Isolation of purified copper protein from horse liver

1. A procedure is described for isolating a copper protein from horse liver in pure form, in a state that appears to be crystalline. 2. This copper protein is colored blue-green, and its copper content varied between 0.3 and 0.4 per cent. No indication of any catalytic properties was obtained in the limited number of tests made. Because of its variable copper content, the protein may function for the storage of copper in the body. 3. The copper could be removed partly from the protein by dialysis against cyanide, and a colorless crystalline protein was obtained by lowering the pH to 5.0, followed by dialysis. The original blue-green color of the protein could be restored by dialysis against copper acetate solution.     

PROTEIN COAGULATION AND ITS REVERSAL : THE REVERSAL OF THE COAGULATION OF HEMOGLOBIN

1. The preparation from completely coagulated hemoglobin of crystalline soluble hemoglobin is described. 2. This soluble hemoglobin by all the tests tried has been indistinguishable from normal native hemoglobin which has never been coagulated. 3. The coagulation of hemoglobin is probably reversible. 4. Since hemoglobin is a typical coagulable protein, protein coagulation in general is probably reversible.     

ISOLATION OF A CRYSTALLINE PROTEIN COMPOUND OF TRYPSIN AND OF SOYBEAN TRYPSIN-INHIBITOR

A crystalline protein compound has been isolated from a solution containing crystalline trypsin and crystalline soybean inhibitor. The protein consists of about equal weights of trypsin and of the inhibitor. Denaturation by heat or by alkali resolves the compound into its components.     

CRYSTALLINE PEPSIN : V. ISOLATION OF CRYSTALLINE PEPSIN FROM BOVINE GASTRIC JUICE

1. A method has been described for isolating a crystalline protein with high proteolytic activity from bovine gastric juice by means of precipitation with magnesium sulfate and fractionation of the precipitate with acetone and magnesium sulfate. 2. The crystalline protein obtained in this way has the same crystalline form, optical activity, and specific activity, as determined by a number of methods, as does the crystalline protein previously isolated from swine gastric mucosa. 3. The solubility of the two preparations, however, is additive so that they are different although very closely related proteins.     

Novel crystalline sheets of Na,K-ATPase induced by phospholipase A2

Treatment of purified preparations of Na,K-ATPase by phospholipase A2 has led to the formation of two-dimensional crystals of the protein. Control tests with another phospholipase and two detergents have shown that crystallization occurs as the result of hydrolysis and/or solubilization of the phospholipids in the enzyme vesicles. Experimentation with various buffer systems has indicated that reduction in the amount of phospholipids alone is sufficient for inducing the formation of crystalline sheets. Inclusion of crystal inducing ions in the buffer facilitates the crystallization process, resulting in more extensive arrays. The new crystalline sheets are exclusively dimeric with average unit cell dimensions: a = 15.8 +/- 0.4 nm, b = 4.9 +/- 0.2 nm, and gamma = 64 +/- 3 degrees. Examination of the micrographs shows that the initial intermolecular interaction leading to the formation of sheets is between the alpha subunits. Results from this study suggest that removal and/or modification of phospholipids by phospholipases could prove successful in crystallizing those membrane proteins in which excess lipid is the main barrier to the formation of two-dimensional arrays.     

Stability of crystalline proteins

By using two model proteins, glucose oxidase and lipase, we demonstrate that dry crystalline formulations are significantly more stable than their amorphous counterparts. The results of Fourier-transform infrared spectroscopy indicate that crystalline proteins better maintain their native conformation in accelerated stability studies. The lower tendency of crystalline proteins to aggregate is confirmed by size-exclusion chromatography. The data suggest that protein crystallization may significantly improve some aspects of protein handling, and change the way biopharmaceuticals are produced, formulated, and delivered.     

Morphological observations on the formation and stability of the crystalline arrays in the plasma membrane of Saccharomyces cerevisiae

Two-dimensional crystalline arrays of freeze-fracture particles are known to occur in abundant quantities in the plasma membrane of stationary state yeast cells. Although these crystalline arrays are seen only infrequently in cells during mid-exponential growth, we now observe that formation of crystalline arrays can be induced in such cells by a “metabolic starvation” protocol. Surprisingly, starvation-induced formation of crystalline patches can be prevented by inhibition of new protein synthesis during the starvation period. The size and quantity of crystalline arrays can be increased by removal of the cell wall prior to starvation. Induction of crystalline arrays in protoplasts has made it possible to investigate the surface morphology of the crystalline particles in isolated membranes as well as at the extracellular surface of intact protoplasts. The stability of isolated crystalline arrays to several detergents has been investigated and conditions have been found that result in improved morphological purity of the isolated crystalline patches.     

THE PRESENCE OF A GELATIN-LIQUEFYING ENZYME IN CRUDE PEPSIN PREPARATIONS

A protein fraction has been isolated from crude pepsin preparations which is about 400 times as active as crystalline pepsin in the lique-faction of gelatin. The activity as measured by the digestion of casein, edestin or egg albumin is less than that of crystalline pepsin. It is more resistant to alkali than the crystalline pepsin.     

ULTRASTRUCTURE OF THE MATURE UNGERMINATED RICE (ORYZA SATIVA) CARYOPSIS. THE STARCHY ENDOSPERM

The structure of the starchy endosperm of rice (Oryza sativa) was studied by using light and transmission electron microscopy coupled with proteolytic enzyme digestions. The starchy endosperm was divided into two regions, the subaleurone and central, based on the number and types of protein bodies observed. The subaleurone region contained three different types of membrane bounded protein bodies—large spherical, small spherical, and crystalline protein bodies. The small spherical protein bodies were most numerous and the large spherical ones were least numerous. The crystalline protein bodies displayed crystal lattice fringes and were a composite of smaller angular components. The central region lacked both the small spherical and crystalline protein bodies. The large spherical protein bodies of this region were located in pockets of densely stained proteinaceous material. In contrast to the relatively well preserved cytoplasm of the subaleurone region, the central endosperm zone consistently was poorly preserved.     

Catalytic activity of non-cross-linked microcrystals of aspartate aminotransferase in poly(ethylene glycol).

The molar activity of crystalline mitochondrial aspartate aminotransferase is decreased to 10% of that of the enzyme in solution. The activity was measured in suspensions of non-cross-linked microcrystals (average dimensions 22 microns X 5 microns X 0.8 microns) in 30% (w/v) poly(ethylene glycol). Kinetic tests ruled out the possibility that diffusion of the substrate in the crystals is rate-limiting. The observed decrease in catalytic efficiency can be attributed exclusively to crystal-packing effects. A direct inhibition by poly(ethylene glycol) is excluded because poly(ethylene glycol), with average Mr 6000, cannot penetrate the liquid channels of the crystals, owing to its large Stokes radius. The crystals examined were triclinic and of the same habit as those used for high-resolution X-ray-crystallographic analysis [Ford, Eichele & Jansonius (1980) Proc. Natl. Acad. Sci. U.S.A. 77, 2559-2563]. The catalytic competence of crystalline aspartate aminotransferase confirms the relevance of the spatial model of this protein for the elucidation of its mechanism of action.     

Characterization of the cellulose-binding domain of the Clostridium cellulovorans cellulose-binding protein A.

Cellulose-binding protein A (CbpA), a component of the cellulase complex of Clostridium cellulovorans, contains a unique sequence which has been demonstrated to be a cellulose-binding domain (CBD). The DNA coding for this putative CBD was subcloned into pET-8c, an Escherichia coli expression vector. The protein produced under the direction of the recombinant plasmid, pET-CBD, had a high affinity for crystalline cellulose. Affinity-purified CBD protein was used in equilibrium binding experiments to characterize the interaction of the protein with various polysaccharides. It was found that the binding capacity of highly crystalline cellulose samples (e.g., cotton) was greater than that of samples of low crystallinity (e.g., fibrous cellulose). At saturating CBD concentration, about 6.4 mumol of protein was bound by 1 g of cotton. Under the same conditions, fibrous cellulose bound only 0.2 mumol of CBD per g. The measured dissociation constant was in the 1 microM range for all cellulose samples. The results suggest that the CBD binds specifically to crystalline cellulose. Chitin, which has a crystal structure similar to that of cellulose, also was bound by the CBD. The presence of high levels of cellobiose or carboxymethyl cellulose in the assay mixture had no effect on the binding of CBD protein to crystalline cellulose. This result suggests that the CBD recognition site is larger than a simple cellobiose unit or more complex than a repeating cellobiose moiety. This CBD is of particular interest because it is the first CBD from a completely sequenced nonenzymatic protein shown to be an independently functional domain.     

PARTICLE SIZE OF INSECTICIDAL SUSPENSIONS AND THEIR CONTACT TOXICITY III. TEMPERATURE COEFFICIENTS AND TESTS BY INJECTION

Aqueous DDT suspensions containing particles of different sizes gave different temperature coefficients of mortality in dipping tests against adult Oryzaephilus surinamensis and Tribolium castaneum. In the range 12–30°C., colloidal DDT showed a large negative coefficient, but the coefficient for a suspension of DDT crystals of 400μ was smaller ( T. castaneum ) or negligible ( O. surinamensis ). Thus, although a suspension of crystalline DDT was more toxic than colloidal DDT to warm insects, it was less toxic than the colloid to cool insects.
A test was made of rotenone suspensions against adult milkweed bugs ( Oncopeltus fasciatus Dall.) by application of measured droplets under the wings. Colloidal rotenone was more toxic than a crystalline suspension. Milkweed bugs could not be killed in this way by DDT suspensions.
Tests were also made by injecting suspensions of rotenone, DDT and its fluorine analogue (DFDT) into milkweed bugs. A suspension of rotenone crystals was equitoxic with colloidal rotenone against insects kept for 3 days at 27°C. after treatment. The same was true of crystalline and colloidal DDT suspensions. If the bugs were kept at 10°C., colloidal rotenone was much more toxic than crystalline rotenone 2 days after treatment. But if the insects were kept for about 3 weeks and inspected at intervals, the first apparent difference in toxicity grew smaller, and in the end it nearly disappeared. With DDT the time for this to occur was about 10 days; and suspensions of colloidal and crystalline DFDT were equitoxic after only 2 days, even at 10°C.
In the application test, the difference between colloidal and crystalline rotenone was a real difference in toxicity. It did not disappear with the lapse of time; but in the injection tests the two forms of each poison differed only in speed of action, and not in ultimate toxicity.     

Use of a Cellulase-Derepressed Mutant of Cellulomonas in the Production of a Single-Cell Protein Product from Cellulose

A cellulase-derepressed mutant of a Cellulomonas species was used to produce single-cell protein from crystalline cellulose. In preliminary tests, maximum yield of single-cell protein was obtained at 30°C (pH 7.0) with urea as the nitrogen source. A continuous-flow foam flotation procedure was developed for rapid and efficient separation of bacteria from the culture liquid and cellulose residue. A pH of 4.5 was optimum for foam flotation of this organism. In preliminary trials, recovery was 85% of the cells with the flotation procedure. Cellulomonas was 68% true protein and had an essential amino acid profile featuring a high lysine content (6.5% of protein). The Cellulomonas product was evaluated nutritionally with weanling rats. The net protein utilization value for the protein supplemented with methionine was 50.4% Weight gain of rats on the Cellulomonas diet was similar to that of rats fed a casein diet.     

鸡肉粉完全替代鱼粉饲料中补充包被氨基酸对凡纳滨对虾生长、体成分及组织氨基酸含量的影响

为了探讨鸡肉粉完全替代鱼粉时饲料氨基酸的平衡性以及外源氨基酸的添加方式与凡纳滨对虾生长、体成分、血浆游离氨基酸及肌肉氨基酸含量的关系, 本试验采用26因子试验设计进行了为期56d的饲养试验。2个饲料蛋白质水平分别为40%和32%, 6个饲料处理分别为鱼粉组(对照组)、鸡肉粉组、鸡肉粉+晶体EAA组、鸡肉粉+晶体EAA+晶体NEAA组、鸡肉粉+包被EAA组、鸡肉粉+包被EAA+包被NEAA组, 配制12组饲料。将凡纳滨对虾(0.300.01) g随机分配到36个圆桶(150 L)中, 每桶30尾, 每3个桶为一个处理组, 饲喂一种饲料, 每天饱食投喂三次。在每一饲料蛋白质水平下, 无论是补充晶体氨基酸(CAA)组还是包被氨基酸组对虾的增重率均显著高于鸡肉粉组(P0.05), 且在32%蛋白质水平下, 包被EAA组对虾增重率达到了鱼粉组水平(P0.05); 补充晶体EAA+NEAA组对虾增重率与补充晶体EAA组无差异(P0.05), 但均显著低于补充包被氨基酸组(P0.05); 补充包被EAA组对虾增重率显著高于补充包被EAA+NEAA组(P0.05)。饲料系数的变化正好与增重率变化相反(P0.05)。饲喂高蛋白质水平饲料较之饲喂低蛋白质饲料明显提高对虾增重率、虾体蛋白含量(P0.05), 但降低虾体脂肪含量(P0.05)。包被氨基酸组凡纳滨对虾血浆游离氨基酸含量总体显著低于CAA组(P0.05)。除谷氨酸、甘氨酸以及脯氨酸外, 各组对虾肌肉氨基酸含量无显著差异(P0.05)。结果表明, 在32%饲料蛋白质水平下, 用鸡肉粉完全替代鱼粉时, 饲料中补充包被EAA可明显促进凡纳滨对虾的生长, 且达到了鱼粉组的饲喂效果。     

Evaluation of Type A Botulinal Toxin Assays that Use Antitoxin to Crystalline Toxin

The type A botulinal toxin assay by the reverse passive hemagglutination procedure which uses antitoxin to crystalline toxin was examined for specificity. The analysis was based on the fact that crystalline type A toxin is a complex of neurotoxic protein (Aalpha) and a nontoxic protein (Abeta). By using these components, obtained in essentially pure forms, it was shown that the antitoxin to crystalline toxin has a significantly higher titer to Abeta than to Aalpha. When Formalin-treated red blood cells were sensitized with this antitoxin, the antibodies coupled to the cells were, for practical results, only anti-Abeta. When the suspension is reacted with dilutions of type A toxic solutions, the limiting dilutions are determined by Abeta and not by the neurotoxin, which should be the determinant if the assay is to measure toxicity. These observations may be pertinent to the development of serological assays for other botulinal toxin types.     

Biological and immunological properties of crystalline proteins from B. thuringiensis var. galleriae and var. insectus

The antigenic and toxic properties of the crystalline proteins from two varieties of B. thuringiensis (var. galleriae and var. insectus) were studied. Methods of double diffusion in agar and two-dimensional disc immunoelectrophoresis revealed four antigens in the crystalline protein from var. galleriae and three in that from var. insectus. A definite correlation was found between the toxicity of the crystalline protein and its capacity to interact with antiserum in a precipitation reaction in agar. It was established that the total protein content cannot be used as the criterion for an evaluation of the activity of preparations from B. thuringiensis. Immunodiffusion methods are recommended for an evaluation of the quality of these preparations.     

Effect of high-glucose levels on protein oxidation in cultured lens cells,and in crystalline and albumin solution and its inhibition by vitamin B6 and N-acetylcysteine: its possible relevance to cataract formation in diabetes

Diabetic patients have elevated levels of glucose in their blood and other body fluids. This project studied the effect of high-glucose concentrations (HG) on the protein oxidation in cultured lens cells and in crystalline protein solution. In addition, we also examined the effect of HG on the oxidation and turbidity (aggregation) of albumin protein solution. This study also examined whether vitamin B₆ [pyridoxine (P), pyridoxamine (PM)] or n-acetylcysteine (NAC) is capable of preventing protein oxidation similar to that seen in cataracts. For cell culture studies, rabbit lens cells were cultured in control or HG medium at 37°C for 2 d. For studies with protein solution, a buffered solution of serum albumin or crystalline protein was incubated with normal glucose (5 mM) or HG (50–100 mM) in a water bath at 37°C for 4 d. All treatments were carried out with and without the addition of P, PM, or NAC. We found significantly higher levels of carbonyl protein (an index of protein oxidation) in HG-treated compared with normal glucose-treated lens cells and in crystalline protein solution. P, PM, and NAC significantly decreased the protein oxidation in lens cells and crystalline protein solution. We also found significantly higher levels of protein oxidation and turbidity (an index of protein aggregation) and its inhibition by P, PM, and NAC in HG-treated compared with normal glucose-treated albumin solution. This suggests that HG can cause the oxidation and modification of proteins in the lens, and that vitamin B₆ and NAC supplementation may be helpful in slowing the oxidation of lens proteins. This study explains the cause of early cataract development and the potential benefit of supplementation with vitamin B₆ and NAC in the prevention of the development of cataract among the diabetic population.     

Crystalline inclusions of the mouse thyroid. Effect of chronic treatment with lithium gluconate

Intracytoplasmic crystalline bodies of various sizes are found in thyroid cells of 10-month-old mice and in younger animals under chronic lithium treatment. They are frequently surrounded by small microvesicles and dense bodies or enclosed in larger vesicles having a dense content. The crystalline skeleton is a network of protein fibers assembled in a characteristic axis with a periodicity of 8 nm. A deficiency of thyroid cell metabolism related to aging or lithium gluconate treatment would lead to an accumulation of substances of a crystalline pattern.     

Membranous cytochrome c oxidase. A freeze-fracture electron microscopic analysis

Suspensions of membranous cytochrome c oxidase prepared from beef heart mitochondria by Triton extraction were ultra-rapidly cooled (in excess of 10,000 deg.C/s) and analyzed using freeze-fracture and freeze-fracture-etch techniques. The preparations contained non-crystalline and crystalline vesicles as isolated vesicles, vesicles inside other vesicles and stacks of vesicles. In non-crystalline vesicles the particles (about 100 Å diameter) are probably formed by the deviation of hydrophobic fracture planes of the membranes around the large transmembrane enzymes. The intramembrane particles thus formed are compared to particles (about 80 Å diameter) in a vesicle reconstituted from purified enzyme and lipid. Crystalline membranous cytochrome c oxidase vesicles display an unusual fracture pattern in which adjacent crystalline surfaces are separated from each other and from the surrounding ice by fracture steps that are approximately the thickness of a single membrane (100 to 120 Å). In addition adjacent crystalline fracture surfaces have similar low-relief textures, both of which differ significantly from the hydrophobic surfaces normally exposed in membrane fractures. This fracture morphology is interpreted in terms of fractures along hydrophilic surfaces of the membranes. Images of etched crystalline vesicles provide support for this interpretation because etching exposes no new surfaces. It is concluded that the crystalline lattices are derived from the portions of enzymes that protrude from the membrane bilayers and that the interdigitation of the enzymes on the inside surfaces of the vesicles or between vesicles determines the appearance of the crystalline surfaces. The arrangement of the tails of the y-shaped molecules on the cytoplasmic sides of the crystalline membranes can be visualized in micrographs directly and in reconstructions of filtered images. The more complex pattern of arms protruding on the matrix side is obscured by the unidirectional shadowing. Fragmentation of the crystalline membranes during fracturing is indicated by particles sometimes present at the edges of fractured membranes and by deep, irregular pits observed in crystalline surfaces. Particles resting on some crystalline surfaces may be fragments of crystalline membranes removed during fracturing. In other crystalline membranes non-protein is removed during fracturing, leaving globular particles embedded in the lattice, which measure about 118 Å diameter. Comparing these particles to the 3-dimensional arrangement of protein described in the accompanying paper (Frey et al., 1982) suggests that such particles are composed of 2 dimers paired along the a-axis. Intramembrane and fragmentation particles of similar size may also have this protein composition.     

Multiplication of Nosocomial Pathogens in Intravenous Feeding Solutions

A major problem in total parenteral nutrition is sepsis, particularly that caused by Candida. Studies of four solutions, a casein hydrolysate, a fibrin hydrolysate, and two crystalline amino acid solutions, show that the protein hydrolysate solutions appear to be highly selective for Candida over bacteria, whereas the crystalline amino acid solutions are not. These findings suggest that the crystalline amino acid preparations may offer a partial solution to the infection problem by minimizing the contribution of the solution as a reservoir for organism multiplication, because they retard the growth of both bacteria and Candida.