Carbonic anhydrase molecular weight

Carbonic anhydrase (CA) exists in three known soluble forms in humans. All three isozymes (CA I, CA II, and CA III) are monomeric, zinc metalloenzymes with a molecular weight of approximately 29,000. The enzymes catalyze the reaction for the reversible hydration of C02. The CA I deficiency is known to cause renal tubular acidosis and nerve deafness. Deficiency of CA II produces osteopetrosis, renal tubular acidosis, and cerebral calcification. More than 40 CA II-defi-cient patients with a wide variety of ethnic origins have been reported. Both syndromes are autosomal recessive disorders. Enzymatic confirmation can be made by quantitating the CA I and CA II levels in red blood cells. Normally, CA I and CAII each contribute about 50% of the total activity, and the CAI activity is completely abolished by the addition of sodium iodide in the assay system (S22). The cDNA and genomic DNA for human CA I and II have been isolated and sequenced (B34, M33, V9). Structural gene mutations, such as missense mutation, nonsense... 

Figure 14.4 Gel image of proteins extracted from a mixed carbonic anhydrase lysozyme tissue surrogate. Lane M, molecular weight marker lane 1, a 1 2 mol ratio mixture of native, non-formalin-treated carbonic anhydrase and lysozyme lane 2, mixed surrogate with 1 2 mol ratio carbonic anhydrase lysozyme, solubilized and retrieved in 20mM Tris-HCl, pH 4.0, with 2% SDS lane 3, mixed surrogate with 1 2 mol ratio carbonic anhydrase lysozyme, solubilized and retrieved in 20mM Tris-HCl, pH 6.0, with 2% SDS. Protein bands corresponding to lysozyme monomer (a), carbonic anhydrase monomer (b), and the putative lysozyme-carbonic anhydrase heterodimer (c) are indicated. For more detail, see Reference 25.

This reaction is essential in maintaining a constant pH in blood by the bicarbonate buffer system. Carbonic anhydrase, which contains a single zinc atom in its structure, has a molecular weight of about 30,000. In this structure, zinc is surrounded tetrahedrally by three histidine molecules and one water molecule. The exact role of the catalyst is not known, but it is believed to involve hydrolysis that can be represented as... 

Fig. 3 SDS-PAGE Photograph Separation (Lane Mr and A) and activity staining (Lane B and C) of the crude filtrate of Funalia trogii. Lane Mr standard molecular weight markers ([3-galactosi-dase, 118.0 kDa bovine serum albumin, 79.0 kDa ovalbumin, 47.0 kDa carbonic anhydrase, 33.0 kDa P-lactoglobulin, 25.0 kDa and lysozyme, 19.5 kDa). Relative mobilities of the standard markers vs. common logarithms of their molecular masses were plotted.With the linear regression output, the molecular masses of the proteins in the crude filtrate were estimated (taken from [18])...

Figure 7.1 (a) The denatured conformation of the zinc metalloenzyme carbonic anhydrase and the ESI mass spectra obtained under acidic denaturing conditions, (b) The ESI mass spectra obtained under native-state conditions. The decon-voluted ESI mass spectra of carbonic anhydrase reveals the protein molecular weight. The three dimensional structure is protein Data Bank ID IBNl. 

Fig. 2.1. Semilogarithmic plot of molecular weight (Mr) of marker proteins vs relative mobility (Rf) of marker proteins in gels of different acrylamide concentrations %T. Proteins 1 aprotinin (6.5 kD) 2 lysozyme (14.5 kD) 3 soybean trypsin inhibitor (21.5 kD) 4 carbonic acid anhydrase (31 kD) 5 hen ovalbumin (45 kD) 6 bovine serum albumin (66 kD) 7 phosphorylase b (97.4 kD) 8 8-galactosidase (116 kD) 9 myosin (205 kD)...

Figure B3.1.1 A 15% SDS-polyacrylamide gel stained with Coomassie brilliant blue. Protein samples were assayed for the purification of a proteinase, cathepsin L, from fish muscle according to the method of Seymour et al. (1994). Lane 1, purified cathepsin L after butyl-Sepharose chromatography. Lane 2, cathepsin L complex with a cystatin-like proteinase inhibitor after butyl-Sepharose chromatography. Lane 3, sarcoplasmic fish muscle extract after heat treatment and ammonium sulfate precipitation. Lane 4, sarcoplasmic fish muscle extract. Lanes M, low-molecular-weight standards aprotinin (Mr 6,500), a-lactalbumin (Mr 14,200), trypsin inhibitor (Mr 20,000), trypsinogen (Mr 24,000), carbonic anhydrase (Mr 29,000), gylceraldehyde-3-phosphate dehydrogenase (Mr 36,000), ovalbumin (Mr 45,000), and albumin (Mr 66,000) in order shown from bottom of gel. Lane 1 contains 4 pg protein lanes 2 to 4 each contain 7 pg protein.

The most studied carbonic anhydrase is the one isolated from bovine or human red blood cells. It contains one zinc per 30 000 molecular weight, and exists as two major isoenzymes, B and C. Each isoenzyme has a distinct amino acid composition with about 60% sequence homology. 

The most important aspect of the study of Co(II) metalloenzymes is the possibility of using the metal ion as a functional, built-in reporter of the dynamics of the active site. The spectral and magnetic properties of Co (II) carbonic anhydrase have given valuable clues to the catalytic function of this enzyme. The recent studies of Co(II) alkaline phosphatase and Co (II) carboxypeptidase A indicate the general applicability of this approach to enzymes where the probe properties of the constitutive metal ion are poor. The comparison of the absorption spectra of these enzymes and low-molecular weight models have shown that the proteins provide irregular, and in some cases nearly tetrahedral environments. It is obvious, however, that a knowledge of the crystal structures of the enzymes is necessary before the full potential of this method can be exploited. 

The enzyme also catalyses the hydrolysis of various esters such as 4-nitrophenyl acetate and sultones. A number of slightly different enzymes, carbonic anhydrases A, B and C occur in different organisms. The most well characterised enzymes are the bovine and human carbonic anhydrases B, which are monomeric and contain one tightly bound zinc per 30000 molecular weight. 

The molecular weight protein standards (tube 5) contain 1 mg/ml each of phosphory-lase (97,400 Da), bovine serum albumin (66,200 Da), ovalbumin (45,000 Da), carbonic anhydrase (31,000 Da), soybean trypsin inhibitor (21,500 Da), and lysozyme (14,400 Da) and will be provided by the instructor. 

Figure 2. SDS gel electrophoresis of the products of partial cystine cleavage for several test proteins. A. molecular weight standards, B. yeast alcohol dehydrogenase. C. P-lactoglobulin, D. hen egg lysozyme, E. ovalbumin, F. calf fetal serum fetuin. Molecular weight standards are indicated by arrows on the left side of the gel and are bovine serum albumin (66,300), bovine liver glutamate dehydrogenase (55,400), porcine muscle lactate ddiydiogenase (36,500), bovine erythrocyte carbonic anhydrase (31,000), soybean trypsin inhibitor (21,500), hen egg lysozyme (14,400), bovine lung aprotinin (6,000), unresolved bovine pancreatic insulin A and B chains.

A. Lane 1, Molecular weight markers lane 2, Purified final product. Molecular mass markers are Novex SeeBlue Pre-Stained Standards and range as follows (from top to bottom) Myosin, 250-kDa BSA, 98-kDa Glutamic dehydrogenase, 64-kDa Alcohol dehydrogenase, 50-kDa Carbonic anhydrase, 36-kDa Myoglobin, 30-kDa Lysozyme, 16-kDa Aprotinin, 6-kDa Insulin B chain, 4 kDa. 

Three genes coding for isoenzymes of carbonic anhydrase I, II, and III, all belonging to the same gene family, are clustered on chromosome 8q22. These are all zinc metalloenzymes, soluble, monomeric, and have molecular weights of 29,000. Isoenzyme I is found primarily in erythrocytes (Chapters 1 and 39) and III in skeletal muscle. There are yet other isoenzymes coded for by different genes. 

For an investigation of the capability of macromolecules to diffuse through the membranes, we used six test proteins ranging in molecular weight from 17000 to 80 000 Da myoglobin (MYO, 17000 Da) soybean trypsin inhibitor (STI, 20 000 Da) carbonic anhydrase (CAR, 29 000 Da) ovalbumin (OVA, 45 000 Da) bovine serum albumin (ALB, 66000 Da) and human transferrin (TFN, 80000 Da). 

Abbreviations are HC, hemocyanin from Helix pomatiar IG, nonspecific human y-im-munogiobulin AD, alcohol dehydrogenase from yeast Hb, human adult carbonmonoxy-hemoglobin CA, human erythrocyte carbonic anhydrase B and LY, hen egg-white lysozyme. The numbers next to the abbreviations give the molecular weights in units of thousands of Daltons. The relaxation rate of the protein-free solvent is indicated by the horizontal dashed line. After Ref. 7. 

FIGURE 7. Determination of the molecular weight of the purified PSPBP from Acanthocardia tuberculatum foot by gel electrophoresis on denaturing conditions. Molecular weight marker proteins were commercial myosine (205 kDa), P-galactosidase (116 kDa), phosphorylase B (97.4 kDa), albumin (66 kDa), ovalbumin (45 kDa) and carbonic anhydrase (29 kDa). The plot represents the relative mobilities of proteins vs. Log (Molecular Weight). 

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