calmodulin-binding kinase conferred by only three amino-acids of OsCaM61

The kinase activity of a Ca(2+)/calmodulin (CaM)-binding serine/threonine protein kinase from rice (Oryza sativa) (OsCBK) has been reported to be unaffected by OsCaM1 binding. In this study, we examined whether other rice CaMs can stimulate OsCBK. It was observed that OsCaM61 stimulated OsCBK in a Ca(2+)-dependent manner. In addition, Ala(111), Gly(123) and Ser(127) were identified as critical residues for OsCBK activation. Mutational study and fluorescent spectroscopy analysis indicated that CaM-binding affinity does not correlate with the kinase activity and that these key amino-acids in OsCaM61 play a vital role in suitable changes of OsCBK conformation for kinase activation.     

Distribution of Pyruvate Dehydrogenase Complex Activities between Chloroplasts and Mitochondria from Leaves of Different Species

Protoplasts from barley (Hordeum vulgare), pea (Pisum sativum), wheat (Triticum aestivum), and spinach (Spinacia oleracea) leaves were fractionated into chloroplast- and mitochondrion-enriched fractions. Pyruvate dehydrogenase complex capacities in mitochondria (mtPDC) and chloroplasts (cpPDC) were measured in appropriate fractions under conditions optimal for each isozyme. The total cellular capacity of PDC was similar in barley and pea but about 50% lower in wheat and spinach. In pea a distribution of 87% mtPDC and 13% cpPDC was found on a cellular basis. In barley, wheat, and spinach the subcellular distribution was the opposite, with about 15% mtPDC and 85% cpPDC. cpPDC activity was constant at about 0.1 nmol cell-1 h-1 in cells from different regions along the developing barley leaf and showed no correlation with developmental patterns of photosynthetic parameters, such as increasing Chl and NADP-glyceraldehyde-3-phosphate dehydrogenase activity. Similarly, the capacity of the mitochondrial isoform did not change during barley leaf development and had a developmental pattern similar to that of citrate synthase and fumarase. Differences in subcellular distribution of PDCs in barley and pea are proposed to be due to differences in regulation, not to changes in isozyme proportions during leaf development or to species-specific differences in phosphorylation state of mtPDC after organelle separation.     

Calmodulin mRNA in Barley (Hordeum vulgare L.) : Apparent Regulation by Cell Proliferation and Light

Calmodulin is encoded by a 650-nucleotide mRNA in higher plants. This messenger was identified in barley and pea by a combination of in vitro translation and blot hybridization experiments using anti-sense RNA produced from an eel calmodulin cDNA probe. In all plant tissues tested, calmodulin mRNA represents between 0.01 and 0.1% of the total translatable mRNA population. Calmodulin mRNA levels are three- to fourfold higher in the meristematic zone of the first leaf of barley. At all other stages of leaf cell differentiation, calmodulin mRNA levels are nearly identical. During light-induced development in barley leaves, the relative proportion of translatable calmodulin mRNA declines about twofold. Cytoplasmic mRNAs that may encode calmodulin-like proteins were also detected. The levels of several of these putative Ca²⁺-binding protein mRNAs are modulated during the course of light-induced barley leaf cell development.     

Occurrence of acid and neutral carboxypeptidases in germinating cereals

High neutral metallocarboxypeptidase activity (EC 3.4.17) has earlier been detected in young seedlings of rice ( Oryza sativa L.) using benzyloxycarbonyl-L-phenylalanyl-L-alanine (Z-Phe-Ala) as substrate at pH 7. This finding was confirmed, and it was observed that the activity could be assayed with higher specificity and sensitivity by using Z-Gly-Ala or Z-Gly-Phe as substrate at pH 6.5–7. No corresponding activity was detected in seedlings of barley ( Hordeum vulgare L. cv. Himalaya), oats ( Avena sativa L.) or maize ( Zea mays L.). The seedlings of the four cereals possessed similar activities of acid carboxypeptidases (EC 3.4.16; hydrolysis of Z-Phe-Ala and Z-Ala-Phe at pH 5.2 and of Z-Ala-Arg at pH 5.7). However, in endosperms of germinating rice and maize these activities were only about 1–5% of those in barley and oats. A corresponding, although less pronounced, difference was evident between the scutella of the two pairs of cereals. The possible relationship between neutral carboxypeptidase activity and ability to grow in anaerobic conditions is discussed.     

The structural organization of the ascidian, Halocynthia roretzi, calmodulin genes. The vicissitude of introns during the evolution of calmodulin genes

Two distinct calmodulin (CaM) genes are isolated from the ascidian, Halocynthia roretzi, (Hr-CaM A and Hr-CaM B) and those structures are determined. There are three nucleotide substitutions, producing two amino acid differences between Hr-CaM A and Hr-CaM B, and those are corresponding to two of the known eight variable residues among metazoan CaMs. Both Hr-CaM A and Hr-CaM B are constructed from six exons and five introns, and the positions of introns are identical. The positions of introns of Hr-CaMs are also identical with those of vertebrate CaMs, except third introns. The third introns of Hr-CaMs are inserted at 28bp upstream when compared with vertebrate CaMs. Thus, sliding of the third intron might have occurred in only the ascidian lineage prior to the gene duplication that also occurred only in that lineage. In addition, with the comparison of the intron positions, we attempt to investigate the vicissitude of introns during the evolution of metazoan CaMs.     

Temporal and spatial distribution of roots and competition for nitrogen in pea-barley intercrops – a field study employing 32P technique

Root system dynamics, productivity and N use were studied in inter- and sole crops of field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.) on a temperate sandy loam. A ³²P tracer placed at a depth of 12.5, 37.5, 62.5 or 87.5 cm was employed to determine root system dynamics by sampling crop leaves at 0, 15, 30 and 45 cm lateral distance. ¹⁵N addition was used to estimate N₂ fixation by pea, using sole cropped barley as reference crop. The Land Equivalent Ratio (LER), which is defined as the relative land area under sole crops that is required to produce the yields achieved in intercropping, were used to compare the crop growth in intercrops relative to the respective sole crops.The ³²P appearance in leaves revealed that the barley root system grows faster than that of pea. P uptake by the barley root system during early growth stages was approximately 10 days ahead of that of the pea root system in root depth and lateral root distribution. More than 90% of the P uptake by the pea root system was confined to the top 12.5 cm of soil, whereas barley had about 25–30% of tracer P uptake in the 12.5 – 62.5 cm soil layer. Judging from this P uptake, intercropping caused the barley root system to grow deeper and faster lateral root development of both species was observed. Barley accumulated similar amounts of aboveground N when grown as inter- and sole crop, whereas the total aboveground N acquired by pea in the intercrop was only 16% of that acquired in the pea sole crop. The percentage of total aboveground N derived from N₂ fixation in sole cropped pea increased from 40% to 80% during the growth period, whereas it was almost constant at 85% in intercropped pea. The total amounts of N₂ fixed were 95 and 15 kg N ha^–1 in sole cropped and intercropped pea, respectively. Barley was the dominant component of the pea-barley intercrop, obtaining 90% of its sole crop yield, while pea produced only 15% of the grains of a sole crop pea. Intercropping of pea and barley improved the utilization of plant growth resources (LER > 1) as compared to sole crops. Root system distribution in time and space can partly explain interspecific competition. The ³²P methodology proved to be a valuable tool for determining root dynamics in intercropping systems.     

Dwarf plants of diploid <Emphasis Type="Italic">Medicago sativa</Emphasis> carry a mutation in the gibberellin 3-β-hydroxylase gene

In this paper we describe the identification of a gene, MsDWF1 coding for a putative gibberellin 3-beta-hydroxylase (GA3ox), whose natural mutation is conditioning a dwarf growth phenotype in Medicago sativa. The dwarf phenotype could not be complemented with grafting, which indicates that the bioactive gibberellin compound necessary for shoot elongation is immobile. On the contrary, exogenously added gibberellic acid restored normal growth. The genetic position of the Msdwf1 gene was mapped to linkage group 2 (LG2) and the physical location was delimited by map-based cloning using Medicago truncatula genomic resources. Based on the similar appearance and behavior of the dwarf Medicago sativa plants to the pea stem length mutant (le) as well as the synthenic map position of the two genes it was postulated that MsDWF1 and pea Le are orthologs. The comparison of wild type and mutant allele sequences of MsGA3ox revealed an amino acid change in a conserved position in the mutant allele, which most probably impaired the function of the enzyme. Our results indicate that the dwarf phenotype was the consequence of this mutation.     

Compared cycling in a soil-plant system of pea and barley residue nitrogen

Field experiments were carried out on a temperate soil to determine the decline rate, the stabilization in soil organic matter and the plant uptake of N from ¹⁵N-labelled crop residues. The fate of N from field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.) residues was followed in unplanted and planted plots and related to their chemical composition. In the top 10 cm of unplanted plots, inorganic N was immobilized after barley residue incorporation, whereas the inorganic N pool was increased during the initial 30 days after incorporation (DAI) of pea residues. Initial net mineralization of N was highly correlated to the concentrations of soluble C and N and the lignin: N ratio of residues. The contribution of residue-derived N to the inorganic N pool was at its maximum 30 DAI (10–55%) and declined to on average 5% after 3 years of decomposition.Residual organic labelled N in the top 10 cm soil declined rapidly during the initial 86 DAI for all residue types. Leaching of soluble organic materials may have contributed to this decline. At 216 DAI 72, 59 and 45% of the barley, mature pea and green pea residue N, respectively, were present in organic N-forms in the topsoil. During the 1–3 year period, residual organic labelled N from different residues declined at similar rates, mean decay constant: 0.18 yr^-1. After 3 years, 45% of the barley and on average 32% of the pea residue N were present as soil organic N. The proportion of residue N remaining in the soil after 3 years of decomposition was most strongly correlated with the total and soluble N concentrations in the residue. The ratio (% inorganic N derived from residues): (% organic N derived from residues) was used as a measure of the rate residue N stabilization. From initial values of 3–7 the ratios declined to on average 1.9 and 1.6 after 2 and 3 yrs, respectively, indicating that a major part of the residue N was stabilized after 2 years of decomposition. Even though the largest proportion of residue N stabilized after 3 years was found for barley, the largest amount of residue N stabilized was found with incorporation of pea residues, since much more N was incorporated with these residues.In planted plots and after one year of decomposition, 7% of the pea and 5% of the barley residue N were recovered in perennial ryegrass (Lolium perenne L.) shoots. After 2 years the cumulative recovery of residue N in ryegrass shoots and roots was 14% for pea and 15% for barley residue N. The total uptake of non-labelled soil N after 2 years of growth was similar in the two residue treatments, but the amount of soil N taken up in each growth period varied between the treatments, apparently because the soil N immobilized during initial decomposition of residues was remineralized later in the barley than in the pea residue treatment. Balances were established for the amounts of barley and mature pea residue N remaining in the 0–10 cm soil layer and taken up in ryegrass after 2 years of decomposition. About 24% of the barley and 35% of the pea residue N were unaccounted for. Since these apparent losses are comparable to almost twice the amounts of pea and barley residue N taken up by the perennial ryegrass crop, there seems to be a potential for improved crop residue management in order to conserve nutrients in the soil-plant system.     

Isolation and identification of an allelopathic substance in Pisum sativum

The residue of peas (Pisum sativum L.) has allelopathic activity and the putative compound causing this inhibitory effect was isolated from a methanol extract of pea shoots. Chemical structure of this compound was determined by high-resolution MS, IR and 1H NMR spectral data as pisatin. Pisatin inhibited growth of cress (Lepidium sativum L.) and lettuce (Lactuca sativa L.) seedlings at concentrations greater than 10 and 30 microM, respectively. The doses required for 50% growth inhibition of roots and hypocotyls of cress were 61 and 91 microM, respectively, and those of lettuce were 78 and 115 microM, respectively. The concentration of pisatin in the pea shoots was 32.7 nmol x g(-1) fresh weight. The effectiveness of pisatin on growth inhibition in cress and lettuce, and its occurrence in pea shoots suggest that it may contribute to the growth inhibitory effect of pea residue, and may play an important role in pea allelopathy.     

Grain yield, symbiotic N2 fixation and interspecific competition for inorganic N in pea-barley intercrops

The effect of mixed intercropping of field pea (Pisum sativum L.) and spring barley (Hordeum vulgare L.), compared to monocrop cultivation, on the yield and crop-N dynamics was studied in a 4-yr field experiment using ¹⁵N-isotope dilution technique. Crops were grown with or without the supply of 5 g ¹⁵N-labeled N m^-2. The effect of intercropping on the dry matter and N yields, competition for inorganic N among the intercrop components, symbiotic fixation in pea and N transfer from pea to barley were determined. As an average of four years the grain yields were similar in monocropped pea, monocropped and fertilized barley and the intercrop without N fertilizer supply. Nitrogen fertilization did not influence the intercrop yield, but decreased the proportion of pea in the yield. Relative yield totals (RYT) showed that the environmental sources for plant growth were used from 12 to 31% more efficiently by the intercrop than by the monocrops, and N fertilization decreased RYT-values. Intercrop yields were less stable than monocrop barley yields, but more stable than the yield of monocropped pea. Barley competed strongly for soil and fertilizer N in the intercrop, and was up to 30 times more competitive than pea for inorganic N. Consequently, barley obtained a more than proportionate share of the inorganic N in the intercrop. At maturity the total recovery of fertilizer N was not significantly different between crops, averaging 65% of the supplied N. The fertilizer N recovered in pea constituted only 9% of total fertilizer-N recovery in the intercrop. The amount of symbiotic N₂ fixation in the intercrop was less than expected from its composition and the fixation in monocrop. This indicates that the competition from barley had a negative effect on the fixation, perhaps via shading. At maturity, the average amount of N₂ fixation was 17.7 g N m^-2 in the monocrop and 5.1 g N m^-2 in the intercropped pea. A higher proportion of total N in pea was derived from N₂ fixation in the intercrop than in the monocrop, on average 82% and 62%, respectively. The ¹⁵N enrichment of intercropped barley tended to be slightly lower than of monocropped barley, although not significantly. Consequently, there was no evidence for pea N being transferred to barley. The intercropping advantage in the pea-barley intercrop is mainly due to the complimentary use of soil inorganic and atmospheric N sources by the intercrop components, resulting in reduced competition for inorganic N, rather than a facilitative effect, in which symbiotically fixed N₂ is made available to barley.Abbreviations MC monocrop - IC intercrop - PMC pea monocrop - BMC barley monocrop - PIC pea in intercrop - BIC barley in intercrop     

Granary trial of protein-enriched pea flour for the control of three stored-product insects in barley

A granary trial was conducted to evaluate the efficacy of protein-enriched pea flour against three common stored-grain insects, Sitophilus oryzae (L.), Tribolium castaneum (Herbst), and Cryptolestes ferrugineus (Stephens). Six 30-t farm granaries were filled with approximately 11 t of barley. The barley was either not treated, treated with protein-enriched pea flour at 0.1% throughout the entire grain mass, or treated at 0.5% throughout the top half of the grain mass. Adult insects were released in screened boxes (two insects per kilogram barley for S. oryzae and T. castaneum 1.4 insects per kilogram barley for C. ferrugineus). Barley was sampled four times during the 70-d trial. The number and mortality of adults and emerged adults in the samples were noted. Four kinds of traps, flight, surface-pitfall, probe-pitfall, and sticky-bar, were placed at different locations in the granaries to estimate the movement of insects. The 0.1% protein-enriched pea flour treatment reduced adult numbers of S. oryzae by 93%, T. castaneum by 66%, and C. ferrugineus by 58%, and reduced the emerged adults by 87, 77, and 77%, respectively. Treating the top half of the barley with 0.5% protein-enriched pea flour had similar effects as treating the entire grain mass with 0.1% pea-protein flour. However, the top-half treatment failed to prevent insects from penetrating into the untreated lower layer. Differences between traps are discussed.     

In situ starch degradation of different feeds in the rumen

The in situ starch degradation of 5 feeds (barley, maize, pea, oats and wheat bran) has been measured (trial 1), and the influence of particle size on starch degradation investigated with 3 feeds (barley, maize, pea) (trial 2). The starch degradability of barley, oats and wheat bran was found to be higher than that of pea, and higher again than that of maize: 98, 97, 96, 90 and 58% respectively. For barley, oats and wheat bran, starch was degraded more rapidly than the other dry matter (pm) components. Maize and pea starches were degraded at the same rate as non-starchy components. The particle size variations between feeds ground on the same screen may partly explain variations in starch degradability. When the particle size increased from 0.8 to 6.0 mm screen grinding, in situ starch degradability decreased; the decrease was higher for maize (13.8 points) than for barley (7.4 points) or pea (10.4 points).     

Nucleotide sequence of the rice cytochrome f gene and the presence of sequence variation near this gene

The rice (Oryza sativa L. var. Labelle) chloroplast (cp) gene encoding cytochrome f has been isolated and sequenced. The coding region of this rice gene displays 95.1%, 85.3% and 85.2% nucleotide sequence homology with that of wheat, pea and spinach, respectively. To examine the cpDNA sequence variation in rice, cpDNA from Labelle and its parents, Belle Patna and Dawn was compared. Using the cytochrome f gene as the probe for hybridization, we found several differences in the size and number of restriction fragments in the cp genome of three rice varieties. An additional restriction fragment found in the Belle Patna cp suggests that this cp genome is either heterogeneous or contains two copies of cytochrome f gene per cpDNA.     

Biomass production, symbiotic nitrogen fixation and inorganic N use in dual and tri-component annual intercrops

The interspecific complementary and competitive interactions between pea (Pisum sativum L.), barley (Hordeum vulgare L.) and oilseed rape (Brassica napus L.), grown as dual and tri-component intercrops were assessed in a field study in Denmark. Total biomass production and N use at two levels of N fertilisation (0.5 and 4.0 g N/m²), were measured at five harvests throughout a growing season. All intercrops displayed land equivalent ratio values close to or exceeding unity, indicating complementary use of growth resources. Whereas both rape and barley responded positively to increased N fertilisation, irrespective of whether they were grown as sole- or intercrops, pea was strongly suppressed when grown in intercrop. Of the three crops barley was the strongest competitor for both soil and fertiliser N, rape intermediate and pea the weakest. Faster initial growth of barley than pea and rape gave barley an initial competitive advantage, an advantage that in the two dual intercrops was strengthened by the addition of N. Apparently the competitive superiority of barley was less strong in the tri-component intercrop, indicating that the impact of the dominantmay, through improved growth of both rape and pea, have been diminished through indirect facilitation. Interspecific competition had a promoting effect on the percent of nitrogen derived from N₂ fixation of pea, and most so at the low N fertilisation level. Results indicate that the benefits achieved from the association of a legume and nonlegume, in terms of N₂ fixed were greatest when pea was grown in association with rape as opposed to barley which could indicate that the benefits achieved from the association of a legume and nonlegume are partly lost if the nonlegume is too strong a competitor.     

转反义蜡质基因水稻亲本后代性状分析

以单质粒转化的粳稻广粳一号和双质粒共转化的籼稻01早5202所获得的转反义蜡质基因水稻后代为供试材料, 通过潮霉素抗性、PCR检测等手段分析了外源基因的遗传分离特性, 同时, 还分析了转基因材料的直链淀粉含量、waxy蛋白含量的变化特性。结果表明, 无论是采用标记基因(hpt)与目的基因(Anti-sense waxy)连锁的单质粒转化, 还是采用双质粒共转化, 其供试的后代植株材料都发生了外源基因分离现象, 且转基因植株材料的直链淀粉含量都有所下降, 有些单株的直链淀粉含量已降至10%(质量百分比)以下, 远低于对照(其直链淀粉含量为22.04%); SDS-PAGE检测结果显示, 供试的转基因材料的waxy蛋白的含量与对应的直链淀粉含量呈正相关性。     

An enzymatic assay for calmodulins based on plant NAD kinase activity

NAD kinase with increased sensitivity to calmodulin was purified from pea seedlings (Pisum sativum L., Willet Wonder). Assays for calmodulin based on the activities of NAD kinase, bovine brain cyclic nucleotide phosphodiesterase, and human erythrocyte Ca2+-ATPase were compared for their sensitivities to calmodulin and for their abilities to discriminate between calmodulins from different sources. The activities of the three enzymes were determined in the presence of various concentrations of calmodulins from human erythrocyte, bovine brain, sea pansy (Renilla reniformis), mung bean seed (Vigna radiata L. Wilczek), mushroom (Agaricus bisporus), and Tetrahymena pyriformis. The concentrations of calmodulin required for 50% activation of the NAD kinase (K0.5) ranged from 0.520 ng/ml for Tetrahymena to 2.20 ng/ml for bovine brain. The K0.5's ranged from 19.6 ng/ml for bovine brain calmodulin to 73.5 ng/ml for mushroom calmodulin for phosphodiesterase activation. The K0.5's for the activation of Ca2+-ATPase ranged from 36.3 ng/ml for erythrocyte calmodulin to 61.7 ng/ml for mushroom calmodulin. NAD kinase was not stimulated by phosphatidylcholine, phosphatidylserine, cardiolipin, or palmitoleic acid in the absence or presence of Ca2+. Palmitic acid had a slightly stimulatory effect in the presence of Ca2+ (10% of maximum), but no effect in the absence of Ca2+. Palmitoleic acid inhibited the calmodulin-stimulated activity by 50%. Both the NAD kinase assay and radioimmunoassay were able to detect calmodulin in extracts containing low concentrations of calmodulin. Estimates of calmodulin contents of crude homogenates determined by the NAD kinase assay were consistent with amounts obtained by various purification procedures.