Jimmy R. Calaycay

Brain Capillary 46,000 Dalton Protein is Cytoplasmic Actin and is Localized to Endothelial Plasma Membrane

The most abundant protein of the brain capillary, which makes up the blood-brain barrier (BBB) in vivo, is a protein that migrates at a molecular weight of approximately 46 kDa on sodium dodecylsulfate–polyacrylamide gel electrophoresis (SDS–PAGE). The bovine brain capillary 46kDa protein was purified by SDS–PAGE and Sephadex G–25 gel filtration. The purified protein migrated as a single band of molecular weight of approximately 42,000 Da on subsequent SDS–PAGE followed by silver staining. The protein was digested by trypsin and tryptic peptides were analyzed by reverse phase high-performance liquid chromatography (HPLC). Two of these peptides, 11 and 18 amino acids in length, were sequenced and found to be identical to amino acid sequences corresponding to portions of cytoplasmic actin. The SDS–PAGE gel-purified 46kDa protein was also subjected to limited proteolysis using S. aureus V8 protease, and this resulted in the formation of a prominent 31kDa doublet as well as smaller proteolytic fragments, and these fragments were of identical molecular weight to those generated from limited proteolysis of bovine actin. Electron microscopic immunoperoxidase studies with primary cultures of bovine brain capillary endothelium showed that immunoreactive actin is intimately associated with the plasma membranes. In conclusion, the brain capillary 46kDa protein is cytoplasmic actin and is localized to the endothelial plasma membrane. Modulations of brain capillary endothelial actin may play a role in the regulation of BBB permeability.

Microsequence analysis of peptides and proteins: IX. An improved, compact, automated instrument

We describe the construction of an improved, compact protein sequencer with a vertical flow path and continuous flow reactor (CFR). Unique features include a hexagonal valve for six fluid inputs to the CFR, which connects vertically to a transfer valve that allows sample, reagent, and solvent input to a conversion flask (CF). The simplified CF contains only two inputs at the top, one for sample, reagent, and solvent input, and the other a vent. The CF drains from the bottom, connecting to a switching valve which allows either delivery to waste or to an on-line HPLC for the analysis of phenylthiohydantoin amino acid derivatives. Approximately 90% of the sample is analyzed by use of a sonic flow detector. The overall vertical flow path of the sequencer is about 16 cm. The size of the instrument (25 w x 38 x 44 d cm) is smaller than that of commercially available sequencers or HPLC systems. The performance of the instrument includes reduced background peaks and high-sensitivity sequence analysis at the 5-10 pmol level. The simplified sequencer is more economical and portable than conventional sequencers.

Predominant low-molecular-weight proteins in isolated brain capillaries are histones

Abstract: Blood‐brain barrier (BBB) function is endowed by the expression of unique proteins within the brain capillary endothelium. In the absence of knowing the function of BBB‐specific proteins, one strategy for identification of these proteins is the purification and amino acid sequencing of proteins within the brain capillary that are not found in other cells. Earlier studies have shown that a 16‐18K triplet of low‐molecular‐weight proteins in isolated brain capillaries is not found in either erythrocytes or in capillary‐free preparations of synaptosomal proteins. Therefore, the present studies describe the purification of the 16‐18K triplet of proteins as well as a 14K protein in isolated brain capillaries using sodium dodecyl sulfate (SDS)‐polyacrylamide gel electrophoresis (PAGE) and C4 reverse‐phase HPLC. Amino acid sequencing of the N‐terminus of the 14K, 17K, and 18K proteins and of two tryptic peptides of the 16K protein showed that these proteins are α‐globin, histone 2B, histone 3, and histone 2A, respectively. SDS‐PAGE of subcellular fractions of bovine brain capillaries demonstrated that the 16‐18K triplet of histone proteins migrated in the nuclear fraction. In addition, a 34K doublet and a 200K protein were localized in the nuclear pellet. Therefore, the present studies demonstrate that the predominant 14‐18K proteins seen on SDS‐PAGE of isolated brain capillaries are known proteins and provide a general scheme for purification of brain capillary proteins isolated following SDS‐PAGE.

Structural Analysis of a 29/38kDa Heparin-Binding Domain of Fibronectin: Evidence that Two Different Subunits of Human Plasma Fibronectin Arise by Alternative mRNA Splicing

Fibronectins comprise a class of high molecular weight, multifunctional glycoproteins present in soluble form in plasma, amniotic fluid; and other body fluids and in insoluble form in extracellular matrices and basement membrane. Multiple functions attributed to this class of proteins include a role in malignancy, cellular adhesion, embryogenesis and development, opsonization and would healing(1–3). These:biological functions of fibronectins are based on their affinities to cell surfaces and a number of macromolecules including collagen, fibrin, heparin, DNA, actin, complement component Clq as well as to certain bacteria.