Sedimentation Properties of Dextran Sulfate-Low Density Lipoprotein Complexes

The nature of interaction between dextran sulfate and the human plasma low density lipoproteins of S_f 0–10 was investigated in high density media of glycine and glucose. The soluble complex formation between the two components was manifested by sedimentation of the lipoproteins along with dextran sulfate in the glycine and glucose media of density 1.063. The addition of sodium chloride to the mixture caused dissociation of the complex: during subsequent ultracentrifugation, flotation of lipoprotein and sedimentation of dextran sulfate occurred. However, when the complex is in the acidic glycine medium (pH 4.0), the addition of sodium chloride did not induce dissociation of the complex.

Both the solubility and the size of the complex were greatly influenced by the ratio of the two components in solution. At low relative concentrations of dextran sulfate, insoluble aggregates were formed; but the aggregates disintegrated into soluble units upon increasing the dextran sulfate concentrations. From the sedimentation patterns of dextran sulfate lipoprotein mixtures at various ratios, it was possible to estimate the ratio of the two components in the complex. In the presence of excess dextran sulfate a composite biphasic Schlieren diagram was produced as a result of the unusual Johnston-Ogston effect.     

Cross-linking of wheat starch and hydroxypropylated wheat starch in alkaline slurry with sodium trimetaphosphate

Wheat starch was cross-linked at 40 °C and pH 11.0 by slurrying the starch (30% solids) in a solution of sodium trimetaphosphate (STMP), sodium hydroxide, and sodium sulfate. The extent of cross-linking was determined by an increase in alkaline fluidity or by a decrease in alkaline clarity. Response surface analysis showed that cross-linking increased with increasing levels of STMP (0.5-1.5%, based on starch, bos) and sodium sulfate (0–4.0%, bos) over a reaction period of 120–720 min. A regression equation with first and second order terms showed that STMP and sodium sulfate concentrations and the reaction time accounted for 99% of the variability in alkaline fluidity. Wheat starch (37% slurry) was hydroxypropylated by reaction with propylene oxide (8%, bos) for 24h at 45 °C in alkali (pH 11.5) containing 16.0% sodium sulfate (bos). The hydroxypropylated (4.5wt%) wheat starch (DS 0.12-0.13) was not isolated but was cross-linked with STMP (0.1-0.5%, bos) over a 10–40 min reaction period. A comparison of pasting curves at pH 3.5 showed that some of the cross-links produced by STMP were less stable than those produced by phosphoryl chloride, indicating a low level of pyrophosphate as well as monophosphate cross-links. The less stable cross-links were diminished by changing reaction conditions with STMP.     

Soluble and membrane-bound forms of lipoprotein lipase in mouse lung tissue

Homogenates of mouse lungs were separated by differential centrifugation into two fractions containing lipoprotein lipase, namely, a soluble and a membrane-bound fraction. Lipoprotein lipase was specifically identified by its inhibition by both protamine sulfate (3 mg/ml) and sodium chloride (0.9 mol/l). The enzymatic activity of each fraction was enhanced when serum was preincubated with the enzyme. Both enzyme fractions showed optimum activity at alkaline pH, but the membrane-bound enzyme showed a higher pH optimum. In addition, the apparent Km of the soluble enzyme was lower than that of the membrane-bound enzyme. It is concluded that there are two different forms of lipoprotein lipase in mouse lung tissue that differ in a number of aspects.     

Dissociation of polyoma virus by the chelation of calcium ions found associated with purified virions.

Analysis of polyoma virions by X-ray fluorometry demonstrated that calcium (Ca2+) was associated with the purified virion. Treatment of purified virions with ethyleneglycol-bis-N,N'-tetraacetic acid (EGTA), which chelates Ca2+, and the reducing agent dithiothreitol caused the virions to dissociate. Electron microscopy revealed that the virions were dissociated to the capsomere level. Incubation of polyoma virions with 150 mM NaCl, 10 mM EGTA, and 3 mM dithiothreitol was optimum for the dissociation reaction. The pH for the dissociation reaction ranged from 7.5 to 10.5. Cesium chloride density gradient centrifugation indicated that both EGTA and dithiothreitol were necessary for dissociation to occur; neither reagent alone dissociated the virus. The major protein product of the dissociated viral particles sedimented at 12S. Relationships between these experiments and the alkaline carbonate-bicarbonate dissociation of polyoma are discussed.     

Alkali-aided protein extraction from chicken dark meat: Chemical and functional properties of recovered proteins

Alkali-aided extraction of proteins from chicken thigh meat has been attempted for improved utilization of this low value raw material. This study focused on chemical and functional properties of proteins recovered after extraction in alkaline conditions at pH values of 10.5, 11.0, 11.5 and 12.0. Solubility of total proteins was significantly reduced (P < 0.0001) for proteins extracted at higher pH (11.5 and 12.0) with a significant increase in protein hydrophobicity. Despite the lower protein solubility, protein recovery yield was found to be higher at pH values of 11.5 and 12.0. Reactive sulfhydryl content also significantly increased (P < 0.05) for these two treatments. Alkali treatments at pH 10.5 and 11.0 were found to have no effect on myofibrillar protein hydrophobicity which was found to be similar to that of raw meat protein, indicating that the proteins retained the native conformation. SDS-PAGE of extracted proteins showed greater amounts of myosin heavy chain relative to raw meat. Emulsification capacity showed relatively little change for samples with different extraction pH values, although it was significantly higher for pH 11.0. The foam expansion property of myofibrillar proteins increased significantly with extraction pH values, and obtained a maximum value at pH 11.5. The proteins extracted at pH 10.5 and 11.0, to some extent, could regain their original conformation after pH readjustment to 6.2. The functionality of the recovered protein suggests that this may provide an opportunity for greater utilization of dark poultry meat.     

Preparation and properties of soluble, immunoreactive apoLDL.

Immunoreactive apo-low density lipoprotein (LDL), soluble in mildly alkaline buffers of low ionic strength, was prepared by attaching LDL to a DEAE-Sepharose column and eluting the lipids with a 0--2% (w/v) gradient of nonionic detergents. Brij-36T, Nonidet P-40, and Triton X-100 gave similar results. After washing the detergent from the column, the bound apoLDL was eluted with 1 M NaCl, pH 7.4, with recoveries up to 85%. This apoLDL could be dialyzed extensively against low ionic strength solutions, and remained soluble as long as the pH was above 7. Spectrophotometric analysis showed that less than 0.1% %w/v) of cholesterol or phospholipids and less than 1% (w/v) of detergent remained associated with the protein. The apoLDL cross-reacted with LDL against antisera prepared vs. intact LDL. Pore-gradient polyacrylamide gel electrophoresis, with SDS and urea, showed that this preparation was less aggregated than organic solvent extracted apolLDL and appeared to be made of oligomers of two monomeric subunits, one with molecular weight around 22,700 and a smaller one of approximately 8000. Isoelectric focusing showed that there also was charge heterogeneity in the soluble apoLDL.     

Alkali-induced conformational transition in different domains of bovine serum albumin

Alkaline pH induced conformational changes in different domains of bovine serum albumin were studied by using domain specific ligands: chloroform, bilirubin and diazepam for domains I, II and III respectively. The effect of alkaline pH on the secondary structure of BSA was monitored by far-UV CD in the range 250 nm to 200 nm. The pH profiles of BSA in the alkaline region showed a two-step change, one corresponding to N<-->B transition (pH 7.5 to 9.0) and the other to B --> U (pH 11.0 to 13.5). Binding of chloroform decreased continuously on increasing pH, whereas binding of diazepam, remained unchanged up to pH 9 and decreased thereafter. In contrast, binding of bilirubin gradually increased up to pH 11.0 and decreased thereafter reaching a value similar to one obtained with native BSA at pH 11.5. Above pH 11.5, bilirubin binding decreased and was abolished completely at pH 12.5. In the pH region 7.5 to 11.0, a continuous decrease in chloroform binding (pH 7.5 to 9.5) and a late decrease in diazepam binding (pH 9.5 to 11.0) suggested major loss of native conformation of domain I followed by domain III during alkaline induced unfolding of BSA. However, a significant increase in bilirubin binding showed a favorable conformational rearrangement in domain II in this pH region (pH7.5 to 11.0). Further, a nearly complete abolishment of bilirubin binding to BSA and significant loss of secondary structure around pH 12.5 indicated that domain II was more resistant to alkaline pH and unfolds only at extreme alkalinity. Taken together, these data suggest that unfolding of three domains of BSA follow the following order of susceptibility towards alkaline denaturation of BSA domain I>domain III>domain II.     

Turnover and tissue distribution of 125-I-labeled low density lipoprotein in swine and dogs.

Swine plasma low density lipoprotein (LDL) isolated ultracentrifugally (d 1.019-1.063) was labeled with 125-I, dialyzed, and reisolated by centrifugation at d 1.063. Over 96% of the radioactivity was shown to be associated with the apoprotein. After reinjection into the donor animal, disapperance of 125-I was followed for up to 122 hr. At all time intervals examined, over 95% of the total plasma 125-I was recovered in LDL (D 1.006-1.063), i.e., there was apparently no transfer of radioactivity to high density or very low density lipoproteins. The disappearance curve was biexponential, with half-lives of 0.83 plus or minus 0.06 and 22.5 plus or minus 1.7 hr for the first and second phases, respectively (13 studies). The mean calculated fractional catabolic rate was 0.041 plus or minus 0.003 hr-minus 1. Similar results were obtained in three dogs using autologous LDL of density 1.020-1.050; fractional catabolic rates were 0.031, 0.031, and 0.029 hr-minus 1. Tissue distribution of 125-I was determined in swine killed at various time intervals after [125-I]LDL injection with corrections for radioactivity in trapped plasma. Of the tissues examined, the liver showed by far the highest concentration. Total hepatic radioactivity, expressed as a percentage of total plasma radioactivity, was rather constant and independent of the time of killing from 3 to 122 hr (15.8 plus or minus 1.9%). The total extravascular LDL pool calculated from analysis of the plasma disappearance curves was about 20-30% of the size of the plasma LDL pool. These data are consistent with the conclusion that the liver accounts for a very large fraction of the total extravascular LDL pool. These data are consistent with the conclusion that the liver accounts for a very large fraction of the total extravascular LDL pool and that it is infairly rapid equilibrium with the plasma pool. To what extent the liver is involved in irreversible degradation cannot be inferred from these findings.     

Different forms of the oxysterol-binding protein. Binding kinetics and stability

Based upon measurements of the sedimentation coefficient and the Stokes radii, three forms of the oxysterol-binding protein were identified. The unliganded binding protein was the largest (7.7 S, Stokes radius = 71.6 A, Mr = 236,000) was relatively asymmetric (f/f0 = 1.7), and was composed of at least three subunits. Binding of 25-hydroxycholesterol was associated with a reduction in the size of the protein (7.5 S, Stokes radius = 50 A, Mr approximately 169,000) and an increase in symmetry (f/f0 = 1.4), due to the loss of a subunit of Mr approximately 67,000. At pH 6 or lower, the Mr = 169,000 sterol-protein complex was altered so that reversible dissociation to give a smaller (4.2 S, Stokes radius = 53 A, Mr = 97,000) more asymmetric (f/f0 = 1.8) sterol-protein complex occurred when it was sedimented in a sucrose gradient buffered at pH 7.4 containing 0.3 M KCl and 2.5 M urea. Irreversible dissociation of the 7.5 S, Mr = 169,000 form to a 4.2 S form occurred spontaneously when the complex in whole cytosol buffered at pH 7.8 was allowed to stand overnight at 0 degree C, or when the partially purified complex was incubated at pH 5.5 at 0 degree C for several days. The partially purified, unliganded binding protein was unstable at 0 degree C (approximately 75% loss of binding activity in 24 h) whereas the liganded protein was stable for 7 days at 0 degree C although irreversible conversion to a 4.2 S form occurred under some conditions. Rates of sterol binding and dissociation were increased in the presence of 2.5 M urea at pH 7.4 or when the pH was lowered to 5.5 Kd values were not greatly altered under the various incubation conditions.     

Determination of the molecular weight of apoprotein subunits from low density lipoprotein by gel filtration

Another method has been developed for obtaining a soluble apoprotein from the low density lipoprotein (LDL) of human plasma in the density class 1.019 < d < 1.063. The approximate molecular weight of the apoprotein subunit from this lipoprotein density class was determined by gel filtration on Sephadex G-200 to be about 80,000. Both on gel filtration and analytical ultracentrifugation the soluble apoprotein showed one peak, but on cellulose acetate electrophoresis it showed two bands, which suggests two differently charged components. Because of the nature of the determination, the value of 80,000 probably represents an upper limit to the molecular weight of the LDL subunits.     

Isolation of plasma lipoproteins by zonal ultracentrifugation in the B14 and B15 titanium rotors

Lipoproteins were isolated from plasma of man, dog, rabbit, rat, and chicken by ultracentrifugation in continuous density gradients using the B14 titanium and B15 titanium zonal rotors. Both the VLDL and the LDL of human plasma were separated easily from the HDL and from the other more plentiful plasma proteins by centrifugation for only 1 or 2 hr in the B14 or B15 rotor, respectively. Satisfactory separation of the HDL from the more dense plasma proteins was not achieved with these rotors. The human LDL achieved isopycnic equilibrium (d 1.04) on prolonged periods (> 24 hr) of centrifugation in a sucrose-KBr density gradient. The pattern of distribution of cholesterol and phospholipid throughout the density gradient coincided with the pattern of distribution of the lipoprotein-protein measured spectrophotometrically or chemically. The concentration of cholesterol and phospholipid in the lipoproteins isolated by zonal ultracentrifugation agreed with analyses reported for lipoproteins isolated by sequential centrifugation in solutions of increasing density. The lipoproteins isolated by zonal ultracentrifugation were characterized further by their electrophoretic behavior. The fractions which were identified as the LDL (d 1.04-1.05) from all species migrated on paper as a beta-globulin; the LDL from plasma of dogs contained an additional component which has been designated as an alpha(2)-globulin. The fractions which were identified as the HDL from all species migrated as an alpha(1)-globulin. Reaction of human LDL with either rabbit antihuman beta-lipoprotein or rabbit antihuman serum resulted in a single immunodiffusion band. The S(f, 1.063) of the human LDL was calculated to be 6.0. When plasma from humans or rabbits was centrifuged in the B15 rotor, the HDL was not visible as a distinct peak and was not separable from the bulk of the more dense plasma proteins; when plasma from dogs or chickens was centrifuged under identical conditions, the HDL was clearly detectable. Even though the mean density of the HDL from dogs or chickens was not different from that of man or rabbits, the visibility of this lipoprotein in dogs and chickens was probably due to its high concentration in the plasma of these species. When plasma from the rat was centrifuged under similar conditions, the HDL was also clearly in evidence. Although rat plasma contained a relatively small concentration of HDL, the lipoprotein had a lower mean density than did the HDL of the other species and was therefore more easily separable from the dense plasma proteins. The procedure of zonal ultracentrifugation for the isolation of lipoproteins by flotation is simultaneously preparative and analytical and should find useful application in the investigation of the soluble lipoproteins from plasma and tissues.     

Lipoprotein(a) binding to other apolipoprotein B containing lipoproteins

A method combining ligand dot blotting and digital imaging was used to determine the apparent dissociation constant (KD) for the binding of lipoprotein(a) to low-density lipoproteins (Lp(a)-LDL2). By use of this approach, the KD for the Lp(a)-LDL2 complex was shown to be in the nanomolar range [(1.05 +/- 0.21) x 10(-8) M, n = 4]. The Lp(a)-LDL2 interaction was both hydrophobic and ionic; however, hydrophobic forces predominated because the interaction was demonstrable at high salt concentration (greater than 2 M NaCl), while no complex was detectable at low salt concentration (less than 0.08 M NaCl). Consistent with the hydrophobic nature of this interaction, the Lp(a)-LDL2 complex was stable over a wide pH range (4-10). Plasminogen did not compete with Lp(a) binding to LDL2 even at a 2.2 X 10(3) molar excess of plasminogen over the LDL2 concentration. The only component identified in plasma and serum that inhibited the binding of LDL2 to Lp(a) was apolipoprotein B containing lipoproteins (apoB-Lp). These studies indicate that the Lp(a)-LDL2 complex could exist in plasma. In fact, up to 72% of purified Lp(a) added to an Lp(a)-negative hypertriglyceridemic plasma floated with apoB-Lp (d less than 1.063 g/mL) following ultracentrifugation, whereas only 9% of the purified Lp(a) added to the apoB-Lp-free 1.12 g/mL infranate floated at d less than 1.063 g/mL. The formation of a complex of Lp(a) with apoB-Lp could increase the amount of cholesterol ester bound per cellular receptor, e.g., LDL receptor, and thus potentially accelerate cholesterol removal from the vascular compartment.     

TEMPO-mediated oxidation of maltodextrins and D-glucose: effect of pH on the selectivity and sequestering ability of the resulting polycarboxylates

Maltodextrins were oxidized to polyglucuronic acids with the ternary oxidation system: NaOCl-NaBr-2,2,6,6-tetramethylpiperidine-l-oxyl (TEMPO). The chemoselective oxidation at the primary alcohol groups was shown to be strongly pH dependent. Oxidation of polysaccharides was best achieved at pH 9.5 in order to minimize depolymerization, whereas oxidation of oligosaccharides required stronger alkaline conditions (pH 11-11.5). The resulting sodium polyglucuronates present interesting sequestering properties, the best of which being obtained from maltodextrins with the highest degrees of polymerization. The same oxidation process allowed the convenient conversion of D-glucose to D-glucaric acid in high yield (> 90%), under strongly basic conditions (pH > 11.5).     

The influence of alkali and heat treatment on yeast protein

The soluble components in disintegrated cells of Saccharomyces cereivisiae have been characterized by means of extraction, centrifugation, dialysis, and gel filtration. The influence of alkali and heat treatment on the protein and RNA in the soluble fraction from disintegrated yeast cells and on functional properties of protein concentrates have been studied. After water extraction and centrifugation at 100000 g 42% of the nitrogen containing components of the disintegrated cells were recovered in the supernatant. By extraction at pH 11.5 an additional 31% of the nitrogen was solubilized. Half of the water-soluble nitrogen-containing components has a molecular weight lower than 5000. In the water- and alkali-soluble fractions about 80% of each amino acid was recovered The water-soluble protein was separated into 3 fractions by gel filtration on Sephadex G 200. The major portion of the protein had a molecular weight about 100,000. The amount of protein in this fraction was decreased after treatment at increasing pH and temperature. No degradation of protein to low molecular peptides occurred. The amount of RNA in the soluble fraction was only slightly influenced by alkali treatment and by heat treatment at pH 7.5 in the presence of 5% NaCl. RNA was not degraded to low molecular components of the treatments. The solubility of protein concentrates decreased after treatment at alkaline pH and after heat precipitation.     

The effect of temperature on the interaction of Yersinia pseudotuberculosis lipopolysaccharide with chitosan

The mechanism of binding of lipopolysaccharide (LPS) from Yersinia pseudotuberculosis to low-molecular-weight chitosan was investigated using sedimentation analysis, centrifugation in glycerol and percoll density gradients, and isopicnic centrifugation in cesium chloride. The LPS interaction with chitosan was shown to be a multistage process that depended on time and reaction temperature. A stable LPS-chitosan complex could be formed only after preliminary incubation of the initial components at an elevated temperature (37 degrees C). This temperature caused the LPS dissociation and promoted its binding to chitosan. The LPS binding to chitosan results in further dissociation of the endotoxin and formation of the complex with a molecular weight that is tens of times less than the initial molecular weight of LPS. The obtained complex remained stable in solutions of high ionic strength.     

Isoionic titration and isopycnic density gradient centrifugation studies of magnesium activation and subunit dissociation in yeast enolase.

Isopycnic density gradient centrifugations were performed on yeast enolase A in cesium chloride and sulfate at the isoionic pH (6.0–6.5) and at pH 8.1. The dissociation of the enzyme appears to be greater at the more alkaline pH. No large effect of the cofactor magnesium or pH or dissociation on the isopycnic point was found. Isoionic titrations were carried out in the presence and absence of magnesium using both salts. The metal reduces net anion binding by isoionic enzyme and net cation binding by pH 8.1 enzyme by about 1.6 – 3.4 equivalents at 0.05 ionic strength and somewhat less at the isopycnic ionic strengths. It is concluded that the metal does not significantly affect net hydration of the enzyme and that subunit dissociation is not accompanied by large changes in hydration or salt binding.     

Some properties of acid and alkaline phosphates from boar sperm plasma membranes

Boar sperm plasma membranes were purified by differential and sucrose density equilibrium centrifugation and were found to yield a single band at a density of 1.14 g/cm3. Both alkaline and acid phosphatase activities were enriched in this fraction. The alkaline phosphatase activity was optimal in 100 mM tris (hydroxymethyl) methylamine (Tris)-NaHCO3 at pH 9.9 with 0.05% Triton X-100 and 1 mM MgCl2. This activity was inhibited by ethylenediaminetetraacetic acid (EDTA), cadmium, zinc or heating at 60 degrees C for 30 min. Also, L-homoarginine caused approximately 70% inhibition and L-phenylalanine or L-leucine caused about 10 to 20% inhibition. Acid phosphatase activity was optimal in 100 mM sodium acetate at pH 5.1 with 0.05% Triton. Sodium dodecyl sulfate, potassium fluoride (KF) or sulfhydryl reagents inhibited the activity, while EDTA or heating at 60 degrees C had no effect. These data for enzymes from boar sperm plasma membranes can be used for future work on the quantitation of the enzymes, distinguishing these two phosphatases from other phosphohydrolases, purification of the enzymes and for comparison to phosphatases in other tissues.     

A method for obtaining protein concentrates from microorganisms

In order to isolate proteins from microalgae, yeasts and bacteria, cell disintegration in a special ball-mill was performed. The degree of disintegration of the different microorganisms was compared. The dependence of disintegration on bead size and on the ratio between the volume of suspension and the volume of glass beads was also investigated. Nondisintegrated and disintegrated cells were extracted with sodium hydroxide and the amount of extractable nitrogen and the amount of nitrogen precipitable at pH 4.0 were determined. The dependence of yield on the sodium hydroxide concentration, extraction time, and temperature was studied. When extracting undisintegrated cells, very low yields were obtained and the nitrogen extracted was mostly nonproteinous. For disintegrated cells high yields were obtained. An optimum was found after extraction with 0.3–0.5% sodium hydroxide; at pH 11.0–11.5. The precipitate obtained represented 60–70% of the cell nitrogen. The nitrogen content of the precipitate was 12–14% of the dry weight.     

Interaction between flavonoids and alpha-tocopherol in human low density lipoprotein

Oxidative modification of low density lipoprotein (LDL) may play an important role in the development of atherosclerosis. Alpha-tocopherol functions as a major antioxidant in human LDL. The present study was to test whether four natural flavonoids (kempferol, morin, myricetin, and quercetin) would protect or regenerate alpha-tocopherol in human LDL. The oxidation of LDL incubated in sodium phosphate buffer (pH 7.4, 10 mM) was initiated by addition of either 5.0 mM CuSO(4) at 37 degrees C or 1.0 mM of 2,2'-azo-bis (2-amidinopropane) dihydrochloride (AAPH) at 40 degrees C. It was found that alpha-tocopherol was completely depleted within 1 hour. Under the same experimental conditions, all four flavonoids demonstrated a dose-dependent protecting activity to alpha-tocopherol in LDL at the concentration ranging from 1 to 20microM. All flavonoids showed a varying protective activity against depletion of alpha-tocopherol in LDL, with kempherol and morin being less effective than myricetin and quercetin. The addition of flavonoids to the incubation mixture after 5 minutes demonstrated a significant regeneration of alpha-tocopherol in human LDL. The protective activity of four flavonoids to LDL is related to the number and location of hydroxyl groups in the B ring as well as the stability in sodium phosphate buffer.     

Proteolytic enzymes in sea urchin eggs: characterization, localization and activity before and after fertilization

The pH versus proteinase activity curve (casein or hemoglobin plus urea substrate) for homogenates of unfertilized Lytechinus eggs reveals two regions of maximum activity: one between pH 3.5 and 4.3, and another of far greater magnitude from pH 8.0 to 11.0. The two classes of proteinases can be separated on a sucrose density gradient. Both the acid and alkaline proteinases in homogenates prepared in isotonic monovalent salt solutions are remarkably stable at pH 7.4 and 0°C. Using synthetic peptide substrates, an enzyme with the specific esterase activity of chymotrypsin was demonstrated; this enzyme accounts for the major part of the proteinase activity at alkaline pH. In addition, an enzyme with specific esterase activity of trypsin was shown to be present, but of low activity. The proteinase activity at acid pH is largely due to an enzyme resembling cathepsin D. The data also suggest the presence of cathepsin B and cathepsin IV (or catheptic carboxypeptidase). When eggs are homogenized in isotonic NaCl plus KCl at pH 7.4, 0.02 M tris buffer at 0°C, all of the alkaline proteinase, and 85–90% of the acid proteinase activity is sedimented at 10,000 g. The presence of any proteinase activity in the supernatant phase represents an artifact of the preparative procedures used. The granules which possess the proteinase activity are contained entirely in the yolk fractions; and the acid proteinase is contained in a population of granules which sediment more readily than those which contain the alkaline proteinase. The acid proteinase resembles the lysosomal acid hydrolases in that it is readily released from the particulates; in contrast, the alkaline proteinase is bound relatively firmly. In contradistinction to reports in the literature, no changes in proteinase activity nor intracellular distribution could be detected following fertilization.