Protein specific activity

Figure 7. Variation of arogenate dehydrogenase levels as a function of the physiological phase of growth in suspension cultures of Nicotiana sil-vestris. A stationary-phase inoculum was diluted into fresh medium and followed throughout the lag (L), exponential (E), and stationary (S) phases of growth. The hatched bar indicates the activity levels of EE cells, i.e., cells maintained continuously in exponential growth for 10 or more generations (53). Profiles are shown in which activity is related to soluble protein (specific activity), to cell number, or to dry weight.

So far, all AFGP analogs prepared in our laboratory have utilized the L-lysine-glycine-glycine tripeptide unit that is radically different in structure than the core repeating tripeptide unit found in native AFGP. The threonine residue in native AFGP was replaced with lysine for two reasons. Firstly, recent work has demonstrated that an alanine-lysine rich polypeptide possessed weak antifreeze protein-specific activity (26). 

The first generation C-linked analogs were assayed using two conventional techniques. These techniques are recrystallization-inhibition (RI) assay (29, 30) and nanoliter osmometry (31). The RI assay offers a distinct advantage in that it is effective at detecting antifreeze protein-specific activity at concentrations typically too low to cause a thermal hysteretic effect (29). 

Protein Specific Activity" (U/me) Molecular Mass (calc.) (daltons) Molecular Mass (obs.) (daltons) Stability (urea, pH 5.0) (kcal/mol) N-terminal Sequence... 

X 1 cm. column of 0-(diethylaminoethyl)ccllulose (washed with 0.002. 1/ Na-K phosphate buffer, pH 6.8) was prepared in a glass tube (having a sintered-glass bottom) which could be cooled to the required temperature. A volume of 6-8 ml. of enzyme solution (dialyzed earlier against 0.002 M phosphate buffer, pH 6.8, to remove ammonium sulfate) containing 10-13 mg. of protein (specific activity, about 5,000) was pipetted onto the top of the column. Elution was carried out wdth the same buffer (7-10 ml./hr.), and fractions were collected every 30... 

Membrane protein calculated as percentage of total mitochondrial protein. Specific activity ( jmoles substrate/minute/mg protein). 

The discussion of the ability of isolated nuclei to synthesize protein can be centered mainly on the elegant work of Allfrey and his coworkers [43-45], They have described their work in numerous review articles therefore, it is summarized only briefly here. A highly purified calf nuclei preparation was used in these experiments. When incubated in the presence of labeled amino acid (lysine, alanine, glycine, etc.), the nuclei rapidly incorporate the labeled amino acid. A curve of protein-specific activity was obtained for each type of amino acid. The curves expressing the counts per minute per milligram of protein versus time presented similar shapes. A lag period for the first 30 minutes was followed by a rapid, almost linear... 

Mole ratio of HNB-(CH3)2SC1 to protein Specific activity units/mg protein Unmodified Tip per mole of enzyme Modified Tip per mole of enzyme... 

Fig. 11. Formation and isolation of a [i- CJacetyl-N-acetyltransferase intermediate from a rapid inactivator rabbit liver. The DEAE-cellulose enzyme fraction (2.5 mg protein, specific activity 0.0395 /imole 2-acetylisonicotinic acid hydrazide formed per min per mg protein) was mixed with 16.4 nmoles of [i- C]acetyl-CoA (58.5 mC/mmole) and 0.01 M potassium phosphate buffer, pH 7.0, in a total volume of 0.40 ml. After 12 min of incubation at 27° the reaction mixture was cooled to 0°, placed on a Sepha-dex G-50 column (L5 cm X 18.5 cm) and eluted with 0.01 M potassium phosphate buffer, pH 7.0, at 0°. A flow rate of 1.0 ml/min was maintained and fractions of 1.0 ml each were collected over a 20-min period. The fractions were analyzed for radioactivity and for absorbance at 280 nm (A). The experiment was repeated with enzyme previously inactivated by heat at 55° for 15 min (B). In C, DEAE-cellulose enzyme fraction (2.5 mg protein, specific activity 0.0395 Ajmole 2-acetylisonicotinic acid hydra-...

Fig 2-Elution profile from a DEAE cellulose column(cm 2 X 30).Elution buffer 10 mM Tris-Cl,pH 8 500 mg proteins. Specific activity= 4moles " C02/hr/g. Insert=chromatographic pattern of active fraction on cellulose thin layer developped in butanol-pyridine -acetic acid-water(45 30 9 36)and sprayed with ninhydrin. 

Fig.3-Elution profile from a Sephadex G 25 superfine column(cml x 200)in ammonium bicarbonate 50 mM. 10 mg of protein.Specific activity=pmoles 002/ h/g.Fractions were collected as depicted in the figure and tested on cellulose thin layer as described in fig.2.

Enzymes are excellent catalysts for two reasons great specificity and high turnover rates. With but few exceptions, all reac tions in biological systems are catalyzed by enzymes, and each enzyme usually catalyzes only one reaction. For most of the important enzymes and other proteins, the amino-acid sequences and three-dimensional structures have been determined. When the molecular struc ture of an enzyme is known, a precise molecular weight could be used to state concentration in molar units. However, the amount is usually expressed in terms of catalytic activity because some of the enzyme may be denatured or otherwise inactive. An international unit (lU) of an enzyme is defined as the amount capable of producing one micromole of its reaction product in one minute under its optimal (or some defined) reaction conditions. Specific activity, the activity per unit mass, is an index of enzyme purity. 

Urokinase (from human urine) [9039-53-6] Mr 53,000, [EC 3.4.21.31]. Crystn of this enzyme is induced at pH 5.0 to 5.3 (4") by careful addition of NaCl with gentle stirring until the soln becomes turbid (silky sheen). The NaCl concentration is increased gradually (over several days) until 98% of saturation is achieved whereby the urokinase crystallises as colourless thin brittle plates. It can be similarly recrystd to maximum specific activity [104K CTA units/mg of protein (Sherry et al. J Lab Clin Med 64 145 1964)]. [Lesuk et al. Science 147 880 1965 NMR Bogusky et al. Biochemistry 28 6728 1989.] It is a plasminogen activator [Gold et al. Biochem J 262 1989 ]. 

Fraction Volume (mL) Total Protein (mg) Total Activity Specific Activity Percent Recovery ... 

The specific activity is die total activity of die fracdon divided by the total protein in die fracdon. This value gives an indication of die increase in purity attained during die course of the purification as die samples become enriched for xandiine dehydrogenase protein. 

Most purification procedures for a particular protein are developed in an empirical manner, the overriding principle being purification of the protein to a homogeneous state with acceptable yield. Table 5.5 presents a summary of a purification scheme for a selected protein. Note that the specific activity of the protein (the enzyme xanthine dehydrogenase) in the immuno-affinity purified fraction (fraction 5) has been increased 152/0.108, or 1407 times the specific activity in the crude extract (fraction 1). Thus, xanthine dehydrogenase in fraction 5 versus fraction 1 is enriched more than 1400-fold by the purification procedure. 

Stage Volume (ml) Total activity (IU) Total protein (mg) Specific activity (IU/mg) Purification factor Bound yield (%)... 

Besides cytoplasmic protein kinases, membrane receptors can exert protein kinase activity. These so-called receptor tyrosine kinases (RTK) contain a ligandbinding extracellular domain, a transmembrane motif, and an intracellular catalytic domain with specificity for tyrosine residues. Upon ligand binding and subsequent receptor oligomerization, the tyrosine residues of the intracellular domain become phosphory-lated by the intrinsic tyrosine kinase activity of the receptor [3, 4]. The phosphotyrosine residues ftmction as docking sites for other proteins that will transmit the signal received by the RTK. 

Finally, a number of studies146"149 have demonstrated that a variety of enzymes purified from tissues of older animals have a decreased specific activity when compared to the enzyme purified from younger animals. These proteins, however, appear to be identical as measured by a variety of physical and chemical parameters. It is possible that one of the differences is due to the accumulation of Met(O) residues in the proteins of the older animals. 

Microelectronic circuits for communications. Controlled permeability films for drug delivery systems. Protein-specific sensors for the monitoring of biochemical processes. Catalysts for the production of fuels and chemicals. Optical coatings for window glass. Electrodes for batteries and fuel cells. Corrosion-resistant coatings for the protection of metals and ceramics. Surface active agents, or surfactants, for use in tertiary oil recovery and the production of polymers, paper, textiles, agricultural chemicals, and cement. 

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