Heat-stable protease from Pseudomonas fluorescens T16: purification by affinity column chromatography and characterization.

A heat-stable extracellular protease from Pseudomonas fluorescens T16, a psychrotroph, was purified by affinity column chromatography on a carbobenzoxy-D-phenylalanine-triethylene tetramine-Sepharose-4B column. The purified enzyme is a monomer with a molecular weight of 38,905 +/- 2,000. In an analytical ultracentrifuge, the Schlieren profile revealed a single symmetrical peak. The sedimentation coefficient was estimated to be 3.93S. Alpha-casein was the preferred substrate, with a Km of 0.05 mM. Heating crude enzyme and purified enzyme in buffer at 50, 90, and 120 degrees C resulted in a rapid initial loss of more than 50% of the initial activity followed by a gradual inactivation which exhibited first-order kinetics. The activation energy for the hydrolysis of casein was calculated to be 3.2 kcal/mol (13.4 kJ/mol).     

Purification and characterization of a protein tyrosine kinase from bovine spleen

Protein tyrosine kinase was purified extensively from a 30,000 X g particulate fraction of bovine spleen by a procedure involving four column chromatographies: DEAE-Sepharose, polyamino acids affinity, hydroxylapatite, and Sephacryl S-200 molecular sieving. The purification resulted in more than 3,000-fold enrichment in [Val5]angiotensin II phosphorylation activity (specific activity 202 nmol/min/mg). All column chromatography profiles showed single protein tyrosine kinase activity peaks with the exception of that of affinity chromatography, where about 50% of the enzyme activity appeared with the breakthrough fraction; only the bound enzyme was further purified. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography of a purified sample phosphorylated in the presence of [gamma-32P]ATP revealed the presence of a single phosphorylated polypeptide of molecular weight 50,000 which represents about 40% of total protein. Analysis by polyacrylamide gel electrophoresis under nondenaturing conditions showed that protein tyrosine kinase activity co-migrated with the phosphoprotein. Stoichiometry of the phosphorylation of the 50-kDa polypeptide was found to be 1.0 mol/mol. The purified sample did not appear to contain phosphotyrosine protein phosphatase activity. Both casein and histone could be phosphorylated by the purified sample, and the phosphorylation occurred only at tyrosine residue, suggesting that there was no protein serine and threonine kinase contamination.     

Purification and Characterization of the polygalacturo nase from Tomato Fruits

The polygalacturonase (PG) isolated fron the pericarp of fully ripe tomato fruits was purified by (NH4)2SO4 precipitation, carboxyme-thylsepharose ion-exchang column and sephadex G-75 gel filtration. Specific activity of purified PG was 1.5 μmol galacturonic acid mg-1 protein min-1, which was 30 times as high as that of the crude extract with 1.7mol NaC1. When the elution separated by second sephadex G-75 column was analyzed by SDS-PAGE, only a single protein band was detected. It was shown by heat and pH experiments of the purified enzyme that the enzyme activity retained 50%, after treatment with heat at 50℃ for 10 min, and that the optimal pH was 4.6.     

Purification and characterization of a membrane-bound nonlysosomal ceramidase from rat brain.

We have purified a membrane bound ceramidase 22,300-fold to apparent homogeneity. The purification scheme included Triton X-100 extraction of membranes followed by Q-Sepharose, blue Sepharose, phenyl-Sepharose, and MonoS column chromatography. The purified enzyme showed an apparent molecular mass of 90 kDa as estimated by SDS-polyacrylamide gel electrophoresis under reducing conditions and 95 kDa by chromatography on Superose 12. Using C(16)-ceramide as substrate, the enzyme showed a broad pH optimum in the neutral to alkaline range. A mixed micelle assay was developed, and using Triton X-100/ceramide mixed micelles, the enzyme exhibited classical Michaelis-Menten kinetics, with a K(m) of 1.29 mol % and a V(max) of 4.4 micromol/min/mg. When dihydroceramide was used as substrate, these values were 3.84 mol % and 1.2 micromol/min/mg, respectively, indicating that the enzyme hydrolyzes ceramides preferentially. The activity of the purified ceramidase did not require cations, and it was inhibited by reducing agents. Phosphatidylcholine and sphingomyelin were without effect on the enzyme activity, whereas phosphatidic acid and phosphatidylserine stimulated the activity 3-fold. Sphingosine acted as a competitive inhibitor with an IC(50) of 5-10 microM. These results indicate that the purified enzyme is a novel ceramidase.     

Partial purification and properties of frog liver tyrosine aminotransferase.

Hepatic tyrosine aminotransferase of the frog Rana temporaria was partially purified by (NH4)2SO4 fractionation and successive chromatography on DEAE-cellulose DE-52, Ultrogel AcA-34, DEAE-cellulose DE-52 again and, finally, hydroxyapatite. During the last step, the enzyme activity separated into two fractions; traces of a third fraction were also found. The major form was purified 6000-fold to a specific activity of 200 units/mg of protein; it was about 50% pure by electrophoretic criteria. It had mol.wt. about 85 000 as determined by gel filtration on a Sephadex G-100 column. It was not activated by added pyridoxal 5'-phosphate. The enzyme was, however, inactivated by the pyridoxal phosphate reactants canaline and amino-oxyacetate. The enzyme was specific for 2-oxoglutarate as the amino group acceptor. Homogentisate inhibited the enzyme and adrenaline was an activator; both effects were seen at low concentrations of the effectors. The relationship between initial rate and tyrosine or 2-oxoglutarate concentration was abnormal and complex. Form-2 enzyme had similar or identical molecular weight, cofactor requirements, oxo acid specificity and kinetics.     

Purification and characterization of an intracellular α-glucosidase with high transglycosylation activity from A. niger M-1

An intracellular α-glucosidase with high transglycosylation activity was purified from a mutant strain of Aspergillus niger M-1 by sequential chromatography using a DEAE-cellulose 52 column, a DEAE-Sepharose CL-6B column, and a Sephadex G-100 column. The molecular mass of the purified enzyme was determined to be 116?kD with no subunits and a pI of 5.23. Maximal α-glucosidase activity occurred at pH 6.0 and 50°C. The N-terminal amino acid sequences were identified as N-SVPGTEYVV-. The presence of Ca(2+) enhanced the enzyme activity by 20%, while the α-glucosidase activity was strongly inhibited by p-chloromercuribenzoate, N-(3-dimethylaminopropyl)-N-ethylcarbodiimide hydrochloride, monochloroacetic acid, and 2-mercaptoethanol. In addition, Ag(+), n-bromosuccinimide, and acetylacetone inhibited enzyme activity by 70%, 50%, and 22%, respectively. K(m) values of 4.32?m?mol?L(-1) and V(max) of 3.10?×?10(-2)?mol?L(-1) min(-1) were found for methyl-α-D-glucopyranoside (α-MG). Maltose was identified as the preferred substrate. The high-performance liquid chromatography (HPLC) analysis indicated that the oligosaccharide products contained 10.54% of isomaltose, 8.08% of panose, and 9.29% of isomaltotriose, and the amount of glucose, maltose, maltotriose, and maltotetrose was dropped from 22.21% to 15.80% using the purified enzyme in the solution of 25% maltose and 3% glucose. This intracellular α-glucosidase has potential applications in the synthesis of sugar derivatives and the investigation of associated mechanisms.     

Purification of NADH-cytochrome b5 reductase from sheep lung and its electrophoretic, spectral and some other properties

1. NADH-cytochrome b5 reductase was purified from sheep lung microsomes in the presence of non-ionic and ionic detergents, Emulgen 913 and cholate, respectively. 2. The purification procedure involved the ion-exchange chromatography of the detergent solubilized microsomes on DEAE-cellulose. 3. Further purification and concentration of lung reductase was carried out with a second DEAE-cellulose column followed by the affinity column chromatography of partially purified reductase on 5'-ADP-agarose column. 4. The specific activity of sheep lung reductase was 638 mumol ferricyanide reduced/min/mg protein and the yield was 6% of the initial activity in microsomes. 5. The SDS-polyacrylamide gel electrophoresis of the purified lung reductase showed one protein band having the monomer mol. wt of 34,500 +/- 1500. In the presence of 0.4% deoxycholate, it existed as an active dimer having a mol. wt of 68,500. 6. Trypsin treated lung reductase showed two extra protein bands of mol. wts of 28,000 and 25,000 on 10% SDS-polyacrylamide gels. 7. The purified enzyme was found to contain FAD as prosthetic group and the absorption spectrum of lung reductase showed two peaks at 390 and 461 nm which were typical for flavoproteins and a shoulder at 490 nm. 8. The maximal activity of lung reductase was observed between pH 6.5-8.0 and at pH 6.8, when ferricyanide and partially purified sheep lung cytochrome b5 was used as electron acceptors, respectively.     

Purification and characterization of nucleoside diphosphatase from rat-liver microsomes. Evidence for metalloenzyme and glycoprotein

Nucleoside diphosphatase was purified from rat liver microsomes more than 3000-fold with a 16% yield using a procedure including concanavalin-A--Sepharose and phenyl-Sepharose column chromatography. The purified enzyme had a specific activity of 2500 units/mg protein and appeared homogeneous by gel electrophoresis. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation. The enzyme had a sedimentation coefficient of 6.5 S by sucrose-density gradient centrifugation, and a Stokes' radius of 4.8 nm was estimated by the gel filtration technique. Its molecular weight is 130,000, but only one single band of Mr 65,000 was detected after sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme seems thus to be composed of two identical subunits. The purified enzyme was confirmed to be a glycoprotein containing approximately 9% carbohydrates. The enzyme had a pH optimum of 7.5, an isoelectric point of 4.85 and a Km of 2.5 mM for UDP. On the basis of direct measurement of metal content in the native enzyme, the rat liver nucleoside diphosphatase was found to be a metalloenzyme containing 0.9 mol zinc and 0.1 mol manganese/mol 65,000-Mr subunit. Metal-free nucleoside diphosphatase has been prepared. The activity of the metal-free enzyme was restored by the addition of several divalent cations, zinc being the most effective.     

Purification and properties of a specific collagenase from rabbit synovial fibroblasts.

1. A specific collagenase from the culture medium of rabbit synovial fibroblasts was purified by gel filtration and ion-exchange chromatography. 2. The enzyme was homogenous on polyacrylamide-gel electrophoresis and showed only traces of contaminants when tested in gels with a non-specific antiserum. 3. The rabbit fibroblast collagenase could hydrolyse collagen both in solution and in fibrillar form. Viscometry showed that at 35 degrees C the purified enzyme could hydrolyse greater than 50 nmol of collagen/min per mg of enzyme. 4. The purified collagenase cleaved collagen in solution at either 24 degrees or 35 degrees C into the characteristic 1/4 and 3/4-length fragments. However, as compared with the impure enzyme, the purified enzyme at 35 degrees C had a much decreased capacity to further degrade the initial specific cleavage products. 5. The specific rabbit collagenase had a mol. wt. of approx. 32000 as estimated by sodium dodecyl sulphate-polyacrylamide-gel electrophoresis, and 35000 by gel filtration.     

花生根瘤菌类菌体氢酶的分离提纯及特性

花生根瘤菌类菌体经超声波破碎,ＴｒｉｔｏｎＸ－１００溶解,正已烷－硫酸铵处理后,再经ＤＥＡＥ－纤维素和Ｓｅｐｈａｃｒｙｌ凝胶柱层析等纯化步骤,获得凝胶电泳纯的膜结合态氢酶,比活为７１．４μｍｏｌＨ２ｍｇ－１Ｐｒｏｔｍｉｎ－１,为类菌体吸Ｈ２活性的２１１倍。纯化的氢酶分子量为１１０ｋＤ。经ＳＤＳ－ＰＡＧＥ后,呈现两个蛋白带,分子量分利为６５ｋＤ和３５ｋＤ。纯酶的Ｎｉ含量为０．６２ｍｏｌＮｉ／ｍｏｌ氢酶。在磷酸缓冲液中其活性的最适ｐＨ为６．５。ＤＣＩＰ、亚甲蓝、铁氰化钾、细胞色素Ｃ均可作为氢酶的电子受体,其中以ＤＣＩＰ为最适。     

Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane

The Ca²⁺/Mg²⁺ ATPase, which is activated by millimolar concentrations of Ca²⁺ or Mg²⁺, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, Nal, EDTA and Tris-HCI at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN₃, but was markedly inhibited by 20 µM gramacidin S and 50 µM trifluoperazine. Analysis of the purified Ca²⁺/Mg²⁺ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 µmol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 µmol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Cau2+/Mg^ATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neurominidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca²⁺/Mg²⁺ ATPase and was found to bind to the purified enzyme with a K_D of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca²⁺/Mg²⁺ ATPase is a glycoprotein and contains a large amount of lipids.     

Purification of the CaATPase of sarcoplasmic reticulum by affinity chromatography

Proteins from sarcoplasmic reticulum vesicles solubilized by a nonionic detergent were fractionated by use of a reactive red-120 agarose column. The Ca-ATPase was obtained in pure form by eluting the column with 400 microM adenyl 5'-yl imidodiphosphate, yielding an enzyme of almost twice the starting specific activity in a fraction containing half the initial protein. The conclusion that the ATPase comprises 50% of the sarcoplasmic reticulum vesicle protein agrees with estimates gained from densitometry using 7 1/2% Laemmli slab gels but not from densitometry using 7% Weber and Osborn slab gels. The mechanism of purification was found to be affinity chromatography, with the ATPase binding the reactive red-120 ligand in its nucleotide-binding site. The steady-state concentration of phosphorylated intermediate relative to the specific activity was found to be lower in the purified enzyme as compared to the starting vesicular enzyme.     

Purification and partial characterization of glycogen synthase kinase-3 from rabbit liver

Summary Glycogen synthase kinase-3 (GSK-3) was purified from rabbit liver to homogeneity by ultracentrifugation, ion-exchange chromatography on DEAE-cellulose, Cellulose phosphate, CM-Sephadex and Fast Protein Liquid Chromatography (FPLC) on Mono-S column. The enzyme was purified approximately 20,000 fold with an approximate 2% recovery. The purified enzyme showed a single band on SDS-polyacrylamide gel electrophoresis. GSK-3 is a monomeric enzyme with a molecular weight of 50,000–52,000 as derived from SDS-polyacrylamide gel electrophoresis and gel filtration. The purified enzyme was indeed a GSK-3 since it phosphorylated three sites, i.e., 3a, 3b, and 3c on liver glycogen synthase. GSK-3 incorporated up to 2.6 mol Pi/mol glycogen synthase subunit with a concomitant inactivation of glycogen synthase activity.     

Purification and properties of the insulin-stimulated cyclic AMP phosphodiesterase from rat liver plasma membranes.

The peripheral high-affinity cyclic AMP phosphodiesterase from rat liver plasma membranes was purified to apparent homogeneity. The procedure used involved the initial purification of liver plasma membranes and the solubilization of the enzyme by using a high-ionic-strength medium. This was followed by chromatography of the enzyme on DEAE-cellulose, Affi-Gel Blue, a novel affinity column and Sephadex G-100. A 9500-fold purification of the enzyme with a 24% yield was achieved by this procedure. The purified enzyme was apparently monomeric (Mr 52000) as it exhibited identical molecular weights on analysis by gel filtration, sedimentation and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It is suggested that the non-Michaelis kinetics exhibited by the enzyme are due to it obeying a mnemonical mechanism, where it displays Km 0.7 micrometer, Vmax. 9.1 units/mg of protein and Hill coefficient (h) 0.62. Cyclic GMP acts as a poor substrate for the enzyme, with Km 120 micrometer and Vmax. 0.4 unit/mg of protein, and also as an inhibitor of the enzyme, with I50 (concentration giving 50% inhibition) 150 micrometer when assayed at 0.4 micrometer-cyclic AMP. Inhibition by 5'-AMP is unlikely to be of physiological importance, as it is only a weak inhibitor of the enzyme (I50 47 mM assayed at 0.4 micrometer-cyclic AMP).     

Phosphofructokinase from Ascaris suum. Purification and properties

A rapid and efficient procedure has been developed to purify phosphofructokinase from the muscle of the parasitic roundworm, Ascaris suum. The procedure can be accomplished in 1 day with a 420-fold purification and a 60% yield. The enzyme was shown to be homogeneous by two-dimensional electrophoresis, Sepharose 6B column chromatography, and high performance liquid chromatography utilizing a size exclusion column. The subunit molecular weight of the enzyme was found to be 95,000 by electrophoresis in the presence of sodium dodecyl sulfate. In solutions of low ionic strength, the native enzyme aggregated to species of higher molecular weight than did the rabbit muscle phosphofructokinase. In the presence of 0.2 M (NH4)2SO4, the minimum native molecular weight was determined to be 398,000 by high performance liquid chromatography and Sepharose 6B column chromatography. Therefore, the enzyme appears to be a tetramer with identical or near-identical subunits. The apparent isoelectric point of the enzyme was shown to be 7.3 to 7.4 by both column and gel isoelectric focusing. Amino acid analysis revealed a lower number of the aromatic residues Phe, Tyr, and Trp than in the rabbit muscle enzyme and this is in agreement with the lower extinction coefficient of E1%280 nm = 6.5. Analysis of the purified enzyme revealed 7.4 +/- 0.6 mol of phosphate/mol of enzyme.     

Preparation of enantiomerically pure (S)-flurbiprofen by an esterase from Pseudomonas sp. KCTC 10122BP

Esterase PF1-K from Pseudomonas sp. KTCC 10122BP was overproduced by the fed-batch culture of Escherichia coli. The soluble expression of esterase PF1-K was achieved by shifting the culture temperature from 37 to 25 °C at the time of IPTG induction. The enzyme was partially purified to about 75% purity by a single-step hydrophobic interaction column chromatography. The purified enzyme exhibited a fairly high enantioselectivity towards the hydrolysis of rac-flurbiprofen ethyl ester. The enzymatic chiral resolution was further improved by optimizing the reaction conditions in terms of reaction rate and enantioselectivity. The optimal reaction conditions were found to be 40 °C, pH 10.5 and 600 mM of initial rac-flurbiprofen ethyl ester. After 90 min of batch reaction under the optimal conditions, 50% of the initial rac-flurbiprofen was hydrolyzed with an enantiomeric excess of 99%.     

非水体系中脂肪酶催化合成乳酸乙基糖苷酯的工艺研究

在非水体系中 ,通过固定化脂肪酶催化合成一种新型α 羟基酸衍生物 乳酸糖苷酯。考察了常压下有机溶剂、酰基供体、不同种固定化酶、乙基糖苷的浓度、酶量和反应温度对反应的影响。研究表明在无溶剂体系中以乳酸丁酯作为酰基供体可有效地合成乳酸糖苷酯 ,固定化酶Novozym435和来源于Candida sp .菌株的细胞固定化酶 ,化学修饰的干酶粉均是合适的催化剂。最佳反应条件为 :酶浓度 75g L ,乙基葡萄糖苷的浓度为 0.4mol L ,温度为 70℃ ,转速 200r min ,反应 50h ,转化率可达 71%。在真空度为 0.09MPa的压力下 ,反应温度 65℃ ,酶浓度 75g L ,乙基葡萄糖苷 0.35mol L时 ,反应初速率可达到 607(mmol·L^-1·h^-1 ) ,40h后转化率可达到 90%。反应产物经过萃取法和硅胶柱层析方法分离 ,纯度达到 95 % (W/W)。     

Isolation and characterization of a monoclonal antibody against pig kidney sodium- and potassium-activated ATPase

A hybridoma cell line producing mouse monoclonal antibody against pig kidney Na,K-ATPase was established. The antibody, named 38 (mAb38, IgG1), was purified from mouse ascites fluid by chromatography on a protein A-Sepharose column. Antigens immobilized on microplate wells with p-benzoquinone were used for titer assays. mAb38 cross-reacted with both dodecyloctaethyleneglycol monoether (C12E8)-solubilized enzyme and membranous sodium dodecyl sulfate (SDS)-treated enzyme from kidney with high affinity (50% binding = 0.6 nM). However, the antibody bound to neither alpha- nor beta-subunit separated by preparative SDS-polyacrylamide gel electrophoresis (PAGE). The stoichiometry of antibody binding to the purified enzyme was estimated to be about 0.86 mol of IgG per mol of alpha beta-protomer. Na,K-ATPase proteins were recovered from a column of mAb38-coupled Affi-Gel by elution with pH 3 buffer when C12E8-solubilized kidney enzyme or detergent extracts of brain microsomes were applied to it, confirming that the mAb is directed to Na,K-ATPase. mAb38 at saturation level concentrations had no effect on kidney Na,K-ATPase activity or on ouabain-sensitive Rb uptake in erythrocytes. In an immunofluorescence study, the antibody bound to intact erythrocytes much more strongly than control IgG1 (mAb50c), but the extent of the antibody binding to inside-out vesicles under hypotonic conditions was lower than that of the control. Most of the antibody binding activity remained when the kidney enzyme was treated with sialidase. These results suggest that this mAb38 was raised against an intact conformation of a cell-surface-exposed site of Na,K-ATPase.     

N-terminal sequence of soybean beta-amylase

The blocked N-terminus and N-terminal sequence of soybean beta-amylase were determined by analyzing the acidic peptides derived on peptic digestion of the enzyme. The acidic peptides were separated from the digest on a Dowex 50 X 2 column and purified by reversed phase-high performance liquid chromatography (RP-HPLC). The major acidic peptide, Pep-4, was a heptapeptide with a molecular weight of 766. Forty-eight hundredths mol acetyl group and 0.61 mol acetyl-Ala per mol of Pep-4 were detected on RP-HPLC analysis. The N-terminal 9 amino acid sequence of soybean beta-amylase was deduced to be acetyl-Ala-Thr-Ser-Asp-Ser-Asn-Met-(Gly-Leu) from the results of sequence analysis of Pep-4 and amino acid analysis of other acidic peptides.     

Purification and Characterization of Sperm Creatine Kinase and Guanylate Cyclase of the Sea Urchin Hemicentrotus pulcherrimus

Creatine kinase and guanylate cyclase were purified from Hemicentrotus pulcherrimus spermatozoa. The molecular weight of the purified sperm tail creatine kinase was estimated to be 137,000 by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Sperm tail guanylate cyclase was purified by chromatography on a WGA-Sepharose column connected to a Concanavalin A-Sepharose column, and a Superose 12 HR column. The molecular weight of the tail guanylate cyclase was estimated to be 128,000 by SDS-PAGE. The specific activity of the purified enzyme was 8.25 μmol of cGMP formed/min/mg protein. Sperm-activating peptide I (SAP-I) causes an electrophoretic mobility shift of H. pulcherrimus sperm guanylate cyclase from 131 kDa to 128 kDa. The 131 kDa form of guanylate cyclase was co-purified with a 76 kDa protein, whose molecular mass is similar to that of a SAP-I receptor. The purified 131 kDa form of guanylate cyclase had higher activity than the 128 kDa form. The 131 kDa and 128 kDa forms of guanylate cyclase contained 23.83 ± 0.65 and 4.16 ± 0.45 moles of phosphate per mol protein (mean ± S.D.; n = 3), respectively. The activities of guanylate cyclase and creatine kinase increased during the testis development. During spermatogenesis, sperm tail creatine kinase was detected immunohistochemically only in mature spermatozoa.