Teflon Pestle Research Articles

Immunological and biochemical study on tissue and subcellular distributions of protein kinase FA (an activating factor of ATP.Mg-dependent protein phosphatase): A simplified and efficient procedure for high quantity purification from brain

Although protein kinase FA/GSK-3 alpha (an activating factor of ATP.Mg-dependent protein phosphatase) has been established as a cytosolic enzyme in mammalian nonnervous tissues involved in the metabolic regulation, immunological and biochemical studies on tissue and subcellular distributions demonstrate that kinase FA/GSK-3 alpha is in fact a membrane-associated enzyme and most abundantly exists in brain particulate membrane fractions depending on the tissue homogenization conditions. For instance, when brain was homogenized in Polytron without 0.32 M sucrose, approximately 40% of the total FA/GSK-3 alpha was found in the cytosol. However, when brain was homogenized in buffer containing 0.32 M sucrose and in a glass homogenizer with Teflon pestle, more than 80% of the total FA/GSK-3 alpha was found associated with the particulate membrane fractions. By manipulating these findings, we have developed a simplified procedure for purification of homogeneous kinase FA/GSK-3 alpha in high recovery and in a substantial amount from brain tissue. The data explain why kinase FA/GSK-3 alpha cannot be isolated in a reasonable amount from most mammalian tissues for the past years. The specific pure antibody that can specifically recognize kinase FA/GSK-3 alpha from crude tissue extracts together with the high quantity purification of the enzyme as presented in this report provides an initial key step for studies on the role of kinase FA/GSK-3 alpha in the regulation of brain functions especially in the brain particulate membrane fractions.

In vitro reduction of peroxidation in UVC‐irradiated cell cultures by concurrent exposure with Bowman‐Birk protease inhibitor

Bowman-Birk protease inhibitor (BBI), extracted from soybean flour, has been shown to protect cells from damage after UVC irradiation. BBI also reduces the number of transformed foci following exposure to the same doses of UVC irradiation. Proteins have been reported to act as antioxidants by their sheer intracellular bulk. In this study we assessed BBI's ability to attenuate UVC-irradiation-induced peroxidation. Balb c/3T3 cells, clone A31, subcloned by this laboratory were used. Treatment groups consisted of cells suspended in Hanks' Balanced Salt Solution with and without 100 micrograms/ml of a crude soybean extract containing the Bowman-Birk protease inhibitor. Cells were exposed to irradiation in suspension by using GE (G8T5) germicidal lamps which deliver predominantly 254 nm light at 0.385 J/m2/s as measured at the cell surface. Aliquots of UVC-exposed cells were homogenized in HBSS in the presence and absence of 100 micrograms/ml BBI with a motor-driven Teflon pestle and assayed for peroxidation. The results showed significant (P less than 0.0001) protection afforded by BBI against peroxidation at 1.16 (65%), 1.93 (60%, and 2.70 (48%) J/m2 of UVC irradiation. Transformation was reduced to 0.33 foci per dish at all levels of radiation exposure.

Isozyme Analysis ofXanthomonas campestrispv.citri

Isozyme Analysis of<i>Xanthomonas campestris</i>pv.<i>citri</i>

Purification and characterization of plasma membranes from Physarum polycephalum amoebae

In this study we describe a method for preparing plasma membranes from Physarum polycephalum amoebae. The cells were swollen in hypotonic medium (1 mM ZnCl 2, 10 mM Tris-HCl, pH 8.0) and then broken in a Thomas tissue grinder fitted with a teflon pestle. The plasma membranes were collected by differential centrifugation and purified by centrifugation on a continuous 20–50% sucrose gradient. The membranes sedimented in a single band having a density of 1.16 g/cm 3. They were found by enzymatic assay and by electron microscopy to be free of lysosomes, mitochondria, and nuclei and minimally contaminated by endoplasmic reticulum. The membrane proteins were analysed by sodium dodecyl sulphate polyacrylamide gel electrophoresis; 10 major and 20 minor bands were seen. Periodic acid Schiff's staining of electrophoretically separated proteins revealed five major and six minor bands containing glycoproteins. Radioiodinated cell surface proteins were separated by sodium dodecyl sulphate polyacrylamide gel electrophoresis into six bands with apparent weights greater than 68 000.

Isozyme Banding Patterns in Two Stands of Cetraria arenaria Kärnef.

Isozyme Banding Patterns in Two Stands of Cetraria arenaria Kärnef.

61] Purification of chick intestinal calcium-binding protein

This chapter describes the purification of chick intestinal calcium-binding protein. Intestinal calcium-binding proteins (CaBP) have been identified and purified from several species of animals. The method described in this chapter enables isolation of chick intestinal CaBP in an electrophoretically homogeneous state. The procedure employed is a modification of the method of Wasserman; it utilizes an extra (NH 4 ) 2 SO 4 fractionation step and substitutes two anion-exchange steps for the originally reported preparative acrylamide disc gel electrophoresis procedure. In first step, duodenum obtained from vitamin D-repleted chickens was immediately cooled to 4°, slit open, and rinsed with cold 0.12 M NaCl. The mucosal tissue was scraped from the underlying muscle layers with a glass slide and then homogenized in a chilled buffer mixture (20% w/v) with a Potter-Elvehjem homogenizer utilizing a Teflon pestle. The composition of the buffer utilized was 13.7 mM Tris, 0.12 M NaC1 with 0.01% B-mercaptoethanol; this solution was adjusted to pH 7.4 with HCl. Other steps in this method that include (NH 4 ) 2 SO 4 precipitation, gel filtration, and anion exchange are also discussed in detail.

Pneumocystis carinii: New separation method from lung tissue

A new method of separating Pneumocystis carinii from infected rat, human, and mouse lung tissue has been developed, based, in part, on techniques used for the separation of lymphocytes from hematopoietic and lymphoid organs. The lungs are homogenized with a Teflon pestle and fine wire-mesh screen, digested with collagenase and hyaluronidase, and centrifuged on a discontinuous Ficoll-Hypaque density gradient. The method produces no morphologic alterations in P. carinii, as judged by light and electron microscopy. The method has been adapted for the quantitation of P. carinii in tissues and the production of antisera in rabbits.

An improved method for isolation of mitochondria from chick breast muscle using Nagarse

An improved procedure to isolate mitochondria from chick pectoralis muscle is described. The muscle is digested with the proteinase Nagarse and homogenized using a Teflon pestle. Mitochondria are isolated by differential centrifugation. These organelles are able to utilize a variety of substrates with a higher degree of respiratory control than mitochondria isolated without Nagarse. Contrary to previous reports, mitochondria isolated by this method from chick pectoralis muscle (αW fibers) are able to oxidize the substrate β-hydroxybutyrate with good respiratory control.

19] Mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase from chicken liver

This chapter discusses the determination of mitochondrial 3-hydroxy-3-methylglutaryl-CoA synthase from chicken liver. The thermodynamically favorable condensation of acetyl-CoA and acetoacetyl-CoA to form 3-hydroxy-3-methylglutaryl-CoA is catalyzed in chicken liver mitochondria by 3-hydroxy-3-methylglutaryl-CoA synthase (HMG-CoA synthase). This enzyme is localized in the mitochondrial matrix and occupies a potentially favorable position for the regulation of hepatic ketogenesis. The distinctly different properties of this enzyme from those of the cytoplasmic HMG-CoA synthase are consistent with independent regulation of ketogenesis and cholesterogenesis. 3-Hydroxy-3-methylglutaryl-CoA synthase activity can be conveniently determined by a radiochemical assay, which monitors the incorporation of [1- 14 C]acetyl-CoA (volatile 14 C activity when taken to dryness at 95° in 6 N HCl) into HMG-CoA (nonvolatile 14 C activity when taken to dryness at 95° in 6 N HCl). A spectrophotometric assay can be used to measure the acetyl-CoA–dependent disappearance of acetoacetyl-CoA absorbance at 300 nm. In the course of purification procedure, fresh liver from laying hens is ground in a Universal No. 1 meat chopper and then homogenized in a loose-fitting Potter-Elvehjem tissue homogenizer with a Teflon pestle. One kilogram of liver is homogenized in 2 liters of media containing 0.25 M sucrose, 0.1 m M ethylenediaminetetraacetic acid (EDTA), and 2 m M N -2-hydroxyethylpiperazine- N -2-ethanesulfonic acid (HEPES) brought to pH 7.2 with potassium hydroxide.

Labeling of Sporozoites of Plasmodium berghei with Tritiated Purines

Sporozoites were labeled in vivo. Mosquitoes (Anopheles stephensi) infected with the NK-65 strain of P. berghei were allowed to feed ad lib. for 14 days subsequent to an infective blood meal on 5% Karo syrup solution containing different levels of tritium-labled purines and pyrimidines. Sporozoites were harvested on day 16 by dissection of mosquito salivary glands. Isotope incorporation in sporozoites was determined by autoradiography. Each of the purines tested, including adenine-8-3H, adenosine-3H (G), deoxyadenosine-3H (G), and deoxyguanosine-3H (G), was incorporated into both nuclear and cytoplasmic regions of sporozoites when fed to mosquitoes at 5 to 100 ,uCi/ml. Similar levels of the pyrimidines tested, including thymine-methyl-3H, thymidine-methyl-3H, or orotic-5-3H acid, failed to label sporozoites. The incorporation of preformed purines, but not pyrimidines, by developing sporozoites suggests that the parasite may require exogenous sources of purine during its development in the mosquito but relies on de novo synthesis of pyrimidines. The adenine8-3H-labeled sporozoites were shown to be infective by intravenous inoculation into A/J mice. These results demonstrate the feasibility of labeling developing sporozoites and should provide a means for further studies on the developmental cycle of malaria in the vertebrate host. After the discovery of exoerythrocytic bodies of malaria in the liver of monkeys and man (Shortt and Garnham, 1948) it was generally assumed that relapses were caused by a continual cycling of histiotrophic merozoites in the liver. More recently, Garnham (1967) has proposed that relapses may be caused by sporozoites which remain latent in parenchymal cells of the liver for various periods of time before undergoing development. To date, these latent forms have not been demonstrated, possibly because of their small size and similarity to the cytoplasm of liver cells in stained preparations. Radionuclide labeling of sporozoites would provide a means for recognizing these latent forms in tissue if, indeed, they exist. In the present study a method for labeling sporozoites in vivo with tritiated purines was developed. In addition, the labeled sporozoites were shown to retain their infectivity when inoculated into susceptible hosts. MATERIALS AND METHODS Anopheles stephensi mosquitoes were allowed to feed on hamsters infected with the NK-65 strain of Plasmodium berghei and maintained at Received for publication 18 September 1973. * This work was presented in part at the 57th Annual Meeting of the Federation of American Societies for Experimental Biology, Atlantic City, New Jersey (1973, Fed. Proc. 32: 704). 21 C as described by Yoeli et al. (1965). Subsequent to the infective blood meal, mosquitoes were allowed to feed from the mouth of a plastic dropping pipette that contained tritium-labeled purines or pyrimidines in 5% Karo syrup solution. The mouth of the pipette was mounted in contact with the mesh on top of the cage. Fresh isotope solution was supplied every other day for a period of 16 days. Sporozoites were harvested on day 18 by dissection of mosquito salivary glands into a 1:1 mixture of saline and mouse plasma. The glands were gently homogenized in a 3-ml tissue grinder fitted with a Teflon pestle in an ice bath. Sporozoite yield was estimated from counts in a Neubauer hemocytometer. Viability of adenine-8-3H-labeled sporozoites was evaluated by the intravenous inoculation of approximately 10,000 sporozoites into 6to 8-weekold A/J mice (Jackson Laboratories, Bar Harbor, Maine) within 1 hr after dissection of salivary glands. Infectivity and prepatent period were determined by daily Giemsa-stained blood films. Isotope incorporation by sporozoites was determined by autoradiography using Kodak NTB-2 nuclear track emulsion (Eastman Kodak Co.). Infected salivary glands were crushed with a glass cover slip on slides previously coated by dipping in 1% bovine serum albumin. Sporozoites were then fixed with methanol and allowed to air-dry. Slides were coated by dipping into undiluted photographic emulsion at 40 C. After exposure to the emulsion for various intervals at 4 C, slides were processed with Kodak D-19 developer and fixer. The preparation was then stained through the emulsion with 7.5% Giemsa for 1 hr at pH 7.2. The degree of labeling was determined microscopically under oil immersion by counting silver grains associated with individual sporozoites.

19] Adenylate cyclase from kidney and bone

Publisher Summary Male Sprague-Dawley rats, 150–200 g, are killed by decapitation. The kidneys are removed, chilled in 0.25 M sucrose, and the hilar structures are dissected away. The kidneys can be sectioned further into cortical, corticomedullary, and medullary zones. The three zones contain distinct receptors for parathyroid hormone, calcitonin, and vasopressin. The tissue is transferred to 8 volumes (w/v) of Tris.HC1 0.05 M, pH 7.4 containing 10% (v/v) DMSO and homogenized in glass with a motor driven Teflon pestle. The particulate fraction is obtained by centrifugation at 2000 g for 10 minutes; the pellet is resuspended in the original volume of Tris·HC1-DMSO and centrifuged again. The pellet is resuspended in Tris·HC1-DMSO to give a protein concentration of 15 mg/ml. This preparation is stored in small aliquots in liquid nitrogen. The enzyme in this form is stable indefinitely and has been used for in vitro bioassays for parathyroid hormone or calcitonin.

Microsomal metabolism of the hepatotoxin -naphthylisothiocyanate (ANIT) following phenobarbital, chlorpromazine or actinomycin D treatment.

Previous investigations of the mechanism for a-naphthylisothiocyanate (ANIT)-induced hyperbilirubinemia have suggested that micro-somal biotransformation of ANIT must precede the onset of hyperbilirubinemia (1). Direct evidence for ANIT biotransformation as well as a similarity between the disposition of (14C) ANIT and/or its metabolites and temporal aspects of ANIT-induced hyper-bilirubinemia has previously been reported from this laboratory (2). Furthermore, a positive correlation has been found in vivo between the quantitative aspects of pheno-barbital and chlorpromazine potentiation of ANIT-induced hyperbilirubinemia and their effect on the rate of formation of ANIT metabolites (3). Actinomycin D, on the other hand, inhibits ANIT-induced hyperbilirubinemia, inhibits phenobarbital and chlorpromazine potentiation of ANIT-induced hyperbilirubinemia, and alters certain aspects of ANIT metabolism in vivo (1, 3). The present study correlates previously reported in vivo alteration of ANIT disposition and ANIT-induced hyperbilirubinemia (2, 3) by phenobarbital, chlorpromazine and actinomycin D with the effect of these agents on in vitro microsomal metabolism of ANIT. Methods. Livers were obtained from four separate groups of pretreated male Sprague-Dawley rats (150-200 g). The rats were pretreated with either chlorpromazine (20 mg/kg, ip) or phenobarbital (60 mg/kg, ip) daily for 3 days prior to sacrifice, or with actinomycin D (100μg/kg, ip) or actinomycin D plus phenobarbital (60 mg/kg, ip) 24 and 12 hr prior to sacrifice. Control rats were treated with saline. After sacrifice, livers were immediately excised, blotted dry, weighed, placed in cold 1.15% KCl, minced with scissors and the KCl was decanted. Each liver was individually homogenized in 2 vol of 1.15% KCl with a Potter-Elvehjem homogenizer (Teflon pestle). Cell debris, cell nuclei, and mitochondria were removed by centrifugation at 9000g for 20 min. The microsomal fraction was obtained by centri-fugation at 100,000g for 60 min.

Autoradiographische Untersuchung zur nukleären 3H-Thymidin-Inkorporation im Homogenat regenerierender Leber

Introduction: Nuclei of regenerating rat liver are able to incorporate precursors of DNA synthesis under suitable conditions in vitro. To evaluate the number of parenchymal nuclei which contribute to the incorporation of 3H-thymidine, autoradiograms of homogenized and incubated rat liver were analysed 23 hrs after a two third hepatectomy and the labelling index of 3H-TdR incorporating nuclei was determined. The influence of various conditions of incubation was investigated. Material and methods: The residual part of the livers of partially hepatectomized rats (Wistar AF/Han, male, 200–225 g) were homogenized in a Potter homogenizer with a teflon pestle and incubated in 4 volumes of TCM 199 containing 12 mM NaHCO 3 after addition of deoxythymidine-6- 3H and ATP. After 60 min of incubation at 37° C the homogenate was layered over sucrose and centrifuged at 600 × g for 10 min. The remainder was resuspended, recentrifugated and fixed with 10% formalin containing 0,4 mM of inactive deoxythymidine. Autoradiograms were made with stripping film (Kodak AR 10), and the percentage of labelled nuclei was determined. Results: The average labelling index of liver cell nuclei was 15% under the conditions of preparation and incubation as described. Elevating the concentration of ATP in the incubation medium did not significantly influence the amount of 3H-TdR labelled nuclei, but the number of silver grains overlying labelled parenchymal nuclei increased. After replacing TCM 199 by isotonic sucrose solution only a few silver grains were overlying a much reduced number of parenchymal cells. Discussion: The findings are compared with the results of regenerating liver in vivo. It can be assumed that DNA synthesis which has been started in vivo continues under adequate experimental conditions of incubation in vitro. Addition to the incubation medium of deoxyribonucleotides, however, is not required. ATP concentration is apparently not a limiting factor for the number of 3H-TdR-incorporating cells, but for the duration of 3H-TdR incorporation in vitro.

Preparation of hepatic microsomal fraction for drug metabolism studies by rapid decompression homogenization.

SummaryThe applicability of rapid decompression as a method for homogenization of liver in the preparation of a microsomal fraction has been studied. It was shown that this method is more efficient and probably less variable than homogenization with a Teflon pestle and glass tube. Increased efficiency was related to more complete cell disruption and probably to the production of smaller microsomal particles. The specific content and activity of components of the mixed function oxidase system of the microsomal fraction were similar to those produced with pestle and tube.Supported by Grant No. HD-03074, Institute of Child Health and Human Development, NIH.

Biochemical characteristics of insect microsomes: N- and O-demethylation

The N-demethylation of N, N-dimethyl- p-nitrophenol carbamate and the O-demethylation of p-nitroanisole by the microsomes from housefly abdomens were studied. Demethylation activity of male flies rises to a maximum at 4 days after emergence, while the maximum activity of females is not realized until 7–9 days after emergence. Homogenization with a teflon pestle is more effective than a blender, and 0.20 M potassium phosphate buffer (pH 7.8) was found to be superior to all other media tested for the preparation of the microsomes. It was apparent that the ionic strength of the preparative medium is more important than either molarity or pH for obtaining microsomes of maximum N- and O-demethylating activity. During the subsequent incubation, 0.5 to 1.0% bovine serum albumin is required for maximum activity. The pH optimum for both O- and N-demethylation is between 7.7 and 8.1 with added NADPH or with the generating system, and both reactions show the greatest activity at 30–33°. The apparent K m for pNA is 9.0 ± 0.4 × 10 −5 M and that for DpNC is 4.0 ± 1.6 × 10 −4 M.

Metabolism of ATP by testis mitochondria of 25-day-old rats.

ATP is known to play a key role in the storage and utilization of metabolic energy in most mammalian tissues. The enhancement of testicular protein biosynthesis by glucose appears to be related to the production ofATP (Means & Hall, 1968). However, there does not appear in the literature any information regarding the metabolism of this essential nucleotide in the testes. This com¬ munication presents preliminary data relating to the catabolism of ATP by a mitochondrial fraction derived from rat testes. The animals used in these experiments were 25-day-old male SpragueDawley rats. Testes were removed, decapsulated, pooled and homogenized with a motor driven teflon pestle (clearance 0-1 to 0-15 mm) in 0-32 M-sucrose 10:1 (v/w). The homogenization was carried out in a cold room at 4° C. The homogenate was centrifuged at 3000 g for 10 min and the sediment discarded. The supernate was then centrifuged at 10,000 g for 20 min to obtain the mito¬ chondrial fraction. The mitochondrial pellet was resuspended in distilled water and the protein concentration determined (Lowry, Rosebrough, Farr & Randall, 1951). Incubation of the mitochondrial fraction with appropriate co-factors and radioactive ATP was carried out as described elsewhere (Hollinger, 1970). The nucleotides were separated by paper and thin-layer chromatography in a solvent system containing butanol, acetone, glacial acetic acid, water and con¬ centrated ammonium hydroxide (14:10:6:9:1, v/v). Rf values for the migra¬ tion of ATP, ADP and AMP in this solvent system are 0-15, 0-28 and 0-52, respectively. The UV-absorbing spots corresponding to the migration of authen¬ tic samples were cut out and assayed for radioactivity. Recovery of radioactivity ranged from 89 to 95 %. The mitochondria were fixed for 1 hr in 1-6% glutaraldehyde in 0-1 mphosphate buffer at 4° C. They were post-fixed in 1 % Os04, dehydrated in ethanol, and embedded in Epon 812. Ultra-thin sections were cut with diamond

Subcellular Particles of Human Platelets

ABSTRACTHuman platelet subcellular particles have been fractionated by three methods. The isolated fractions have been examined by electron microscopy and assayed for protein, serotonin, lactate dehydrogenase, cytochrome c oxidase, β‐hydroxybutyrate dehydrogenase, 5′‐nucleotidase, acid phosphatase, β‐glucu‐ronidase, β‐galactosidase, β‐N‐acetylgluco‐saminidase, cathepsin, aryl sulphatase and a‐mannosidase. The latter two enzymes had low activities. Homogenization with blendor caused great solubilization of serotonin and lysosomal enzymes but not cytochrome c oxidase. The granules in the homogenate were swollen and showed great loss of osmo‐philia. The particles were isolated by differential centrifugation and separated into 3 zones after centrifugation on discontinuous sucrose gradients. The upper zone contained proteinaceous material, the second contained mostly vesicular membranes and the lowest one contained α‐granules, transected pseudo‐pods plus mitochondria with varying degrees of preservation. Acid phosphatase was present in the membranes but the highest activity was found in the upper part of the granular band together with β‐galactosidase. β‐Glucuronidase, β‐N‐acetylglucosaminidase and cathepsin were mainly located in the lower part of this band. Homogenization with a ‘no‐clearance’ Teflon pestle caused less solubilization than the blendor and gave better organelle preservation. Isolated particles from such homogenates separated into two bands on gradient centrifugation which morphologically and biochemically were equal to those obtained when these homogenates were applied directly on the gradient. The upper particulate band contained membranes and had bound 5′‐nucleotidase. The lower band possessed in its upper part well‐preserved α‐granules and mitochondria whereas in the lower regions more electron dense granules, often with ‘Bull's eye’, were present. Swollen α‐granules were present mainly in the lower part of this band. All lysosomal enzymes were localized at the same sucrose density. Probably, the α‐gra‐nules are the platelet lysosomes, but with pestle homogenization there was not the biochemical heterogenity as found when using the blendor. With pestle homogenization particle bound serotonin was found in fractions of high sucrose density which contained the dense α‐granules with the ‘Bull's eye’.

Phosphorylation of protamine during spermatogenesis in trout testis.

Recent experiments by Kleinsmith, Allfrey, and Mirsky' and Stevely and Stocken2 have clearly demonstrated the incorporation of P32-phosphate into the histones of calf and rat thymus nuclei, the site of linkage of this phosphate being 0-phosphoserinel' 2 or O-phosphothreonine.1 Considerable interest focuses upon these findings as suggesting a means whereby the activity of histones in complexing with DNA may be modulated. Kleinsmith et al.' have reported that an increase in phosphorylation of nuclear proteins correlates with an increase of RNA synthesis in lymphocytes stimulated to grow and divide by phytohemagglutinin, while Stevely and Stocken2 have shown an increased ability of complexes of DNA with phospho-fl-histone to prime in vitro RNA synthesis by RNA polymerase as compared with the nonphosphorylated histone-DNA complex. This paper reports studies on protamines labeled with P32-phosphate or C14_ serine by incubation of dissociated intact cells from testes of steelhead trout (Salmo gairdnerii). Development of the testes and spermatogenesis was induced4 by injecting the fish with an extract of pituitary glands from salmon. Testes were obtained in this way conveniently and reproducibly at various stages of maturation. During the later stages of maturation these cells, which are actively synthesizing the characteristic sperm nuclear protein, protamine,' have been shown to incorporate P32-phosphate into both histone and protamine fractions. The presence of 0-phosphoserine in partial acid hydrolysates of a commercially available preparation of protamine (salmine) was noted by Murray.5 More recently Langan and Smith6 have isolated a kinase from rat liver which catalyzes the phosphorylation of protamine and histones in vitro. In the present work phosphorylation has also been shown to be at serine residues, and several labeled phosphopeptides have been isolated by tryptic digestion of P32-protamine. Parallel incorporation of C'4-serine and P32-phosphate into protamine shows that all newly incorporated serine residues are phosphorylated. Since the total content of 0-phosphoserine in protamine falls during the later stages of maturation of the testis, a rapid turnover of phosphate is indicated. This view is confirmed by the almost complete absence of phosphate in protamine extracted from mature sperm collected from naturally spawning trout and salmon. Methods.-Cells at a stage active in protamine biosynthesis were obtained from the testes of steelhead trout in which spermatogenesis had been induced by injections of salmon pituitary extracts.4 Mature spermatozoa were collected by expressing milt from both steelhead trout and coho salmon (Onchorhynchus kisutch) during annual spawning operations at fish hatcheries on the Skagit River (State of Washington Departments of Game and Fisheries). Incubation procedures: Cell suspensions from the developing trout testes were prepared at 0?C as previously described.3 Minced tissue was suspended in buffer (0.14 M NaCl, 5.4 mM KCI, 0.81 mM MgSO4, 1.26 mM CaCl2, 5.6 mM glucose, 4.2 mM NaHCO3, pH 7.2) and gently homogenized by hand in Potter-Elvehjem tubes with a glass-reinforced Teflon pestle. The cell suspension, centrifuged and resuspended in the same medium, was at a final dilution of 8 ml packed