Sturgeon Muscle Research Articles

Contamination by organochlorine compounds in the edible tissue of four sturgeon species from the Caspian Sea (Iran)

This study focused on accumulation of organochlorine compounds (OCs), including dichloro-diphenyl-trichloroethanes (DDTs), hexachlorobenzene (HCB), hexachlorocyclohexanes (HCHs), and polychlorinated biphenyls (PCBs) accumulation in the muscle of four sturgeon (Persian sturgeon, Acipenser persicus; Stellate sturgeon, Acipenser stellatus; Ship sturgeon, Acipenser nudiventris and Beluga sturgeon, Huso huso) from the southern Caspian Sea. The DDT group was prominent in all of the sturgeon muscle tested constituting almost half or more of the total organochlorine content. Contaminant concentration generally followed this order: DDTs > PCBs > HCHs > HCB. The OCs concentrations in Beluga sturgeon ( H. huso) were the highest and over four times higher than in the next highest species ( A. nudiventris). From an ecotoxicological point of view, the concentrations of OCs in experimental fishes do not reflect a comparatively clean and pollution-free environment; however, results from this study shown that the inflow of organic pollutants into the Caspian Sea has been reduced when compared with prior studies. Levels of measured OCs in sturgeon were relatively low, but the level of some OCs in some of the specimens tested exceeded the guidelines for food; therefore, the maximum allowable daily consumption rate for sturgeon from this watershed may be limited by DDTs and PCBs content for high risk populations.

Analysis of creatine kinase variation in some members of the family Acipenseridae

Creatine kinase (E.C. 2.7.3.2) was examined in stellate sturgeon Acipenser stellatus Pallas, Russian sturgeon A. gueldenstaedtii Brandt, European sterlet A. ruthenus L., Siberian sterlet A. ruthenus marsiglii Brandt, and great sturgeon (beluga) Huso huso L., using polyacrylamide gel electrophoresis. Two loci for creatine kinase were identified: CK-A* in white skeletal muscle and CK-C* in stomach wall muscle. Most species proved to be monomorphic at the CK-A* locus, showing the same phenotype represented by a single band. Heterogeneity and polymorphism in creatine kinase, determined by the CK-A* locus, were found only in Russian sturgeon. Based on the results of densitometric analysis of band staining intensity, we have advanced a hypothesis that synthesis of subunits of the CK-A* product in this species was controlled by eight genes. However, the genotype frequencies in the sample were significantly different from those theoretically expected upon free and independent gene recombination. The results of this study support the hypothesis on the absence of heterodimeric creatine kinase molecules in the skeletal muscle of Russian sturgeon. Locus CK-C* in sterlet was revealed as a single, intensely stained, rapidly migrating fraction, whereas in Russian sturgeon, the enzyme activity in this zone was very weak. No creatine kinase was found in liver, kidneys, spleen, heart, and intestine mucous tunic.

Mercury Concentrations in Gonad, Liver, and Muscle of White Sturgeon Acipenser transmontanus in the Lower Columbia River

This study determined the partitioning of total mercury in liver, gonad, and cheek muscle of white sturgeon (Acipenser transmonatus) in the lower Columbia River. The relationship between tissue mercury concentrations and various physiologic parameters was assessed. White sturgeon were captured in commercial fisheries in the estuary and Bonneville, The Dalles, and John Day Reservoirs. Condition factor (CF), relative weight (Wr), and gonadosomatic index (GSI) were determined for each fish (n = 57). Gonadal tissue was examined histologically to determine sex and stage of maturity. Liver (n = 49), gonad (n = 49), and cheek muscle (n = 57) were analyzed for total mercury using cold-vapor atomic fluorescence spectrophotometry. Tissue protein concentrations were measured by ultraviolet-visible spectroscopy. Plasma was analyzed for testosterone (T), 11-ketotestosterone (KT), and 17ss-estradiol (E2) using radioimmunoassay. Mean tissue mercury concentrations were higher in muscle compared with liver and gonad at all sampling locations, except Bonneville Reservoir where mean liver mercury content was the highest tissue concentration observed in the study. Significant negative correlations between plasma androgens (T and KT) and muscle mercury content and plasma E2 and liver mercury content were found. A significant positive linear relationship between white sturgeon age and liver mercury concentrations was evident. Significant negative correlations between CF and relative weight and gonad and liver mercury content were found. In addition, immature male sturgeon with increased gonad mercury content had decreased GSIs. These results suggest that mercury, in the form of methylmercury, may have an effect on the reproductive potential of white sturgeon.

Residue Analysis for Halofuginone in Sturgeon Muscle by Immunoaffinity Cleanup and Liquid Chromatography

A high-performance liquid chromatography (LC) method was developed for the determination of halofuginone (HFG) in sturgeon muscle. The extracted samples were cleaned up by an immunoaffinity chromatography column that was prepared by covalently coupling polyclonal antibodies against HFG to cyanogen bromide (CNBr) activated Sepharose 4B. The eluate was evaporated to dryness, and residues were determined by LC with absorbance detection at 243 nm. Recoveries of HFG from samples fortified at 20-200 microg/kg levels ranged 74.6-81.1%, with coefficients of variation of 0.7-8.6%. The detection limit was estimated to be 10 microg/kg in a 2 g sample.

Bioconcentration and elimination of avermectin B1 in sturgeon

The bioconcentration and elimination of avermectin B1 in sturgeon muscle were investigated with high-performance liquid chromatography. Mean concentrations of 0.2 and 1 ng x ml(-1) in water were maintained for a 22-d exposure period. The concentrations of avermectin B1alpha in muscle tissues reached steady state within 14 to 18 d. The level of avermectin B1 concentrations in the fish muscles was 7.75+/-0.88 and 38.29+/-1.65 ng x g(-1) for the low and high concentrations, respectively, on day 22. The half-life (t1/2) of the concentrations for the two treated groups was 4.95 and 4.33 d for the low and high concentrations, respectively. Greater than 95% of the tissue concentrations were eliminated from the exposed fish after the 14- and 18-d elimination periods. The estimated values of the bioconcentration factor were 42 L/kg for the low-concentration group and 41 L/kg for high-concentration group, and these values were not significantly different (alpha = 0.05). Avermectin B1alpha does not strongly bioconcentrate in individual aquatic organism and would not be expected to biomagnify in the food chain.

Concentrations of trace elements in muscle of sturgeons in the Caspian Sea

Concentrations of 21 trace elements were determined in muscle of beluga ( Huso huso), Persian sturgeon ( Acipenser persicus), Russian sturgeon ( Acipenser gueldenstaedtii), ship sturgeon ( Acipenser nudiventris) and stellate sturgeon ( Acipenser stellatus) collected from coastal regions of Azerbaijan, Iran, Kazakhstan and Turkmenistan, surrounding the Caspian Sea during 2000–2001. Concentrations of Mn, Co, Cu, Mo, Sn, Hg, Pb and Bi in the muscle were apparently different among the five species of sturgeons. Especially, beluga showed the highest concentrations of Hg, Pb and Mn in all the five species. In addition, more than half of the individuals of beluga exceeded the guideline level (0.3 μg/g wet wt.) of Hg for food in UK. However, V, Cr, Zn, Ga, Rb, Sr and Ba concentrations were similar among five sturgeons. Growth-dependent increase in Hg in beluga, Cu in Persian sturgeon, and Zn in Russian sturgeon were observed, whereas V, Mn, Co, Cu, Ga, Sr, Mo, Sn, Hg and Bi concentrations in Persian sturgeon, Pb in ship sturgeon, and Cr, Mn, and Rb in stellate sturgeon were negatively correlated with body length. All species of sturgeon in Azerbaijan showed the highest concentration of Sr and the lowest concentration of Rb, while the five sturgeons from Iran showed opposite trends. Concentration of V, which is present in oil, in sturgeons in the Caspian Sea was considerably lower than that of fish from Kuwait, but was comparable to that of Cambodia and the Gulf and Gulf of Oman. To our knowledge, this study provides the first extensive data on multielemental accumulation in sturgeons of the Caspian Sea.

Mass Spectrometry as a Novel Approach to Probe Cooperativity in Multimeric Enzymatic Systems

Investigating cooperativity in multimeric enzymes is of utmost interest to improve our understanding of the mechanism of enzymatic regulation. In the present article, we propose a novel approach based on mass spectrometry to probe cooperativity in the binding of a ligand to a multisubunit enzyme. This approach presents the selective advantage of giving a direct insight into all the subsequent ligation states that are formed in solution as the ligand is added to the enzyme. A quantitative interpretation of the electrospray ionization (ESI) mass spectra gives the relative abundance of all the distinct enzymatic species, which allows one to directly deduce the cooperativity of the system. The overall method is described for the addition of the oxidized cofactor nicotinamide adenine dinucleotide (NAD +) to a dimeric mutant of Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase (GPDH). It is then applied to four tetrameric enzymes: sturgeon muscle GPDH, wild type and S48G mutant of GPDH from B. stearothermophilus, and alcohol dehydrogenase (ADH) from Bakers yeast. The results illustrate the possibilities offered by this new technique. First, mass spectrometry allows a control of the enzymes before the addition of NAD +. Second, the cooperative behavior can be drawn from one single ESI mass spectrum, which makes the method highly attractive in terms of the amount of biological material required. Above all, the major benefit lies in the direct visualization of all the enzymatic species that are in equilibrium in solution. The direct measurement of cooperativity readily resolve the inconvenience of the classical approaches employed in this field, which all need to model the experimental data in order to get the cooperative behavior of the system.

Concentrations of heavy and trace metals in muscle and blood of juvenile gulf sturgeon (Acipenser oxyrinchus desotoi) from the suwannee river, Florida

Concentrations of arsenic, chromium, copper, iron, mercury, lead, selenium, and zinc in muscle of juvenile (0.41 to 1.1 kg) Gulf sturgeon, Acipenser oxyrinchus desotoi, from the Suwannee River, Florida, were 13.78, 1.50, 0.98, 25.28, 0.18, 0.49, 6.43, and 17.03 μg/g dry weight, respectively. Concentrations of As (r = 0.51), B (r = 0.68), Ba (r = 0.72), Be (r = 0.73), Cd (r = 0.70), Cu (r = 0.56), Mg (r = 0.50), and Ni (r = 0.65) increased in muscle tissue with an increase in length of juvenile Gulf sturgeon. Mean concentrations of As, Cr, and Mg (10.99, 1.38, and 1122 μg/g, respectively) were higher in the muscle tissue than in blood (1.76, 0.96, and 677 μg/g, respectively). However, blood samples contained significantly high concentrations of Fe, Mn, Ni, Pb, and Zn (1777, 4.16, 2.68, 2.37 and 35.3 μg/g, respectively). Juvenile Gulf sturgeon are accumulating heavy and trace metals while in the Suwannee River before migrating to marine waters of the Gulf of Mexico. In addition to identifying contaminants of concern, these data represent background conditions and may have future value in assessing the impact on sturgeon of spills or operational practices affecting water quality in the Suwannee River.

Characterization of acetylcholinesterase and its molecular forms in organs of five freshwater teleosts

The activity and molecular forms of acetylcholinesterase (AChE, EC 3.1.1.7) were characterized in the brain, heart, white skeletal muscle and liver of the Siberian sturgeon (Acipenser baeri), European eel (Anguilla anguilla), wels (Silurus glanis), common carp (Cyprinus carpio) and silver carp (Hypophthalmichthys molitrix), belonging in four families. The brain had the highest (between 183.2±10.6 and 1361.2±189.7 mU mg-1 protein and between 4.7±0.4 and 23.5±2.7 U g-1 wet tissue) and the liver the lowest (between 25.3±1.9 and 126.5±22.7 mU mg-1 protein and between 1.0±0.1 and 4.5±0.5 U g-1 wet tissue) activity in all these fish, with the exception of the wels, where the heart contained the smallest amount of AChE (26.2±7.5 mU mg-1 protein and 1.2±0.3 U g-1 wet tissue). The highest tissue AChE activity was found in the carp and lowest in the sturgeon. The solubility properties and molecular forms of AChE in the four tissues were studied by extraction in high-salt medium (1.0 M NaCl) with and without the detergent Triton X-100 (0.5%, v/v). The proportions of detergent-soluble (DS) and salt-soluble (SS) forms of AChE varied considerably from one species to another, but a general tendency could be observed: the proportion of DS AChE was generally higher in the brain (between 70.4±4.1 and 82.5±1.2%) and lower in the heart (between 12.0±2.0 and 51.3±2.3%) and skeletal muscle (between 16.0±2.0 and 64.4±0.7%). Velocity sedimentation centrifugation revealed that most tissues contained the G4 or A4 form. Three tissues (brain, heart, and liver) of the Siberian sturgeon contained a relatively high proportion of the G1 form (between 24.3±2.4 and 28.6±4.9%), while the skeletal muscle of the wels contained only the most complex type of AChE, i.e., A12. Amongst the tissues studied, the brain and liver mostly contained amphiphilic globular forms of the G4 type, while the heart and skeletal muscle were rich in asymmetric forms (A4 and A12). It was concluded that the differences revealed in the activities of AChE and in the distribution of its molecular forms may be connected with the variation in behavioral habits (feeding and swimming patterns), and the developmental stage of the nervous system.

Differentiation of Cultured and Wild Sturgeon (Acipenser oxyrinchus desotoi) Based on Fatty Acid Composition

ABSTRACTSturgeon species have attracted interest for aquaculture due to high value of the flesh, caviar and wild stock depletion. Lipid was extracted from sturgeon muscle using the Bligh and Dyer procedure. Fatty acids from total lipid were methylated using boron‐trifluoride in methanol. Fatty acid methyl esters were analyzed by gas chromatography as weight %. The fatty acid profiles of lipids were different between cultured and wild sturgeon. Wild sturgeon had higher levels of 16:0, 16:1ω7, 18:1ω9, 22: 4ω6 and 22:5ω6. Cultured fish had higher levels of 18:2ω6, C20 and C22 monoenes, 20:5ω3 and 22:6ω3. Stepwise discriminant analysis (SDA) was used to develop a mathematical model to distinguish the two populations; the levels of 16:2ω6, 22:5ω6 and phytanic acid accurately identified the two fish populations.

Amino acid composition of whole body, egg and selected tissues of white sturgeon ( Acipenser transmontanus)

The amino acid composition of whole bodies, muscle, gill, gastrointestinal tract, liver, eggs, vitellogenin and lipovitellin of hatchery-raised white sturgeon was determined. The essential amino acid ratios ( A E ratios) of the whole body, muscle and eggs were calculated and found to be significantly ( P < 0.05) different, but the whole body amino acid composition of sturgeon of four different sizes (19.5 ± 0.4, 57.8 ± 0.9, 179.5 ± 0.3 and 535.4 ± 9.7 g; mean ± s.e., n = 3) was found to be very similar. Muscle contained significantly higher concentrations of histidine and lysine, and liver had the highest levels of cystine and the branched-chain amino acids. Gills contained the lowest isoleucine, leucine and valine but were significantly higher in glycine and proline. Differences were also observed between the amino acid composition of sturgeon eggs, vitellogenin and lipovitellin. The A E ratios will provide a valuable index for formulating amino acid test diets and for screening potential protein sources for white sturgeon.

White Sturgeon Tissue Fatty Acid Compositions Are Affected by Dietary Lipids

Juvenile white sturgeon (Acipenser transmontanus) were fed eight isonitrogenous and isoenergetic purified diets for 9 wk to study their ability to utilize different dietary lipids. Each diet contained 15% of control oil mixture (corn oil-cold liver oil-lard, 1:1:1), corn oil, cod liver oil, lard, linseed oil, soybean oil, safflower oil or canola oil. No significant (P > 0.05) differences in percentage of body weight increase, feed efficiency or body composition were observed among sturgeon fed the different lipids. Tissue fatty acid compositions most sensitive to dietary lipids were those of muscle and liver, whereas brain fatty acid composition was the least sensitive. Results of this study indicate that it is possible to increase the levels of (n-3) polyunsaturated and highly unsaturated fatty acids in sturgeon muscle by feeding the fish lipids high in these fatty acids for 9 wk.

4-Chloroacetylpyridine adenine dinucleotide, a chromophoric reagent for the thiol group—Study with glutathione and with aldehyde dehydrogenase

We have shown that 4-chloroacetylpyridine adenine dinucleotide (clac 4PdAD +) inactivated the glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle by reacting with the catalytic cysteine residue 149. We observed that the modification of the thiol group gave rise to the appearance of an absorption band in the visible region, the λ max depending on the pH of the medium. To test the suitability of clac 4PdAD + as a specific cysteine reagent we studied the modification of reduced glutathione and the mitochondrial and cytoplasmic aldehyde dehydrogenases of beef liver with clac 4PdAD +. We found that the reaction of reduced glutathione and mitochondrial and cytosolic aldehyde dehydrogenases with clac 4PdAD + also gave rise to a chromophoric species whose absorption occurred at around 400 nm depending on the pH of the medium. We observed that the two modified isoenzymes had a different spectroscopic behavior at high pH values. The mitochondrial enzyme, like reduced glutathione, presented a new chromophore absorbing at 474 nm; the former was absent in the case of the cytoplasmic enzyme. This behavior and the results obtained with the glyceraldehyde-3-phosphate dehydrogenase suggest that the spectroscopic properties of the chromophore depend on the environment of the thiol group. We observed that clac 4PdAD + had similar inactivation properties toward aldehyde dehydrogenase isoenzymes such as disulfiram.

NAD+ analogue binding to glyceraldehyde-3-phosphate dehydrogenase.

A series of NAD+ analogues, modified on the pyridinium ring, have been tested for their enzymic properties in reactions with D-glyceraldehyde-3-phosphate dehydrogenase form sturgeon muscle, rabbit muscle and Bacillus stearothermophilus. The observed activity, inhibition and binding data are correlated to the structure of the enzyme and coenzyme analogue by model building on a Vector General interactive graphic display system using coordinates from the B. stearothermophilus holoenzyme structure. Most of the analogues with substituents in the pyridinium-3 position could be bound to glyceraldehyde-3-phosphate dehydrogenase, either in manner similar to NAD+ or in a completely different way with the substituted pyridinium ring rotated 110 degrees or more around the glycosidic bond. This indicates different possible modes of binding of NAD+ analogues within the pyridinium binding subsite. Analogues with substituents in the pyridinium-4 position are shown to be weakly bound to glyceraldehyde-3-phosphate dehydrogenase. This is explained by a strong interaction of the substituent in the 4 position with the residues Asn-313 and Cys-149.

Applicability of the induced-fit model to glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle. Study of the binding of oxidized nicotinamide adenine dinucleotide and nicotinamide 8-bromoadenine dinucleotide.

A new method of calculation, based on a direct fitting of the protein fluorescence intensity observed upon coenzyme binding (H.-P. Lutz, unpublished results), is used to study the negative cooperative behavior of glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle. The calculation procedure simultaneously elaborates data obtained for four different protein concentrations, and it is able to compare different models by computing the minimal and critical sum of squares. Using this approach, it is shown that the induced-fit model [Koshland, D. E., Jr., Nemethy, G., & Filmer, D. (1966) Biochemistry 5,365] and the dimer of dimer model [Malhotra, O. P., & Bernhard, S. A. (1968) J. Biol. Chem. 243, 1243-1252] can both be applied for explaining the negative cooperativity observed upon coenzyme binding to sturgeon glyceraldehyde-3-phosphate dehydrogenase. In addition to the progressive modification of the binding affinity during ligand binding, different maximal fluorescence quenchings for the binding steps must be postulated; and furthermore, the binding capability decreases by decreasing the protein concentration. The fact that the induced-fit model can also be applied is rather in contradiction with the view generally accepted of a dimer of dimer structure of sturgeon glyceraldehyde-3-phosphate dehydrogenase. By use of the same approach, nicotinamide 8-bromoadenine dinucleotide is shown to bind to glyceraldehyde-3-phosphate dehydrogenase from sturgeon in a negative cooperative manner.

Close resemblance between muscle pyruvate kinase from a primitive vertebrate, the river sturgeonAcipenser fulvenscens, and the ancestral type K isozyme

Pyruvate kinase (PK) exists as at least two electrophoretically distinguishable, tissue specific form in the river sturgeonAcipenser fulvenscens. In contrast with the PK isozyme system in other vertebrates, the isozyme present in muscle is also present in non-musclce tissues, notably the spleen. The presence of a five membered set of hybrids in liver, gills, eyes and intestine suggests that the synthesis of this isozyme occurs in other tissues as well. Thus, in this primitive vertebrate, the specialization of one PK isozyme for function in muscle tissues is not complete. Sturgeon muscle PK had slightly cooperative phosphoenolpyruvate (PEP) saturation curves (S0.5=0.33 mM) and a Hill coefficient,nH, of 1.24, and was strongly activated by fructose 1,6 bisphosphate (PEP S0.5=0.098 mM;nH=1.0). The enzyme was strongly inhibited by phenylalanine, alanine and MgATP. Fructose 1,6 bisphosphate (FBP) not only totally reversed these inhibitory effects, but also activated the enzyme to the same extent as in the absence of the inhibitors. These kinetic properties closely resemble those of vertebrate type K pyruvate kinases, and are quite distinct from those of higher vertebrate type M isozymes. Nevertheless, immunotitration studies indicate structural homology among the sturgeon, frog and dog muscle PK's as they are all precipitated by antisera to the other forms. This maintenance of structural homology, despite a marked degree of functional differentiation, suggests that only small structural modifications were required to generate the functionally specialized forms of muscle PK found among the vertebrates.

Properties of the charge-transfer transition observed in glyceraldehyde-3-phosphate dehydrogenase from sturgeon muscle alkylated by 3-chloroacetylpyridine--adenine dinucleotide. Characterisation of the modified amino acid.

An absorption band at 340 nm is shown to be formed concomitantly with the covalent bond between the affinity label 3-chloroacetylpyridine--adenine dinucleotide (clac3PdAD+) and glyceraldehyde-3-phosphate from sturgeon. This band corresponds to a charge-transfer transition. Its intensity depends upon the pH and the ionic strength but is almost independent of the nature of the anions present in the medium. The pH dependence shows an inflection point at pH 7.1. This result suggests the participation of a residue with a pKa of 7.1 within the active site of the enzyme in the formation of this transition. Using various techniques, the amino acid alkylated by clac3PdAD+ is shown to be the essential Cys-149, thus excluding the participation of this residue in the formation of the charge-transfer transition. On the other hand, the modification of Cys-153 seems not to affect this charge-transfer band. Other possible donors are proposed, such as the invariant His-176 or Tyr-317 residues. These amino acids might be implicated in the formation of the Racker band.

Fructose 1,6-bisphosphate aldolase of Drosophila melanogaster: Comparative sequence analyses around the substrate-binding lysyl residue

The amino acid sequence of a 103 residue segment encompassing the substrate-binding active site lysyl residue of fructose 1,6-bisphosphate aldolase from Drosophila melanogaster is determined. The sequence is identical to more than 70% with the structure of rabbit muscle aldolase and with the known partial sequences of the sturgeon muscle, trout muscle, and ox liver enzymes. The homology of the insect enzyme with the vertebrate aldolases strongly implies a similar tertiary structure folding.

Comparative studies of kinetic and optical properties of rabbit muscle, sturgeon muscle, and yeast pyruvate kinase

The kinetic and optical properties of pyruvate kinase isolated from rabbit muscle, sturgeon muscle, and yeast were compared using various activating divalent metal ions as probes for functional features and using ultraviolet circular dichroism (cd) measurements for conformational features, respectively. All three preparations of pyruvate kinase were similar in many aspects, such as activating efficiencies of the four activating metal ions, Mg(II), Co(II), Mn(II), and Ni(II) and pH-rate profiles, suggesting the presence of a similar metal binding locus of these enzymes as well as a common underlying mechanism of action. L-Phe inhibited the rabbit muscle enzyme and turned the hyperbolic kinetics into a sigmoidal kinetic with respect to phosphoenolpyruvate at alkaline pH, while fructose-1,6-biphosphate activated the sturgeon muscle and yeast enzymes and turned the sigmoidal kinetics into hyperbolic kinetics with respect to phosphoenolpyruvate. The ultraviolet cd spectral changes qualitatively correlated well with kinetic observations of all three native enzymes in the presence and absence of allosteric effectors. Our results suggested that there are at least two conformational states of pyruvate kinase which are inducible by the binding of substrate and (or) allosteric effectors. The conformational changes from one form to another in these enzymes are very similar, especially between the rabbit and sturgeon muscle enzymes.

Preparation and properties of rabbit-muscle glyceraldehyde-phosphate dehydrogenase with equal binding parameters for the third and fourth NAD + molecules

1. 1. A method of preparing rabbit-muscle glyceraldehyde-phosphate dehydrogenase ( d-glyceraldehyde-3-phosphate:NAD + oxidoreductase (phosphorylating), EC 1.2.1.12) is described which yields a preparation differing in important respects from those previously described and resembling the enzyme isolated from sturgeon muscle. 2. 2. Direct binding measurements at 25°C by equilibrium gel filtration fit dissociation constants for the first two molecules that are too low to be measured by this technique and 0.9 μM for the third and fourth molecules. The dissociation constant of the fourth molecule is much lower than that previously reported for the rabbit-muscle enzyme. 3. 3. In contrast to previous results with the rabbit-muscle enzyme, the increase in absorbance at 360 nm between three and four molecules of NAD + bound to the enzyme was, within experimental error, the same as that with each of the first three molecules. 4. 4. Data on the quenching of the protein fluorescence by NAD + at 15°C at different enzyme concentrations closely fit dissociation constants of 0.028 μM for the first two molecules and 0.27 μM for the third and fourth molecules.